Astaxanthin ameliorates behavioral and biochemical alterations in in-vitro and in-vivo model of neuropathic pain

Accepted Manuscript Title: Astaxanthin Ameliorates Behavioral and Biochemical Alterations In In-Vitro and In-Vivo Model of Neuropathic Pain Authors: Kuhu Sharma, Dilip Sharma, Monika Sharma, Nishant Sharma, Pankaj Bidve, Namrata Prajapati, Kiran Kalia, Vinod Tiwari PII: DOI: Reference:

S0304-3940(18)30207-6 https://doi.org/10.1016/j.neulet.2018.03.030 NSL 33489

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

20-1-2018 3-3-2018 16-3-2018

Please cite this article as: Kuhu Sharma, Dilip Sharma, Monika Sharma, Nishant Sharma, Pankaj Bidve, Namrata Prajapati, Kiran Kalia, Vinod Tiwari, Astaxanthin Ameliorates Behavioral and Biochemical Alterations In In-Vitro and In-Vivo Model of Neuropathic Pain, Neuroscience Letters https://doi.org/10.1016/j.neulet.2018.03.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Astaxanthin Ameliorates Behavioral and Biochemical Alterations In In-Vitro and In-Vivo Model of Neuropathic Pain Kuhu Sharmaa, Dilip Sharmaa, Monika Sharma1, Nishant Sharma1, Pankaj Bidve1,

1Department

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Namrata Prajapati1, Kiran Kalia1, Vinod Tiwari1,2

of Pharmacology and Toxicology, National Institute of Pharmaceutical

Education and Research (NIPER)-Ahmedabad, Gandhinagar-382355, Gujarat, India 2Department

of Anesthesiology and Critical Care Medicine, Johns Hopkins University

equal contribution

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School of Medicine, Baltimore, MD, USA

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*Corresponding authors: Address for correspondence Assistant Professor, Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER)-Ahmedabad, Gandhinagar-382355,

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Gujarat, India. Phone: +91-79-27401190; Fax: +91-79-27401192

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Visiting Professor, Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

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E-mail: [email protected]; [email protected]; [email protected]

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Highlights: 

Astaxanthin fits into the inhibitory binding pocket of NMDA receptor particularly with NR2B protein which is involved in nociception. This shows that astaxanthin

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has antagonistic properties for NR2B receptors and thus, can be effective against neuropathic pain. 

Decrease in oxido-nitrosative stress and GFAP expression in LPS treated C6 glial cells shows that astaxanthin reduces the astrocytic activation.

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Astaxanthin showed decrease in mechanical and thermal hypersensitivity in CCI-

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induced neuropathic rats, which shows its important role in attenuation of

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neuropathic pain.

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Abstract

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Despite considerable advances in understanding mechanisms involved in chronic pain,

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effective treatment remains limited. Astaxanthin, a marine natural drug, having potent anti-oxidant and anti-inflammatory activities is known to possess neuroprotective

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effects. However, effects of astaxanthin against nerve injury induce chronic pain remains unknown. Overactivity of glutamatergic NMDARs results in excitotoxicity which

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may participate in astrocytic and microglial activation during pathology which further

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contribute to the development of neuropathic pain. In this study, we investigate the effects of astaxanthin on oxido-inflammatory and NMDA receptor down-regulation pathway by using in-silico, in-vitro and in-vivo models of neuropathic pain. In-silico molecular docking study ascertained the binding affinity of astaxanthin to NMDA receptors and showed antagonistic effects. Data from in-vitro studies suggest that 2

astaxanthin

significantly

reduces

the

oxidative

stress

induced

by

the

lipopolysaccharides in C6 glial cells. In male Sprague dawley rats, a significant attenuation of neuropathic pain behavior was observed in Hargreaves test and von Frey

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hair test after astaxanthin treatment. Findings from the current study suggest that astaxanthin can be used as potential alternative in the treatment of chronic neuropathic pain. However, more detailed investigations are required to further probe the in-depth

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mechanism of action of astaxanthin.

