European Journal of Pharmacology 720 (2013) 16–28

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Minocycline modulates neuroprotective effect of hesperidin against quinolinic acid induced Huntington's disease like symptoms in rats: Behavioral, biochemical, cellular and histological evidences Anil Kumar n, Tanya Chaudhary, Jitendriya Mishra Pharmacology Division, University Institute of Pharmaceutical Sciences, UGC-Centre of Advanced Study (UGC-CAS), Panjab University, Chandigarh 160014, India

art ic l e i nf o

a b s t r a c t

Article history: Received 31 July 2013 Received in revised form 25 October 2013 Accepted 29 October 2013 Available online 5 November 2013

Emerging evidences indicate hesperidin, a citrus flavanone, attenuates neurodegenerative processes and related complications. Besides its anti-oxidant properties, the other probable mechanisms which underpin its neuroprotective potential are still not clear. In light of emerging role of flavonoids in modulating oxidative stress and neuro-inflammation, the study has been designed to explore the possible neuroprotective effect of hesperidin and its combination with minocycline (microglial inhibitor), against quinolinic acid (QA) induced Huntington's disease (HD) like symptoms in rats. Unilateral intrastriatal administration of QA (300 nmol/4 ml) significantly reduced body weight, impaired behavior (locomotor activity, beam balance and memory performance), caused oxidative damage (increased lipid peroxidation, nitrite concentration, depleted super oxide dismutase and reduced glutathione), demonstrated mitochondrial dysfunction (decreased Complex-I, II, III, and IV activities), increased striatal lesion volume and altered the levels of TNF-α, caspase-3 as well as BDNF expression, as compared to sham group. Meanwhile, chronic hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/kg, p.o.) treatment for 21 days significantly attenuated the behavioral, biochemical and cellular alterations as compared to QA treated (control) animals, whereas hesperidin (50 mg/kg, p.o.) treatment was found to be non-significant. However, treatment of hesperidin (50 mg/kg) in combination with minocycline (25 mg/kg) potentiated their neuroprotective effect, which was significant as compared to their effects per se in QA treated animals. Taken altogether, the results of the present study suggest a possible interplay of microglial modulation and anti-oxidant effect in neuroprotective potential of hesperidin against QA induced HD like symptoms in rats. & 2013 Elsevier B.V. All rights reserved.

Keywords: Apoptosis BDNF Microglia Mitochondrial dysfunction Neuro-inflammation

1. Introduction Huntington's disease (HD) is an autosomal neurodegenerative disorder caused by an expansion of the cytosine–adenine–guanine (CAG) repeat in the gene coding for the N-terminal region of the huntingtin protein (htt), leading to the formation of a polyglutamine stretch (mhtt) (Bano et al., 2011). The behavioral deficits manifest as typical involuntary choreiform movements, cognitive impairment and mood disorders, eventually compromising a person's daily functional abilities (Raymond et al., 2011). Unilateral quinolinic acid (QA) induced striatal lesions are highly reminiscent of histological (selective loss of GABAergic and cholinergic neurons) and neurochemical characteristics of HD in experimental animals. It is a well documented fact that over excitation of N-methyl-D-aspartate (NMDA) receptor following QA administration

n

Corresponding author. Tel.: þ 91 172 2534106; fax: þ 91 172 2541142. E-mail address: [email protected] (A. Kumar).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.057

results in profound oxidative damage, lipid peroxidation, mitochondrial dysfunction and apoptosis (Estrada Sanchez et al., 2008; PerezDe La Cruz et al., 2012) The role of microglial activation in the pathogenesis of HD has recently been addressed by clinical studies demonstrating a stark correlation between abnormal microglial activity and disease progression (Tai et al., 2007a). According to several study reports, microglial activation was found to be evident in pre-symptomatic HD gene carriers and can be detected up to 15 years before the onset of disease (Tai et al., 2007b). However the mechanism(s) by which mhtt causes the detrimental changes in microglial physiology are not clear yet. The localization of QA specifically to microglial cells following brain damage and subsequent increased expression of neuroinflammatory cytokines (interleukins, tumor necrosis factors) (Dihne et al., 2001; Ryu et al., 2005) has also been demonstrated in several in vivo studies (Lehrmann et al., 2001). However, neuroprotective potential of minocycline, a second generation semi-synthetic tetracycline derivative has recently been reported in various neurodegenerative conditions including

A. Kumar et al. / European Journal of Pharmacology 720 (2013) 16–28

HD (Lee et al., 2003; Wu et al., 2002; Chen et al., 2000). Besides, studies from our laboratory also demonstrated the neuroprotective potential of hesperidin in several neurodegenerative conditions including QA induced neurotoxicity in wistar rats (Kalonia et al., 2012; Kumar et al., 2012a). However, the mechanisms by which minocycline exerts neuroprotective effects in CNS disorders also incorporate the capacity to inhibit neuronal apoptosis (Lee et al., 2003; Wang et al., 2003) and free radical formation (Jiang et al., 2009). Neuroprotective potential of hesperidin has been reported extensively in both in vitro as well as in vivo studies which is attributed primarily to its anti-oxidant and anti-inflammatory activities (Gaur and Kumar, 2010; Menze et al., 2012; RaineySmith et al., 2008). Besides, the possible involvement of nitric oxide mechanism in the neuroprotective potential of hesperidin has also been demonstrated by our own group (Gaur et al., 2011; Kumar and Kumar, 2010). Recently, microglial pathway in the neuroprotective effect of hesperidin has also been targeted (Koppula et al., 2012; Yamamoto and Saneyoshi, 2009), however, there is lack of convincing data around this hypothesis. Therefore, the present study has been designed to explore microglial pathway in the neuroprotective effect of hesperidin based upon its interaction with minocycline (microglial inhibitor) against QAinduced neurobehavioral, neurochemical, behavioral, and histopathological alterations in rats.

2. Materials and methods 2.1. Animals Male wistar rats (250–300 g) bred in Central Animal House, Panjab University, Chandigarh were used in this study. The animals were kept under standard laboratory conditions, maintained on 12-h light/dark cycle and have free access to food and water. All the experimental procedures were performed between 9:00 and 17:00 hours. The experimental protocol was approved by Institutional Animal Ethics Committee (IAEC) of Panjab University (Protocol no. 282/30/8/12/UIPS-42) and carried out in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA), Government of India and Indian National Science Academy Guidelines for the use and care of experimental animals. 2.2. Intrastriatal administration of QA The rats were anesthetized with thiopental sodium (45 mg/kg, i.p.) and placed in a stereotaxic apparatus. The surface of the skull was exposed by making incision on the scalp. A 1–2 mm diameter hole was made in the skull for microinjection using a small hand drill at anterior þ 1.7 mm; lateral 72.7 mm; ventral  4.8 mm from bregma and dura as described by Paxinos and Watson (2007). QA (300 nmol/4 ml) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in normal saline (pH 7.4) and administered unilaterally in right striatum via a 28-gauge stainless steel needle attached to a 10 μl Hamilton syringe. A total volume of 4 μl of QA was delivered slowly over a period of 2 min and injection needle was left in place for another 2 min to prevent back diffusion of the injected drug solution.