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Abbreviations

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BBB: Blood brain barrier

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AD: Alzheimer’s disease

BSA: Bovine serum albumin

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CNS: Central nervous system

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CCI: Chronic constriction injury DMSO: Dimethylsulphoxide

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DTNB: 5,5'-dithio-bis-[2-nitrobenzoic acid] FBS: Fetal bovine serum FITC: Fluorescein iso-thiocyanate GFAP: Glial fibrillary acidic protein

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GSH: Glutathione IAEC: Institutional Animal Ethics Committee I.p.: Intraperitoneal

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LTD: Long-term depression LTP: Long term potentiation LPS: Lipopolysaccharides MDA: Malondialdehyde

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NCCS: National Centre for Cell Science

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ANOVA: One-way analysis of variance

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NMDARs: N-methyl-D-aspartate receptors

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NBT: Nitroblue tetrazolium

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MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide

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PWT: Paw withdrawal threshold PBS: Phosphate buffer saline

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ROS: Reactive oxygen species

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SEM: Standard error mean SOD: Superoxide dismutase

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TBA: Thiobarbituric acid TCA: Trichloroacetic acid TBS: Tris buffer saline

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Keywords: Neuropathic pain; C6 glial cells; NMDA receptors; Neuroprotection; Rats;

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Mechanical allodynia

1. Introduction

Activated astrocytes and microglia are considered as hall-mark of neurodegenerative diseases which release different factors like, free radicals, pro and anti-inflammatory cytokines, antioxidants and neurotrophic factors during pathological conditions which functionally

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further contribute to neurodegeneration (Singh et al., 2011). Neurons

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depend on the astrocytes for the replenishment of intermediates of various metabolic

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pathways (Sidoryk-Wegrzynowicz and Aschner, 2013). Because of this neuron-

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astrocyte strong functional association, neuronal survival gets impaired due to any

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disruption in astrocytic energy metabolism, falling antioxidant capacity or progressive astrocytic death.(Liu and Chopp, 2016). Astroglial cells react sensitively to oxidative

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stress and bacterial/viral infection with increase in intracellular Ca2+ion concentration,

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mitochondrial potential loss,reduced oxidative phosphorylation (Robb et al., 1999). These glial cells are involved in self-defense against damage mediated by cytokines,

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neurotrophins and heat shock proteins, ultimately promoting neuronal function (Nomura,

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2001).

Neuroinflammation can serve as both a cause and a consequence of chronic

oxido-reductory dysfunction.Oxidative stress is part of an untreated neuroinflammatory reaction, possibly caused due to a combination of mitochondrial functional impairment and pathophysiologic activation of both astrocytes and microglia. The cytokines

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released in response to inflammation stimulate microglia, which then release various reactive oxidative and nitrogen species putting ambient neurons and cells in stress. These cytokines also stimulate transcription of pro-inflammatory genes in glia leading to

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various inflammatory reactions (Mhatre et al., 2004). The overexpressed Reactive Oxygen Species (ROS) and NO directly influence ability of synapse to undergo long term potential (LTP). The initiation of LTP requires synaptic activation, which is caused by stimulation of NMDA followed by post-synaptic Ca2+ entry. NMDA receptors (NMDARs) are important because they contribute to synaptic plasticity, synaptogenesis,

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neuronal circuitry formation, as well as in the molecular pathogenesis of various

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neurological disorders.NR2A and NR2B are the subtypes of NMDA receptors, both

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playing a vital role in modulating LTP, synaptic plasticity and long-term depression

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(LTD)(Rai et al., 2013). As glutamate NMDA receptors overactivity leads to excitotoxicity in neurons, these receptors may have a role in astrocytic and microglial

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activation.

Astaxanthin is a ketocarotenoid naturally occuring in a variety of living organisms

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such as complex plants, microalgae, crustacean shells (crabs, shrimps), salmon, and Asteroidean(Kuedo et al., 2016). It is a deep red-colored phytonutrient synthesized by a

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marine microalgae called Haematococcus pluvialis for commercial use. Previous

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studies have shown the antioxidant, anti-inflammatory and neuroprotective properties of astaxanthin in various models of neurodegenerative disorders(Lee et al., 2010, Ye et al., 2013). We hypothesised that astaxanthin can cure neuropathic pain by a) antagonising the NR2A and NR2B receptor proteins b) modulating the oxidative-

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nitrosative stress mediated astrocyte activation and reverse the behavioral parameters of neuropathic pain. 2. Materials and methods

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Rat C6 glial cells were obtained from National Centre for Cell Science (NCCS), Pune, India. Ham’s F12 K nutrient mixture Kainghn’s modification, fetal bovine serum (FBS), and other tissue culture reagents were purchased from Gibco (Grand Island, NY, U.S.A.). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich and was initially

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dissolved in sterile PBS (1 mg/ml stock) and subsequently diluted with media.

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Astaxanthin was purchased from Sigma-Aldrich and initially dissolved in DMSO and

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subsequent dilutions were made in medium as per the required concentrations (0.1 %).

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3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitroblue tetrazolium (NBT), and Folin ceocalteau’s Phenol reagent were obtained from Himedia. Protease

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inhibitor cocktail was obtained from Sigma-Aldrich (India). Glutathione was purchased

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from Sigma-aldrich. Griess reagent was purchased from Fluka analytical (Germany). 2.1. In-silico studies

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2.1.1. Molecular docking: Molecular modeling studies were performed using

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automated docking software Glide 5.0 (Schrodinger-Maestro) that utilizes a scoring process to sort out the best orientations and conformations of the ligand i.e. poses

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based on its interaction pattern with the receptor (Vijesh et al., 2013). The following steps are involved in docking; 2.1.1.1. Ligand Preparation: Ligprep module was used to generate the three dimensional coordinates of the ligands, isomeric, ionization and tautomeric states. The

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ligands were assigned partial atomic charges according to the OPLS-2005 force field (Elokely and Doerksen, 2013). 2.1.1.2. Protein Preparation: The x-ray crystal structure of NMDA receptor protein

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NR2B (5EWJ) were obtained from RCSB protein data bank (PDB). Protein were prepared using the protein preparation wizard of Glide module of Schrodinger-Maestro. The hydrogens were added and water molecules beyond 5A° of the ligand were removed from the protein complex. Then, using the Receptor Grid Generation wizard, a grid was generated within the center defined by the co-crystallized ligand. OPLS_AA

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2005 force field helps assign partial atomic charges (Hasanat et al., 2017).

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2.1.1.3. Ligand-Receptor Docking: The test compound astaxanthin was docked into

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the active site of NR2B receptor proteins using Schrodinger’s Glide module (5.0) to

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ascertain the direct binding affinity of the ligand with NMDA receptor (Halgren et al., 2004).

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Astaxanthin, along with native bound ligand and standard drug in crystal

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structure were subjected to docking using previously generated grid with default option.

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Extra-precision docking mode (XP- Dock) was used for docking. The test and standard were then observed for their docking score and appropriate docking pose in the cavity.

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2.2. In-vitro studies

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2.2.1. Cell culture: Cells were cultured in 75cm2 and 25 cm2 culturing flasks in Ham’s F12K nutrient medium containing 10% heat-inactivated fetal bovine serum (FBS), 100μg/mL streptomycin, and 100U/mL penicillin at 37°C and 5% CO2. The medium was replenished every alternative day after the initial seeding (Liu et al., 2001). After 8

reaching to 85–95% confluency, cells were trypsinized and centrifuged at 2000 rpm for 10 mins at 25°C, supernatant was discarded and the pellet was re-suspended in media. The cells were reseeded into culture dishes. After 24 hrs of seeding, the cells were

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pretreated with astaxanthin with various concentrations (0.1, 1, 5, and 10μM) 30 min before LPS treatment (100ng/mL)(Choi et al., 2008). Astaxanthin was dissolved in1% (v/v) DMSO. LPS treatment included200μL/well and 2mL/well in nutrient medium for 96 (1×104 cells/mL) and 6 well (3.5×105 cells/mL) plates respectively. For the control group, C6 glial cells were cultured with media alone, and for vehicle control group, 1% DMSO

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was added to the cell culture.