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Fig. 1. Experimental design.

volume of 0.5 ml per 100 g of body weight. Each group received treatment daily in the morning 10:00 hours, for 21 days starting from day 1 after recovery from experimental procedure (Fig. 1). Doses of hesperidin and minocycline were selected on the basis of our previous study reports (Gaur et al., 2011; Kalonia et al., 2012; Kumar and Kumar, 2010). The entire protocol involves seven treatment groups with eleven animals (n ¼11) in each treatment arm (Table 1). Several studies in our laboratory reported that, hesperidin (100 mg/kg, p.o.) and minocycline (100 mg/kg, p.o.) per se groups did not exhibit any significant difference in the behavioral, biochemical and mitochondrial parameters as compared to the naive/sham animals (Kalonia et al., 2012; Kumar and Kumar, 2010). Therefore, per se groups for hesperidin and minocycline at the above mentioned doses have been excluded from the present study protocol in order to minimize the use of experimental animals as per CPCSEA guidelines and protocol has been designed with an assumption that hesperidin and minocycline do not possess any per se effect in rats. 2.4. Measurement of body weight The body weights of the animals were recorded after intrastriatal administration of QA (day 1) and on the last day of the study (21st day). Percentage change in body weight was calculated as Percentage change in body weight ¼ body weight

ðday21  day1Þ  100 ðday1Þ

2.5. Behavioral assessments 2.5.1. Assessment of gross behavioral activity (locomotor activity) The locomotor activity was assessed using actophotometer (IMCORP, Ambala, India) on weekly intervals. Animals were placed individually in the activity chamber for 3 min as a habituation period before recording actual motor activity for next 5 min. The instrument consisted of a closed arena equipped with 12 infrared light-sensitive photocells in two rows (six in each row), at a distance of 3 cm and 9 cm respectively and values expressed as counts per 5 min. The beams in actophotometer cut by the animals were taken as the measure of movements. The values were expressed as counts per 5 min (Kumar et al., 2012b).

2.3. Drug and treatment schedule Hesperidin (25, 50, and 100 mg/kg) (Sigma-Aldrich, St. Louis, MO, USA) and minocycline (25 mg/kg) (Wyeth Ltd., Mumbai, India) were suspended in 0.25% (w/v) sodium carboxy methyl cellulose (CMC) solution and administered per oral in a constant

2.5.2. Balance beam walking (hind limb functioning) test This behavioral test assesses the motor performance of animals by their ability to walk across an elevated tapered beam of increasing the difficulty provided with an under-hanging ledge which serves as a crutch for the animal to use when there is a deficit. The setup

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Table 1 Grouping of experimental animals. Sl. no.

Group

Treatment

1 2 3 4 5 6 7 8

Naive Sham Control Mino (25) Hesp (25) Hesp (50) Hesp (100) Hesp (50)þ Mino (25)

No treatment Surgery performed and only vehicle administered Single unilateral intrastriatal administration of QA (300 nmol/4 μl) Minocycline (25 mg/kg, p.o.) þQA (300 nmol/4 μl) Hesperidin (25 mg/kg, p.o.) þ QA (300 nmol/4 μl) Hesperidin (50 mg/kg, p.o.) þ QA (300 nmol/4 μl) Hesperidin (100 mg/kg, p.o.) þQA (300 nmol/4 μl) Hesperidin (50 mg/kg, p.o.) þ Minocycline (25 mg/kg, p.o.) þ QA (300 nmol/4 μl)

consists of a flat rigid wooden beam of 165 cm length, equally divided into three color coded regions of 45 cm each, supported between two platforms elevated 65 cm above the ground. The tapered end of the beam hosts a dark wooden box (goal-box) to act as a reinforcer. Prior to the surgery, the animals were habituated to the set-up by progressive beam training with the aim of completing the task by 3 min without turning around and without making any foot faults. In the test phase, number of foot slips (placement of limb on the ledge crutch) was counted and motor performance was assessed on a scale ranging from 0 to 4 based upon the distance traveled on the beam. A score of 4 was assigned to animal that could readily traverse the beam. Scores 3, 2 and 1 were assigned to animals demonstrating mild, moderate and severe impairment, respectively, whereas score 0 was assigned to the animals completely unable to walk on the beam (Wang et al., 2006). 2.5.3. Elevated plus maze test for spatial memory performance Elevated plus maze consists of two opposite open arms (50  10 cm), crossed with two closed arms of same dimensions with 40 cm high walls. The arms are connected to a central square (10  10 cm) elevated at a height of 50 cm from ground level. Acquisition memory was assessed on 20th day after intrastriatal administration of QA. Rats were placed individually at one end of an open arm facing away from the central square. The time taken by the animal to move from open arm and enter into one of the closed arms was recorded as initial transfer latency (ITL). Rats were allowed to explore the maze for 30 s after recording initial acquisition latency and returned to its home cage. Retention latency was noted again on the 21st day (Kumar et al., 2006). Percentage retention of memory was calculated by the following formula: Percentage retention of memory ¼ transferlattency

ðday20  day21Þ ðday21Þ

100 2.6. Dissection and homogenization After behavioral assessments on day 21, the animals were randomized into different groups. The first group of animals was used for estimation biochemical parameters, second group for mitochondrial enzyme complex activities and third for striatal lesion volume measurement. The animals were sacrificed by decapitation. Whole brains were rapidly removed and placed on ice. For the biochemical and mitochondrial enzyme complex estimations, striatum regions were traced out by removing the cortex and other brain tissues.

were centrifuged at 10,000g at 4 1C for 15 min. Aliquots of supernatants were separated and used for biochemical estimations. 2.7.1. Estimation of lipid peroxidation The quantitative measurement of lipid peroxidation in brain was performed according to the method of Wills (1966). The amount of malondialdehyde (MDA), a measure of lipid peroxidation was measured by reaction with thiobarbituric acid at 532 nm using a Perkin-Elmer lambda 20 spectrophotometer (Norwalk, CT, USA). The values were calculated using molar extinction coefficient of chromophore (1.56  105 M  1 cm  1) and expressed as nanomoles of malondialdehyde per milligram of protein. 2.7.2. Estimation of nitrite The accumulation of nitrite in the supernatant, an indicator of the production of nitric oxide (NO), was determined with a colorimetric assay with Greiss reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, 1% sulfanilamide and 2.5% phosphoric acid) as described by Green et al. (1982). Equal volume of supernatant and Greiss reagent was mixed. The mixture was incubated for 10 min at room temperature and the absorbance was determined at 540 nm. The concentration of nitrite in the supernatant was determined from a sodium nitrite standard curve and expressed as micromole per litre. 2.7.3. Estimation of superoxide dismutase (SOD) activity Superoxide dismutase (SOD) activity was accessed according to Kono (1978), wherein reduction of nitrobluetetrazolium (NBT) was inhibited by superoxide dismutase and measured at 560 nm using spectrophotometer. Briefly, the reaction was initiated by addition of the hydroxylamine hydrochloride to the mixture containing NBT and sample. The results were expressed as units/milligram of protein, where one unit of enzyme is defined as the amount of enzyme inhibiting the rate of reaction by 100%. 2.7.4. Estimation of reduced glutathione (GSH) Reduced glutathione in brain was estimated as described by Ellman (1959). One milliliter supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4 1C for 1 h. The sample was centrifuged at 1200 rpm for 15 min at 4 1C. To 1 ml of this supernatant, 2.7 ml of 0.1 M phosphate buffer (pH 8) and 0.2 ml of 5,5-dithiobis-2-nitrobenzoic acid (DTNB) was added. The absorbance was read immediately at 412 nm and results were calculated using molar extinction coefficient of chromophore (1.36  l04 M  1 cm  1) and were expressed as micromole GSH per milligram protein.