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2.2.2. Morphological changes and Cell viability assay:

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Change in the cell morphology was observed after LPS and astaxanthin treatment in

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various groups using Zeiss axiocam inverted fluorescence microscope at 20μm scale and 100X resolution. An MTT assay was performed to measure the effect of astaxanthin

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on cell-viability. C6 glial cells were treated with astaxanthin at concentrations of 0.1 μM,

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1μM, 5μM, and 10 μM (Showalter et al., 2004) for 24 h. 20 μL of MTT solution (0.5

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mg/mL) was added into each cultured well containing cell suspension and incubated at 37°C for 4-5 hrs. The reaction mixture was aspirated and formazan crystals formed reactionwere

solublized

with

200

μL

of

DMSO

and

quantified

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during

spectrophotometrically by determining the absorbance at 570 nm using micro-plate

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reader(Thakkar et al., 2018). 2.2.3. Nitrite assay: 3.5×105 cells/well C6 Glial cells were seeded in the 6 well plates and were incubated for 24 hrs to make it adhere to wells. After the treatment period, cells were lysed using

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RIPA buffer and then centrifuged at 12,000×g for 20 mins at 4°C. Pellet was discarded and supernatant was collected.100 μL of cell supernatant (lysate) was added with 100 μL

of

Griess

reagent

(1%

sulfanilamide/0.1%

N-(1-naphthyl)-

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ethylenediaminedihydrochloride in 2.5% v/v phosphoric acid) and 50 μL of deionized water in to 96-well plate in triplicates and the mixture was incubated in dark for 30 mins at room temperature and the absorbance was measured at 540 nm using a spectrophotometric microplate reader(Ishola et al., 2013). 2.2.4. Lipid peroxidation assay:

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C6 cells were seeded at the cell density of 3.5×105 cells/well. Followingthe treatment

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period, cells were lysed by same above given procedure using RIPA buffer. In an

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eppendorf tube, 100μL supernatant, 100μL of 0.1M Tris HCL was added and incubated

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at 37°C for 2hrs. 200μL of ice cold 10% w/v TCA (trichloroacetic acid) and 200 μL of 0.67% w/v TBA (Thiobarbituric acid) was then added and heated at 95°C for 10mins.

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After heating, samples were rapidly cooled down and 200μL of miliQ and

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1mLbutanol:pyridine mixture in the ratio of 15:1 was added to the tube and then again

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centrifuged at 3000 rpm for 10 mins. The upper pink layer was then transferred to a micro plate and absorbance was recorded at 532 nm. Malondialdehyde (MDA) level

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was denoted as μM of MDA/mg of protein (Colado et al., 1997). 2.2.5. Superoxide Dismutase assay:

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C6 cells were seeded at the cell density of 3.5×105 cells/well and50μL of cell supernatant, 100μL 0.2M pyrogallol solution and then, 100μL of Tris EDTA buffer (pH 8.2) was added into 96 well plate in triplicates. Absorbance was then taken at 420nm for

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2 minutes at 30 seconds interval using spectrophotometric microplate reader. Tris EDTA buffer was taken as blank (Marklund and Marklund, 1974). 2.2.6. Reduced Glutathione estimation:

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Reduced glutathione levels were estimated to measure oxidative damage indirectly (Anderson et al., 1985). C6 glial cells at the cells density of 4*10 5 cells/well were seeded in the six well plate and incubated for 24 hrs at 37˚C in the CO2 incubator to make it adherent. After the incubation, the treated cells (astaxanthin 0.1, 1, 5 and 10 μM) were washed with ice-cold phosphate buffer saline (PBS) and scrapped in sodium phosphate

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buffer containing 0.1% triton-X. 100 μL of this cell lysate was deproteinised by adding

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100 µL of pre-cooled 10% trichloroacetic acid (TCA) and then incubated for 1hour at

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4˚C, centrifuged at 5000 g for 5 mins at 4˚C to separate supernatant. 100 µL of 0.1M

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phosphate buffer (pH 8.0) and 50 µLof DTNB(5,5'-dithio-bis-[2-nitrobenzoic acid])(0.1% in 0.1M phosphate buffer pH 8.0) was added to 75 μL of the above supernatant.