2.7. Biochemical estimations For the biochemical estimations, 10% (w/v) tissue homogenates were prepared in 0.1 M phosphate buffer (pH 7.4). The homogenates

2.7.5. Protein estimation The protein was measured by biuret method using bovine serum albumin as standard (Gornall et al., 1949).

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2.7.6. Estimation of tumor necrosis factor-alpha (TNF-α) The quantifications of TNF-α was done using rat Quantikine TNF-α immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instruction. The assay employs a 4.5 h solid phase sandwich enzyme immunoassay technique. Monoclonal antibody specific for rat TNF-α is pre-coated in the microplate. Standard, control, and samples were pipetted into the wells and any rat TNF-α present was bound by the immobilized antibody. The intensity of the color measured is in proportion to the amount of rat TNF-α bound in the initial steps. The sample values were then read off the standard curve. 2.7.7. Caspase-3 colorimetric assay Caspase-3, also known as CPP-32, Yama or Apopain, is an intracellular cysteine protease that exists as a pro-enzyme, becoming activated during the cascade of events associated with apoptosis. The tissue lysates/homogenates can then be tested for protease activity by the addition of a caspase specific peptide that is conjugated to the color reporter molecule p-nitroanaline (pNA). The cleavage of the peptide by the caspase releases the chromophore pNA, which can be assessed spectrophotometrically at a wavelength of 405 nm. The level of caspase enzymatic activity in the cell lysate/homogenate is directly proportional to the color reaction. The enzymatic reaction for caspase activity was carried out using caspase-3 colorimetric kit (R&D Systems, Minneapolis, MN, USA). 2.7.8. BDNF estimation BDNF assay was done using rat ChemiKineTM BDNF Sandwich ELISA kit (Millipore, USA) according to the manufacturer's instruction. In the assay system, rat monoclonal antibodies generated against human BDNF are coated onto a microplate and are used to capture BDNF from a sample. Standard, control, and test samples were pipetted into the wells and the color produced was immediately read at 450 nm. The sample values were then read off from the standard curve. 2.8. Mitochondrial enzyme complex estimation 2.8.1. Isolation of rat brain mitochondria Second group of animals were used for mitochondrial isolation as described in the method of Berman and Hastings (1999). The brain regions were homogenized in isolated buffer. Homogenate was centrifuged at 13,000g for 5 min at 4 1C. Pellet was resuspended in isolation buffer with ethylene glycol tetra acetic acid (EGTA) and spun again at 13,000g at 4 1C for 5 min. The resulting supernatant was transferred to new tubes and topped off with isolation buffer with EGTA and again spun at 13,000g at 4 1C for 10 min. Pellets containing pure mitochondria was resuspended in isolation buffer without EGTA. 2.8.2. NADH dehydrogenase (Complex I) activity NADH dehydrogenase activity was measured spectrophotometrically by the method of King and Howard (1967). The method involves catalytic oxidation of NADH to NAD þ with subsequent reduction of cytochrome C. The reaction mixture contained 0.2 M glycyl glycine buffer (pH 8.5), 6 mM NADH in 2 mM glycyl Glycine buffer and 10.5 mM cytochrome C. The reaction was initiated by addition of requisite amount of solubilized mitochondrial sample and absorbance change was read at 550 nm for 2 min. 2.8.3. Succinate dehydrogenase (SDH) (Complex II) activity Succinate dehydrogenase was measured spectrophotometrically according to method of King (1967). The method involves oxidation of succinate by an artificial electron acceptor, potassium

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ferricyanide. The reaction mixture contained 0.2 M phosphate buffer (pH 7.8), 1% BSA, 0.6 M succinic acid, and 0.03 M potassium ferricyanide. The reaction was initiated by the addition of mitochondrial sample and absorbance change was read at 420 nm for 2 min. 2.8.4. Mitochondrial redox (Complex III) activity The MTT assay was based on the reduction of (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-H-tetrazolium bromide) (MTT) by hydrogenase activity in functionally intact mitochondria. The MTT reduction rate was used to assess the activity of the mitochondrial respiratory chain in isolated mitochondria by the method of Liu et al. (1997). Briefly, 100 μl mitochondrial samples were incubated with 10 μl MTT for 3 h at 37 1C. The blue formazan crystals were solubilized with dimethyl sulfoxide and measured by an ELISA reader at 580 nm filter. 2.8.5. Cytochrome oxidase (Complex IV) assay Cytochrome oxidase activity was assayed in brain mitochondria according to the method as described by Sottocasa et al. (1967). The assay mixture contained 0.3 mM reduced cytochrome C in 75 mM phosphate buffer. The reaction was started by the addition of solubilized mitochondrial sample and absorbance change was recorded at 550 nm for 2 min. 2.9. Striatal lesion volume measurement At the end of drug administration, animals were sacrificed for TTC (2,3,5-triphenyltetrazolium chloride) staining. Brains were quickly removed, placed in ice-cold saline solution and were then sectioned at 2-mm intervals using the rat brain matrix. Sections were then stained with 2% TTC prepared in normal saline at 37 1C for 30 min in the dark, then removed and placed in 4% formaldehyde, in 0.1 M phosphate buffer (pH 7.4). For measurement of lesion volume, serial, coronal sections (25 mm) were cut throughout the entire striatum using a microtome (Maragos et al., 2004). Using computer-based image analysis (Image J 1.42q, NIH, USA), lesion volume was calculated by multiplying lesion volume from each section and the distance between the sections (Kim et al., 2005). 2.10. Statistical analysis A group of eleven animals (n¼ 11) was assigned to a specific drug treatment. Results were expressed as mean 7S.E.M. The data were analyzed by analysis of variance (ANOVA) followed by Tukey's test. The non-parametric data (neurological score) was analyzed by Kruskal Wallis test followed by Dunn's multiple comparison test. P o0.05 was considered to be statistically significant. Graph Pad Prism (Graph Pad Software, San Diego, CA) was used for all statistical analysis.

3. Results 3.1. Effect of hesperidin, minocycline and their combination on body weight in QA treated rats Single intrastriatal QA (300 nmol/4 μl) administration significantly reduced body weight on day 21 as compared to sham group. Sham treatment did not show any significant effect on body weight as compared to naive group of animals. Unlike hesperidin (50 mg/kg, p.o.), hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/kg, p.o.) treatment for 21 days significantly attenuated the loss in body weight as compared to QA treated group (Fig. 2). However, hesperidin (50 mg/kg) treatment in combination with

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Fig. 2. Effect of hesperidin, minocycline and their combination on body weight (percentage reduction) in QA-treated rats. Values are expressed as mean7 S.E.M. #P o0.05 as compared to sham. nP o0.05 as compared to QA (300). @Po 0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼ Quinolinic acid; Mino ¼Minocycline; and Hesp ¼ Hesperidin.