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Absorbance was read at 412 nm after 10 mins (Monostori et al., 2009, Sharma et al.,

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2016).

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2.2.7. Catalase assay:

Cell lysate was treated with 100μl of cell supernatant, 100μl of 50 mM sodium,

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potassium phosphate buffer of pH 7.4, 100μl of hydrogen peroxide(65 mM) dissolved in 50 mmol/L sodium and potassium phosphate buffer in an eppendorf which were mixed

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together by vortex and incubated at 37°C for 3 min. 400 μl of Dichromate/acetic acid was then added and the tubes were kept at 100°C for 10 min. Tubes were then cooled with tap water followed by centrifugation (2500g for 5 min) to remove precipitated

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protein, the changes in absorbance were recorded at 570 nm against the reagent blank(Hadwan, 2016). 2.2.8. Quantification of ROS production:

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Nitro Blue Tetrazolium (NBT) test was used to evaluate percentage of ROS production inside glial cells. Cells (1×104 cells/mL) were seeded in a 96-well plate. Cells were incubated with NBT after astaxanthin treatment in the 96 well plate for 2 hours. 2 M potassium hydroxide(KOH) solution in DMSO (100μl 2M KOH+ 100Μl DMSO) was used to dissolve the formazan crystals, and the resulting colour reaction was measured

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spectrophotometrically on a microplate reader at 630 nm (Vergallo et al., 2014).

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2.2.9. Immunocytochemistry of GFAP expression:

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Cells were seeded in 6-well plate and treated with different concentrations of

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astaxanthin, incubated at 37°C for 24 hrs. After 24 hrs, medium was aspirated, cells were washed three times with tris buffer saline (TBS pH 7.4) and then fixed with 4%

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paraformaldehyde for 5-10 mins, washing was given again three times with TBS and

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permeabilized with 10% H2O2 in TBS and kept for 10 mins. C6 glial cells were then

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blocked by adding 3% bovine serum albumin (BSA), 0.3% Triton-X in TBS and kept at room temperature for 1hr. After that, the cells were incubated with rabbit polyclonal anti

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GFAP antibody (Merck milipore; diluted 1:500 in dilution buffer) overnight at 4°C on horizontal shaker, washed 3 times with TBS, incubated with FITC conjugated anti rabbit

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Ig-G (1:1000 dilution) for 1hr at room temperature. Images were visualized under fluorescence microsope (Zeiss) at 100X magnification (Goswami et al., 2015). 2.3. In-vivo studies

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2.3.1. Animals: Adult male Sprague dawley rats (220-250 grams) obtained from Zydus Research Centre (ZRC), Ahmedabad were used in the study. The animals were housed under standard laboratory conditions, maintained on a 12:12 h light:dark cycle and had

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free access to food (Hylasco Bio-Technology Pvt. Ltd, Hyderabad, India) and water. Animals were acclimatized to laboratory conditions before the tests. All experiments were carried out between 09:00 and 17:00 h. The experimental protocols were approved (Approval No.: NIPERA/IAEC/2017/014/R) by the Institutional Animal Ethics Committee (IAEC) of NIPER-Ahmedabad and performed in accordance with the

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guidelines of Committee for Control and Supervision of Experimentation on Animals

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(CPCSEA), Government of India on animal experimentation.

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2.3.2. Induction of neuropathic pain and drug treatment schedule: Chronic

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constriction injury model was used for the induction of neuropathic pain in rats. Rats were anaesthetized with intra-peritoneal administration of ketamine (80 mg/kg) and

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xylazine (10 mg/kg). Left hind leg of the rats was shaved and they were placed onto a

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thermo-regulated heating mat at 37°C. Lubricating ophthalmic ointment was applied to

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the eyes (Bennett, 1993). Shaved area was sterilized with three alternate applications of 70% isopropyl alcohol and iodine solution. With the rat lying on its chest/thorax, left hind