Fig. 3. Effect of hesperidin, minocycline and their combination on locomotor activity in QA-treated rats. Values are expressed as mean 7 S.E.M. (percentage of sham). # P o0.05 as compared to sham. nPo 0.05 as compared to QA (300). @P o0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼ Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin.

minocycline (25 mg/kg) for 21 days potentiated their protective effect (improved body weight), which was significant as compared to their effects per se in QA treated animals (one way ANOVA followed by Tukey's test; P o0.001; DFn ¼7; DFd ¼80; and F¼32.35). Moreover, hesperidin (25 mg/kg, p.o.) treatment for 21 days did not exhibit any protection in attenuating the loss in body weight as compared to QA treated group (data not shown). 3.2. Effect of hesperidin, minocycline and their combination on locomotor activity in QA treated rats Locomotor activity in all treatment arms was invariable prior to surgery (day-1). Single intrastriatal QA (300 nmol/4 μl) administration significantly increased locomotor activity on day 7 (1st week), however with progression of disease, a substantial decrease in locomotor activity in 2nd (day 14) and 3rd week (day 21) was observed in the QA treated animals as compared to sham treated group. No significant difference in locomotor activity was observed between sham and naive group in all treatment weeks. Animals treated with hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/ kg, p.o.) for 21 days displayed a significant improvement in locomotor activity at the end of 2nd and 3rd week as compared

to QA treated group, whereas, animals treated with lower dose of hesperidin (50 mg/kg, p.o.) displayed significant improvement in locomotor activity by end of 3rd week only (Fig. 3). Meanwhile, hesperidin (50 mg/kg) treatment in combination with minocycline (25 mg/kg) for 21 days potentiated their protective effect (improved locomotor activity), which was significant as compared to their effects per se in QA treated animals (two-way ANOVA followed by Bonferroni's test; P o0.001; DFn ¼7.3; DFd ¼320; and F¼11.38). However, hesperidin (25 mg/kg, p.o.) treatment for 21 days did not display any significant protective effect on locomotor activity throughout the treatment period as compared to QA treated group (data not shown). 3.3. Effect of hesperidin, minocycline and their combination on balance beam test in QA treated rats Single intrastriatal QA (300 nmol/4 μl) administration significantly increased number of foot slips as well as decreased the distance traveled (assessed by neurological scoring) on balance beam as compared to sham group, whereas sham treated animals did not display any significant effect on balance beam performance as compared to naive animals. Chronic treatment with hesperidin

A. Kumar et al. / European Journal of Pharmacology 720 (2013) 16–28

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Fig. 4. (a) Effect of hesperidin, minocycline and their combination on neurological score in QA-treated rats. Values are expressed as mean 7S.E.M. #P o 0.05 as compared to sham. nPo 0.05 as compared to QA (300). @P o0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼ Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin. (b) Effect of hesperidin, minocycline and their combination on number of slips in QA-treated rats. Values are expressed as mean7 S.E.M. #Po 0.05 as compared to sham. nP o 0.05 as compared to QA (300). @Po 0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA¼ Quinolinic acid; Mino¼Minocycline; and Hesp ¼ Hesperidin.

(100 mg/kg, p.o.) as well as minocycline (25 mg/kg, p.o.) significantly attenuated number of foot slips (Fig. 4a) but could not restore the neurological score as compared to QA treated group. However, hesperidin (50 mg/kg, p.o.) treatment for 21 days could not reverse the balance beam test parameters albeit its combination with minocycline (25 mg/kg), caused potentiation in their protective effect (decreased number of foot slips as well as improved neurological score), which was significant as compared to their effects per se in QA treated animals [No. of slips: one way ANOVA followed by Tukey's test; Po 0.001; DFn ¼ 7; DFd¼ 80; F¼34.04 and Neurological score: Kruskal Wallis test followed by Dunn's multiple comparison test; P o0.001; Kruskal Wallis statistic ¼ 33.99]. However, hesperidin (25 mg/kg, p.o.) treatment for 21 days did not show any significant effect on beam walk performance as compared to QA treated group (data not shown). 3.4. Effect of hesperidin, minocycline and their combination on memory retention in elevated plus maze paradigm in QA treated rats In elevated plus maze performance task, single intrastriatal QA (300 nmol/4 μl) administration significantly delayed retention transfer latency as compared to sham treatment, whereas 21 days treatment with hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/kg, p.o.), significantly improved mean transfer latency (retention memory) as compared to QA treated group (Fig. 5). In contrast, administration of

hesperidin (50 mg/kg, p.o.) did not demonstrate any significant effect on transfer latency, whereas co-administration of hesperidin (50 mg/kg) and minocycline (25 mg/kg) for 21 days potentiated their protective effect (improved memory retention) which was significant as compared to their effects per se in QA treated animals (one way ANOVA followed by Tukey's test; Po0.001; DFn¼7; DFd¼ 80; and F¼16.04). However, hesperidin (25 mg/kg, p.o.) treatment for 21 days did not show any significant improvement in memory performance as compared to QA treated group (data not shown). 3.5. Effect of hesperidin, minocycline and their combination on striatal oxidative damage (lipid peroxidation, nitrite, superoxide dismutase and reduced glutathione) in QA treated rats Single intrastriatal QA administration (300 nmol/4 μl) significantly increased lipid peroxidation and nitrite levels but conversely depleted endogenous anti-oxidant enzymes (SOD, GSH) in the striatum as compared to the sham group. However, sham treatment did not show any significant effect on these parameters as compared to naive group animals. Further, 21 days treatment with hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/kg, p.o.), significantly attenuated oxidative stress (decreased lipid peroxidation, nitrite concentration, restored SOD and GSH enzyme activities) as compared to QA treated group, whereas hesperidin (50 mg/kg, p.o.) treatment could not completely restore the QA

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Fig. 5. Effect of hesperidin, minocycline and their combination on percentage memory retention in QA-treated rats. Values are expressed as mean 7S.E.M. #Po 0.05 as compared to sham. nP o0.05 as compared to QA (300). @Po 0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼ Quinolinic acid; Mino ¼Minocycline; and Hesp ¼ Hesperidin. Table 2 Effect of hesperidin, minocycline and their combination on striatal oxidative damage (lipid peroxidation, nitrite, SOD and reduced glutathione) in QA treated rats. Group

MDA nmol/mg protein (% of sham)

Nitrite level μmol/mg protein (% of sham)

SOD nmol/mg protein (% of sham)

GSH nmol/mg protein (% of sham)

Naive Sham QA (300) Mino (25) Hesp (50) Hesp (100) Hesp (50)þ Mino (25)

96.0 7 5.9 100.0 7 6.1 186.3 7 5.9a 150.4 7 6.1b 152.6 7 7.6b 141.6 7 6.5b 111.2 7 9.5c,d

95.2 7 4.5 100.0 7 4.5 175.6 7 9.4a 141.5 7 9.6b 140.5 7 5.6b 130.5 7 7.3b 113.5 7 7.9c,d

105.8 76.0 100.0 76.3 47.6 77.2a 77.8 76.9b 65.2 75.1 83.6 75.7b 107.5 77.5c,d

103.3 7 5.6 100.0 7 6.4 41.5 7 7.8a 76.5 7 7.5b 55.9 7 7.6b 85.6 7 9.1b 110.5 7 6.2c,d

Values are expressed as mean7 SEM (percentage of sham). QA¼ Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin. a

P o0.05 as compared to sham. Po 0.05 as compared to control. c Po 0.05 as compared to Mino (25). d Po 0.05 as compared to Hesp (50). b

induced oxidative damage (reduced SOD and GSH levels) (Table 2). Interestingly, 21 days treatment of hesperidin (50 mg/kg) in combination with minocycline (25 mg/kg), significantly potentiated their protective effect (decreased oxidative stress), which was significant as compared to their effects per se in QA treated animals one way ANOVA followed by Tukey's test; MDA (P o0.001; DFn ¼7; DFd ¼24; and F¼ 23.72); Nitrite (Po0.001; DFn ¼7; DFd ¼24; and F¼16.44); SOD (P o0.001; DFn ¼7; DFd¼ 24; and F¼11.79); and GSH (P o0.001; DFn ¼ 7; DFd ¼24; and F¼ 11.88). However, hesperidin (25 mg/kg, p.o.) treatment for 21 days was found to have non-significant effect on QA induced oxidative stress parameters as compared to QA treated group (data not shown).

attenuated the increase in levels of capase-3 [one way ANOVA followed by Tukey's test; (Po0.001; DFn¼7; DFd¼24; and F¼23.39)]; (Fig. 6a) and TNF-α (one way ANOVA followed by Tukey's test; Po0.001; DFn¼7; DFd¼ 80; and F¼25.81) (Fig. 7), and conversely restored the BDNF levels [one way ANOVA followed by Tukey’s test; Po0.001; DFn¼7; DFd¼ 24; and F¼15.87] (Fig. 6b) as compared to QA treated group. Further, treatment of hesperidin (50 mg/kg) in combination with minocycline (25 mg/kg), potentiated their protective effect, which was significant as compared to their effects per se in QA treated animals. However, hesperidin (25 mg/kg, p.o.) treatment for 21 days did not show any significant effect as compared to QA treated group (data not shown).

3.6. Effect of hesperidin, minocycline and their combination on caspase-3, BDNF and TNF-α level in the striatum of QA treated rats

3.7. Effect of hesperidin, minocycline and their combination on mitochondrial enzyme complexes (I–IV) activities in the striatum of QA treated rats

Single intrastriatal QA administration (300 nmol/4 μl) resulted in significant increase in the levels of apoptotic marker (caspase-3) and pro-inflammatory cytokine (TNF-α) but conversely reduced the levels of BDNF in the striatum, as compared to sham group. However, sham treatment did not show any significant effect on these parameters as compared to naive group. Unlike the lower dose of hesperidin (50 mg/kg, p.o.), hesperidin (100 mg/kg, p.o) and minocycline (25 mg/kg, p.o.) treatment for 21 days significantly

Single intrastriatal QA (300 nmol/4 μl) administration significantly impaired mitochondrial enzyme complex activities (I–IV) in the striatum as compared to the sham group, whereas the sham treatment did not show any significant effect on these alterations as compared to naive group. However, hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/kg, p.o.) administration for 21 days, significantly restored mitochondrial enzyme complex activities (I–IV), while, hesperidin (50 mg/kg, p.o.) treatment could not

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Fig. 6. (a) Effect of hesperidin, minocycline and their combination on caspase-3 levels in the striatum of QA-treated rats. (b) Effect of hesperidin, minocycline and their combination on BDNF levels in the striatum of QA-treated rats. Values are expressed as mean 7S.E.M. (percentage of sham). #Po 0.05 as compared to sham. nPo 0.05 as compared to QA (300). @P o0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin.

Fig. 7. Effect of hesperidin, minocycline and their combination on TNF-α levels in the striatum of QA-treated rats. Values are expressed mean7 S.E.M. (percentage of sham). # P o 0.05 as compared to sham. nPo 0.05 as compared to QA (300). @P o0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼ Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin.

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attenuate the decrease in mitochondrial enzyme complex activities (Table 3) as compared to QA treated group. Meanwhile, treatment of hesperidin (50 mg/kg) in combination with minocycline (25 mg/kg), caused potentiation in their protective effect on mitochondrial enzyme complex activity, which was significant as compared to their effects per se in QA treated animals [one way ANOVA followed by Tukey's test; Complex I (P o0.001; DFn ¼7; DFd¼ 24; and F ¼13.15); Complex II (P o0.001; DFn ¼7; DFd ¼24; and F¼13.53); Complex III (Po 0.001; DFn ¼ 7; DFd ¼24; and F¼11.60); and Complex IV (P o0.001; DFn ¼ 7; DFd ¼24; and F¼14.26)]. However, hesperidin (25 mg/kg, p.o.) treatment for 21 days did not restore mitochondrial alterations significantly as compared to QA treated group (data not shown). 3.8. Effect of hesperidin, minocycline and their combination on striatal lesion volume in QA treated rats Single unilateral intrastriatal administration of QA (300 nmol/ 4 μl) resulted in highly reliable striatal lesion (evident from striatal lesion volume and photomicrographs) as compared to the sham group. Unlike hesperidin (50 mg/kg, p.o.), treatment with hesperidin (100 mg/kg, p.o.) and minocycline (25 mg/kg, p.o.) for 21 days, significantly reduced the QA induced striatal lesion volume (Figs. 8 and 9). However, treatment of hesperidin (50 mg/kg) in combination with minocycline (25 mg/kg) potentiated the

neuroprotective effect as evident from the decreased striatal lesion volume, which was significant as compared to their effects per se in QA treated animals (one way ANOVA followed by Tukey's test; Po 0.001; DFn ¼7; DFd ¼16; and F¼23.15). Meanwhile, hesperidin (25 mg/kg) treatment for 21 days did not show any significant decrease in striatal lesion volume as compared to QA treated group (data not shown).

4. Discussion In the present study, single unilateral intrastriatal administration of QA displayed Huntington's-like symptoms in rats as evident from behavioral alterations (impairment in locomotor activity, balance beam walk test and memory performance). The motor abnormalities can be explained on the basis of death of dopaminergic neurons in the striatum due to QA-induced excitotoxicity and other related cellular cascades such as oxidative stress (Perez-De La Cruz et al., 2012), mitochondrial dysfunction (Kalonia and Kumar, 2011; Kalonia et al., 2010) and microglial activation (Guillemin, 2012). Excitotoxicity induced by QA results in oxidative stress due to increased ROS/RNS production and related impairment in mitochondrial oxidative phosphorylation which further leads to oxidative damage. However, formation of pro-oxidant complexes of QA with Fe (II), result in the production of ROS (hydroxyl radicals) suggesting the existence of

Table 3 Effect of hesperidin, minocycline and their combination on mitochondrial enzyme complexes (I–IV) activities in the striatum of QA treated rats. Group

Complex-I (nmol NADH oxidized/ min/mg protein) (% of sham)

Complex-II (nmol/min/mg protein) (% of sham)

MTT assay (% of sham)

Complex-IV (nmol cyt-C oxidized/min/mg protein) (% of sham)

Naive Sham QA (300) Mino (25) Hesp (50) Hesp (100) Hesp (50)þ Mino (25)

108.5 7 5.3 100.0 7 6.1 45.17 6.9a 78.3 7 9.3b 53.8 7 7.6 83.5 7 7.9b 112.0 7 6.4c,d