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leg was elevated and held in the position with the femur at 90° to the spine using masking tape on the foot. An incision was made in the skin, parallel to but 3-4 mm

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below the femur. Skin was kept free from the muscle surrounding the incision by cutting through the connective tissue. Blunt scissors were used to cut through the connective tissue between the gluteus superficial and the biceps femuris muscles. A retractor was used to widen the gap between the two muscles, allowing clear visualization of the

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sciatic nerve. Approximately 10 mm of the sciatic nerve (proximal to the sciatic trifurcation) was made gently free from the surrounding connective tissue using curved blunt-tipped forceps and micro-scissors. Under the microscope and a good light source,

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3 ligatures (silk 4.0) were tied with a double knot, 1 mm apart, proximal to the trifurcation of the sciatic nerve. For each ligature, we started with a single loose loop and then grasped the two ends close to the loop; finally tighten until the loop is just barely snug and the ligature does not slide along the nerve. To hold the loop in its proper position, a second loop was placed on the top of the first to complete the knot.

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Finally, loose ends of the ligature were cut around 1mm. Constriction of the nerve was

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tried to be minimized and immediately stopped if a brief twitch is observed, to prevent

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arresting of the epineural blood flow. Over tightening the ligatures leads to axotomy, and

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autotomy (self-mutilation), both unwanted side-effects which preclude successful pain hypersensitivity testing. Animals were observed for clinical signs of distress post-

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surgery; iodine solution was applied on the incision site to avoid infection. No post-

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operative analgesia was given as there is evidence that local application of anesthetic,

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lidocaine, at time of injury can alter the development of neuropathic pain, and diminish the degree of hyperalgesia in the preceding weeks.

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After baseline pain behavior testing, the animals were randomly divided into five experimental groups with 5–8 animals in each viz. naive group (un-injured), vehicle-

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treated, astaxanthin treated (5 and 10 mg/kg) and gabapentin treated (30 mg/kg) nerve injured (CCI) rats. Astaxanthin was prepared by dissolving in 5% Tween-80 and normal saline while gabapentin was dissolved in saline alone. .Astaxanthin and gabapentin

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were both administered by intra-peritoneal route and testing was done at 30, 60 and 120 min after drug administration. 2.3.3. Mechanical allodynia using electronic von Frey anesthesiometer: Rats were

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placed individually on an elevated mesh (1 cm 2 perforations) in a clear plastic cage and adapted to the testing environment for at least 15-30 min. Electronic von-Frey anesthesiometer (IITC, Woodland Hills, USA) was used to deliver punctuated mechanical stimuli to the plantar surface of the rats hindpaw. Each stimulation was applied 3 times with an inter-stimulus interval of 4–5 s. Care was taken to stimulate

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random locations on the plantar surface. A positive response was noted if the paw was

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robustly and immediately withdrawn. Paw withdrawal threshold was defined as the

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minimum pressure required to elicit a withdrawal reflex of the paw. Voluntary movement

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associated with locomotion was not considered as a withdrawal response. Mechanical allodynia was defined as a significant decrease in withdrawal thresholds to von-Frey

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hair application (Yang et al., 2016; Tiwari et al., 2009).

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2.3.4. Thermal hyperalgesia using Hargreave’s test: Thermal paw withdrawal

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threshold were conducted blinded for different groups of animals. After 30 min of habituations to testing environment, thermal paw withdrawal threshold was measured.

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Latency (in sec) to withdraw paw in response to a radiant heat stimulus applied on the plantar region of the hind paw was evaluated using Hargreaves apparatus (Cheah et al.,

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2017; Tiwari et al., 2016). 2.4. Statistical analysis Data was analysed using one-way analysis of variance (ANOVA) and difference between the control groups versus treated group (control vs astaxanthin) was analysed

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by post hoc Newman-Kuels test. Values are expressed as mean ± SEM (standard error of the mean). P<0.05 was considered for values to be statistically significant(Singh et al., 2017).