105.6 7 5.4 100.0 7 8.4 40.17 7.2a 75.17 6.9b 49.97 7.5 78.17 9.5b 109.8 7 7.6c,d

107.9 76.3 100.0 79.2 42.3 77.3a 78.1 77.1b 58.2 77.9 87.8 78.7b 113.8 78.3c,d

110.5 7 4.5 100.0 7 7.3 42.5 7 7.2a 80.5 7 6.4b 55.9 7 8.1 89.6 7 7.3b 115.9 7 7.2c,d

Values are expressed mean7 SEM (percentage of sham). QA ¼ Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin. a

P o 0.05 as compared to sham. Po 0.05 as compared to QA (300). c Po 0.05 as compared to Mino (25). d Po 0.05 as compared to Hesp (50). b

Fig. 8. Effect of hesperidin, minocycline and their combination on striatal lesion volume in QA-treated rats. Values are expressed mean 7 S.E.M. (percentage of sham). # P o0.05 as compared to sham. nPo 0.05 as compared to QA (300). @P o0.05 as compared to Mino (25). $Po 0.05 as compared to Hesp (50). QA ¼ Quinolinic acid; Mino¼ Minocycline; and Hesp ¼ Hesperidin.

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Fig. 9. Representative photomicrographs of rat brain sections. Sections were stained with TTC. Arrows indicate lesioned portion. (A) Naive: no lesion; (B) sham: no lesion; (C) QA (300 nmol): unilateral lesion with large number damaged cells; (D) Mino (25): mild lesion with less damaged cells; (E) Hesp (50): moderate lesion with appreciable amount of damaged cells; (F) Hesp (100): mild lesion with very less damages cells; and (G) Hesp (50) þMino (25): very mild lesion.

NMDA receptor independent oxidative stress in QA induced neurotoxicity (Perez-De La Cruz et al., 2012). Consistent with these reports, the results of the present study demonstrated oxidative damage (increase in lipid peroxidation, nitrite concentration and decrease in SOD as well as GSH enzyme activities) and mitochondrial dysfunction (Complexes I–IV) following intrastriatal QA administration. However, hesperidin and minocycline administration for 21 days attenuated lipid peroxidation, increased endogenous anti-oxidant defense and restored mitochondrial enzyme complex levels which suggests that the neuroprotective effect can be partially attributed to their antioxidant and bio-energetic properties (Gaur et al., 2011; Jiang et al., 2009; Kalonia et al., 2012). Interestingly, co-administration of minocycline with sub-therapeutic dose of hesperidin potentiated its neuroprotective effect, suggesting the possible involvement of microglial pathway. In line with reports suggesting the excitotoxicity mediated activation of apoptotic pathways (Kalonia and Kumar, 2011; Simonian et al., 1996), intrastriatal QA administration in the present study also resulted in the induction of caspase-3 enzyme activity, a characteristic marker for apoptosis. However, treatment with hesperidin, minocycline and their combination for 21 days

demonstrated significant anti-apoptotic activity, as evident by substantial decrease in levels of caspase-3 enzyme. These results suggest possible involvement of microglial modulation give evidence that caspases are key mediators of microglia mediated neurotoxicity (Burguillos et al., 2011; Surace and Block, 2012) and also are in line with previous study reports demonstrating protective effects of hesperidin and minocycline against several neurological conditions mediated through apoptotic pathways (Trivedi et al., 2011; Wang et al., 2003). These altered caspase-3 levels may be correlated to the alteration in the striatal BDNF levels in the treatment groups as discussed later in this section. Among the neurotrophic factors, BDNF secreted by cortical afferents plays an important role in the development and survival of MSNs in the striatum (Baquet et al., 2004). In the present study, the BDNF levels were drastically reduced following excitotoxin administration, which validates cortical BDNF dependent striatal vulnerability in QA-induced HD like symptoms in rats. However, treatment with hesperidin and minocycline resulted in considerable increase in striatal BDNF levels in QA treated animals, which may be correlated with improved behavior of the animals. The present study results are further supported by the findings of

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Fig. 10. Possible targets in the neuroprotective effect of hesperidin against QA induced neurotoxicity in rats.

Reiner et al. (2012), which suggest that elevated BDNF levels may have a key role in reversal of motor and cognitive dysfunction in neurodegenerative disorders. The improvements caused by hesperidin and minocycline in QA treated animals are in corroboration with several previous findings (Gaur and Kumar, 2010; Menze et al., 2012; Raza et al., 2011) and lends further support to the concept that flavonoid rich food may have a beneficial effect in reversing the motor and cognitive dysfunction in neurodegenerative conditions (Rendeiro et al., 2012). Further, BDNF serves a crucial signaling molecule for cross talk between neurons and microglia (Coull et al., 2005) and therefore, drastic increase in striatal BDNF levels following co-administration of minocycline with hesperidin, suggests the involvement of microglial pathway underlying the observed potentiation. Under neurotoxic conditions, microglia become over activated and presents an upregulated catalog of surface molecules which together induce the release a variety of cytotoxic mediators including ROS/RNS, prostaglandins (arachidonic acid metabolites) QA and glutamate (Espey et al., 1997) as well as pro-inflammatory cytokines such as IL-1, IL-6, TNF-α, and IFN-γ (McGeer and McGeer, 1995; Smith et al., 2012). In line with these, our study results demonstrated significant increase in TNF-α (distinct marker for neuroinflammatory cascade) levels following intrastriatal QA administration (Ryu et al., 2005). Further, 21 days treatment of hesperidin and minocycline was able to attenuate the increase in TNF-α levels, suggesting their anti-inflammatory effect in the neurodegenerative conditions. However, significant attenuation of elevated TNF-α levels following combined treatment with hesperidin and minocycline, suggests a functional link for involvement of microglial pathway in neuroprotective effect of hesperidin. Furthermore, stimulation of NADPH oxidase, a membrane bound enzyme, is implicated as both the predominant source of microglial derived ROS and a mechanism of pro-inflammatory signaling in over activated microglia (Babior, 2000). Additionally, research indicates that inhibiting NADPH oxidase activity would simultaneously down regulate multiple pro-inflammatory factors (including PGs, cytokines, and NO) and might be more efficacious alleviate microglial dysregulation and over activation associated with neurodegenerative conditions (Block et al., 2007). Recently, several peptides, antibiotics and small molecules including hesperidin and hesperetin

analogs (Yamamoto and Saneyoshi, 2009) have been identified as NADPH inhibitors, thus advocating their use in neuroinflammatory conditions with underlying microglial over activation. The possible targets of action involved in the neuroprotective effect of hesperidin against QA induced neurotoxicity in rats has been depicted in Fig. 10. The present hypothesis is further strengthened by the TTC staining data where single intrastriatal QA administration, caused a significant increase in the striatal lesion volume, which was subsequently reduced by the 21 days treatment with hesperidin, minocycline and their combination. These findings are also in consistent with the previous reports of our laboratory suggesting the specific striatal degeneration following QA administration (Kalonia and Kumar, 2011). It is an interesting prospect to see dietary flavonoids as microglial inhibitors, moreover, microglial inhibition by polyphenolic compounds including curcumin, naringenin and silymarin has recently been addressed by several reports (Blaylock and Maroon, 2012; Dirscherl et al., 2010; Karlstetter et al., 2011). Although early in vivo studies were disappointing as they suggested that dietary flavonoids cannot access the brain to interfere with microglial activation in a meaningful way. However, in light of recent evidences demonstrating the detection of flavonoids in brain (Paulke et al., 2006; Youdim et al., 2004), it becomes increasingly necessary to understand the role of flavonoids in the regulation of microglial activity.

5. Conclusion In conclusion, our findings indicate that neuroprotective effect displayed by hesperidin treatment might be due to its inhibitory action on QA mediated neuroinflammatory signaling cascades originating from microglia activation besides its potential to suppress mitochondrial dysfunction due to its powerful antioxidant activity. However, our study results did not rule out the effect of minocycline on other brain cells and involvement of other mechanisms in the neuroprotective effect of hesperidin. Thus, further investigations on interplay of neuro-inflammation and microglial activation in HD and modulatory role of hesperidin in these processes are warranted before approaching to final implications.

A. Kumar et al. / European Journal of Pharmacology 720 (2013) 16–28

Acknowledgment The Major Research Project sanctioned to Professor Anil Kumar by University Grants Commission (UGC), New Delhi is gratefully acknowledged. Jitendriya Mishra is the project fellow in this project.

References Babior, B.M., 2000. Phagocytes and oxidative stress. Am. J. Med. 109, 33–44. Bano, D., Zanetti, F., Mende, Y., Nicotera, P., 2011. Neurodegenerative processes in Huntington's disease. Cell Death Dis. 2, e228. Baquet, Z.C., Gorski, J.A., Jones, K.R., 2004. Early striatal dendrite deficits followed by neuron loss with advanced age in the absence of anterograde cortical brainderived neurotrophic factor. J. Neurosci. 24, 4250–4258. Berman, S.B., Hastings, T.G., 1999. Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria: implications for Parkinson's disease. J. Neurochem. 73, 1127–1137. Blaylock, R.L., Maroon, J., 2012. Natural plant products and extracts that reduce immunoexcitotoxicity-associated neurodegeneration and promote repair within the central nervous system. Surg. Neurol. Int. 3, 19. Block, M.L., Zecca, L., Hong, J.S., 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat. Rev. Neurosci. 8, 57–69. Burguillos, M.A., Deierborg, T., Kavanagh, E., Persson, A., Hajji, N., Garcia-Quintanilla, A., Cano, J., Brundin, P., Englund, E., Venero, J.L., Joseph, B., 2011. Caspase signalling controls microglia activation and neurotoxicity. Nature 472, 319–324. Chen, M., Ona, V.O., Li, M., Ferrante, R.J., Fink, K.B., Zhu, S., Bian, J., Guo, L., Farrell, L.A., Hersch, S.M., Hobbs, W., Vonsattel, J.P., Cha, J.H., Friedlander, R.M., 2000. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat. Med. 6, 797–801. Coull, J.A., Beggs, S., Boudreau, D., Boivin, D., Tsuda, M., Inoue, K., Gravel, C., Salter, M.W., De Koninck, Y., 2005. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438, 1017–1021. Dihne, M., Block, F., Korr, H., Topper, R., 2001. Time course of glial proliferation and glial apoptosis following excitotoxic CNS injury. Brain Res. 902, 178–189. Dirscherl, K., Karlstetter, M., Ebert, S., Kraus, D., Hlawatsch, J., Walczak, Y., Moehle, C., Fuchshofer, R., Langmann, T., 2010. Luteolin triggers global changes in the microglial transcriptome leading to a unique anti-inflammatory and neuroprotective phenotype. J. Neuroinflamm. 7, 3. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Espey, M.G., Chernyshev, O.N., Reinhard Jr., J.F., Namboodiri, M.A., Colton, C.A., 1997. Activated human microglia produce the excitotoxin quinolinic acid. Neuroreport 8, 431–434. Estrada Sanchez, A.M., Mejia-Toiber, J., Massieu, L., 2008. Excitotoxic neuronal death and the pathogenesis of Huntington's disease. Arch. Med. Res. 39, 265–276. Gaur, V., Aggarwal, A., Kumar, A., 2011. Possible nitric oxide mechanism in the protective effect of hesperidin against ischemic reperfusion cerebral injury in rats. Indian J. Exp. Biol. 49, 609–618. Gaur, V., Kumar, A., 2010. Hesperidin pre-treatment attenuates NO-mediated cerebral ischemic reperfusion injury and memory dysfunction. Pharmacol. Rep. 62, 635–648. Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751–766. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal. Biochem. 126, 131–138. Guillemin, G.J., 2012. Quinolinic acid, the inescapable neurotoxin. FEBS J. 279, 1356–1365. Jiang, W., Desjardins, P., Butterworth, R.F., 2009. Minocycline attenuates oxidative/ nitrosative stress and cerebral complications of acute liver failure in rats. Neurochem. Int. 55, 601–605. Kalonia, H., Kumar, A., 2011. Suppressing inflammatory cascade by cyclo-oxygenase inhibitors attenuates quinolinic acid induced Huntington's disease-like alterations in rats. Life Sci. 88, 784–791. Kalonia, H., Kumar, P., Kumar, A., Nehru, B., 2010. Protective effect of montelukast against quinolinic acid/malonic acid induced neurotoxicity: possible behavioral, biochemical, mitochondrial and tumor necrosis factor-alpha level alterations in rats. Neuroscience 171, 284–299. Kalonia, H., Mishra, J., Kumar, A., 2012. Targeting neuro-inflammatory cytokines and oxidative stress by minocycline attenuates quinolinic-acid-induced Huntington's disease-like symptoms in rats. Neurotox. Res. 22, 310–320. Karlstetter, M., Lippe, E., Walczak, Y., Moehle, C., Aslanidis, A., Mirza, M., Langmann, T., 2011. Curcumin is a potent modulator of microglial gene expression and migration. J. Neuroinflamm. 8, 125. Kim, J.H., Kim, S., Yoon, I.S., Lee, J.H., Jang, B.J., Jeong, S.M., Lee, B.H., Han, J.S., Oh, S., Kim, H.C., Park, T.K., Rhim, H., Nah, S.Y., 2005. Protective effects of ginseng saponins on 3-nitropropionic acid-induced striatal degeneration in rats. Neuropharmacology 48, 743–756. King, T.E., 1967. Preparation of succinate dehydrogenase and reconstitution of succinate oxidase. Methods Enzymol. 10, 322–331. King, T.E., Howard, R.L., 1967. Preparations and properties of soluble NADH dehydrogenases from cardiac muscle. Methods Enzymol. 10, 275–294.

27

Kono, Y., 1978. Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Arch. Biochem. Biophys. 186, 189–195. Koppula, S., Kumar, H., Kim, I.S., Choi, D.K., 2012. Reactive oxygen species and inhibitors of inflammatory enzymes, NADPH oxidase, and iNOS in experimental models of Parkinson's disease. Mediators Inflamm. 2012, 1–16. Kumar, A., Vashist, A., Kumar, P., Kalonia, H., Mishra, J., 2012a. Potential role of licofelone, minocycline and their combination against chronic fatigue stress induced behavioral, biochemical and mitochondrial alterations in mice. Pharmacol. Rep. 64, 1105–1115. Kumar, A., Vashist, A., Kumar, P., Kalonia, H., Mishra, J., 2012b. Protective effect of HMG CoA reductase inhibitors against running wheel activity induced fatigue, anxiety like behavior, oxidative stress and mitochondrial dysfunction in mice. Pharmacol. Rep. 64, 1326–1336. Kumar, P., Kumar, A., 2010. Protective effect of hesperidin and naringin against 3nitropropionic acid induced Huntington's like symptoms in rats: possible role of nitric oxide. Behav. Brain Res. 206, 38–46. Kumar, P., Padi, S.S., Naidu, P.S., Kumar, A., 2006. Effect of resveratrol on 3nitropropionic acid-induced biochemical and behavioural changes: possible neuroprotective mechanisms. Behav. Pharmacol. 17, 485–492. Lee, S.M., Yune, T.Y., Kim, S.J., Park, D.W., Lee, Y.K., Kim, Y.C., Oh, Y.J., Markelonis, G.J., Oh, T.H., 2003. Minocycline reduces cell death and improves functional recovery after traumatic spinal cord injury in the rat. J. Neurotrauma 20, 1017–1027. Lehrmann, E., Molinari, A., Speciale, C., Schwarcz, R., 2001. Immunohistochemical visualization of newly formed quinolinate in the normal and excitotoxically lesioned rat striatum. Exp. Brain Res. 141, 389–397. Liu, Y., Peterson, D.A., Kimura, H., Schubert, D., 1997. Mechanism of cellular 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J. Neurochem. 69, 581–593. Maragos, W.F., Young, K.L., Altman, C.S., Pocernich, C.B., Drake, J., Butterfield, D.A., Seif, I., Holschneider, D.P., Chen, K., Shih, J.C., 2004. Striatal damage and oxidative stress induced by the mitochondrial toxin malonate are reduced in clorgyline-treated rats and MAO-A deficient mice. Neurochem. Res. 29, 741–746. McGeer, P.L., McGeer, E.G., 1995. The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegenerative diseases. Brain Res. Brain Res. Rev. 21, 195–218. Menze, E.T., Tadros, M.G., Abdel-Tawab, A.M., Khalifa, A.E., 2012. Potential neuroprotective effects of hesperidin on 3-nitropropionic acid-induced neurotoxicity in rats. Neurotoxicology 33, 1265–1275. Paulke, A., Schubert-Zsilavecz, M., Wurglics, M., 2006. Determination of St. John's wort flavonoid-metabolites in rat brain through high performance liquid chromatography coupled with fluorescence detection. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 832, 109–113. Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, CA. Perez-De La Cruz, V., Carrillo-Mora, P., Santamaria, A., 2012. Quinolinic acid, an endogenous molecule combining excitotoxicity, oxidative stress and other toxic mechanisms. Int. J. Tryptophan Res. 5, 1–8. Rainey-Smith, S., Schroetke, L.W., Bahia, P., Fahmi, A., Skilton, R., Spencer, J.P., Rice-Evans, C., Rattray, M., Williams, R.J., 2008. Neuroprotective effects of hesperetin in mouse primary neurones are independent of CREB activation. Neurosci. Lett. 438, 29–33. Raymond, L.A., Andre, V.M., Cepeda, C., Gladding, C.M., Milnerwood, A.J., Levine, M.S., 2011. Pathophysiology of Huntington's disease: time-dependent alterations in synaptic and receptor function. Neuroscience 198, 252–273. Raza, S.S., Khan, M.M., Ahmad, A., Ashafaq, M., Khuwaja, G., Tabassum, R., Javed, H., Siddiqui, M.S., Safhi, M.M., Islam, F., 2011. Hesperidin ameliorates functional and histological outcome and reduces neuroinflammation in experimental stroke. Brain Res. 1420, 93–105. Reiner, A., Wang, H.B., Del Mar, N., Sakata, K., Yoo, W., Deng, Y.P., 2012. BDNF may play a differential role in the protective effect of the mGluR2/3 agonist LY379268 on striatal projection neurons in R6/2 Huntington's disease mice. Brain Res. 1473, 161–172. Rendeiro, C., Guerreiro, J.D., Williams, C.M., Spencer, J.P., 2012. Flavonoids as modulators of memory and learning: molecular interactions resulting in behavioural effects. Proc. Nutr. Soc. 71, 246–262. Ryu, J.K., Choi, H.B., McLarnon, J.G., 2005. Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acidinjected rat striatum. Neurobiol. Dis. 20, 550–561. Simonian, N.A., Getz, R.L., Leveque, J.C., Konradi, C., Coyle, J.T., 1996. Kainate induces apoptosis in neurons. Neuroscience 74, 675–683. Smith, J.A., Das, A., Ray, S.K., Banik, N.L., 2012. Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res. Bull. 87, 10–20. Sottocasa, G.L., Kuylenstierna, B., Ernster, L., Bergstrand, A., 1967. An electrontransport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol. 32, 415–438. Surace, M.J., Block, M.L., 2012. Targeting microglia-mediated neurotoxicity: the potential of NOX2 inhibitors. Cell Mol. Life Sci. 69, 2409–2427. Tai, Y.F., Pavese, N., Gerhard, A., Tabrizi, S.J., Barker, R.A., Brooks, D.J., Piccini, P., 2007a. Imaging microglial activation in Huntington's disease. Brain Res. Bull. 72, 148–151.

28

A. Kumar et al. / European Journal of Pharmacology 720 (2013) 16–28

Tai, Y.F., Pavese, N., Gerhard, A., Tabrizi, S.J., Barker, R.A., Brooks, D.J., Piccini, P., 2007b. Microglial activation in presymptomatic Huntington's disease gene carriers. Brain 130, 1759–1766. Trivedi, P.P., Tripathi, D.N., Jena, G.B., 2011. Hesperetin protects testicular toxicity of doxorubicin in rat: role of NFkappaB, p38 and caspase-3. Food Chem. Toxicol. 49, 838–847. Wang, X., Zhu, S., Drozda, M., Zhang, W., Stavrovskaya, I.G., Cattaneo, E., Ferrante, R.J., Kristal, B.S., Friedlander, R.M., 2003. Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease. Proc. Natl. Acad. Sci. USA 100, 10483–10487. Wang, X.M., Gao, X., Zhang, X.H., Tu, Y.Y., Jin, M.L., Zhao, G.P., Yu, L., Jing, N.H., Li, B.M., 2006. The negative cell cycle regulator, Tob (transducer of ErbB-2), is involved in motor skill learning. Biochem. Biophys. Res. Commun. 340, 1023–1027.

Wills, E.D., 1966. Mechanisms of lipid peroxide formation in animal tissues. Biochem. J. 99, 667–676. Wu, D.C., Jackson-Lewis, V., Vila, M., Tieu, K., Teismann, P., Vadseth, C., Choi, D.K., Ischiropoulos, H., Przedborski, S., 2002. Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J. Neurosci. 22, 1763–1771. Yamamoto, M., Saneyoshi, A., 2009. NADH/NADPH oxidase inhibitors. JP2009007256. Youdim, K.A., Qaiser, M.Z., Begley, D.J., Rice-Evans, C.A., Abbott, N.J., 2004. Flavonoid permeability across an in situ model of the blood-brain barrier. Free Radical Biol. Med. 36, 592–604.