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3. Results 3.1. Molecular docking:

In-silico data represents NMDA receptor inactivation as astaxanthin very well fits into the binding pocket of NR2B target protein (5EWJ) and shows nearly close docking score as compared to standard ketamine (Table 1). These studies may provide a base

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for assessing the target in-vitro as well as in-vivo in preclinical models for the treatment

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of neuropathic pain (Figure 1).

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3.2. In-vitro studies

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3.2.1. Effect of astaxanthin on LPS-induce morphological change and cell viability:

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Exposure of C6 cells to LPS (100 ng/ml) did not alter the cell viability (Figure 1a).

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Exposure of astaxanthin (0.1-10μM) in presence of LPS (100 ng/mL) for 24hours did not

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alter the cell viability of C6 cells by MTT assay (Figure 2a). Morphological changes of C6 cells were studied for assessing the activation of

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astrocytes induced by LPS. The C6 cells (2 * 10 5 cells) were incubated with astaxanthin (0.1, 1, 5 and 10 μM) in the presence of LPS (100 ng/mL) for 24 hours on the cover

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slide in the six well plate. Induction with LPS caused the formation of reactive astrocytes with more complex and hypertrophic structure and increase in the pseudopodia formation. Astaxanthin treatment decreased the reactive form of astrocytes in a dose dependent manner. The control cells exhibited no change in the morphology after 24

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hours of incubation. These data suggest that astaxanthin prevents the morphological activation of LPS-induced C6 cells (Figure 2b). 3.2.2. Effects of astaxanthin on LPS-induced nitrosative stress:

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In this study, we evaluated the effect of astaxanthin on nitrite level in LPS stimulated C6 glial cells. We observed profound increase (p<0.001 vs control) of nitrite concentration in LPS treated group which was significantly decreased (p<0.01) in a concentration dependent manner on treatment with astaxanthin (Figure 3a).

3.2.3. Effect of astaxanthin on malonaldehyde (MDA) levels in LPS stimulated C6

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glial cells:

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A significant increase in malondialdehyde production was found in LPS treated glia cells

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(p<0.001 vs control) (Figure 3b). However, treatment with astaxanthin resulted in

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significant decrease in LPS-induced malondialdehyde in a concentration dependent manner. Data showed that lower concentration of astaxanthin (0.1 μM) reversed

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(p<0.05 vs control) the effect of LPS on MDA level and higher concentration of

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astaxanthin (5 and10 μM) further lowered MDA levels (P<0.001 vs LPS (100 ng/mL))

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after treatment in C6 glial cells(Figure 3b). 3.2.4. Effect of astaxanthin on LPS-induced decrease in superoxide dismutase

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(SOD) activity:

Superoxide dismutase (SOD) enzyme provides antioxidant defense against ROS by

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catalyzing superoxide anions (O2−) into hydrogen peroxide (H2O2) or oxygen molecule (O2). There is significant decrease in SOD activity (p<0.001 vs control) in LPS treated C6 glial cells group compared to control group. Data showed that astaxanthin improved SOD enzyme activity, here in this study SOD activity got significantly reversed after

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treatment with astaxanthin in a concentration dependent manner. Results suggest that lower dose of astaxanthin (0.1μM) significantly restored decreased enzyme activity {p<0.01 vs LPS(100 ng/ml)} and higher dose (5 and 10 μM) further potentiates this

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effect {p<0.001 vs LPS (100 ng/mL)} as compared to LPS induced group (Figure 3c). 3.2.5. Effect of astaxanthin on LPS induced decrease in intracellular GSH levels:

Glutathione (GSH) is often referred to as the body's master antioxidant. Its concentration is significantly decreased (p<0.001 vs control) in LPS stimulated group while astaxanthin treatment significantly restored the decreased levels of intracellular

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3.2.6. Effect of astaxanthin on catalase activity:

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GSH at 5 and 10μM (p<0.05, p<0.01 vs LPS (100 ng/mL)) (Figure 3d).

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Catalase activity was significantly decreased (p<0.001) in LPS group as compared to

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control (Figure 3e). Data showed that astaxanthin treatment significantly restored LPS induced decrease in catalase activity. Findings suggest that low (0.1μM) concentration

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of astaxanthin didn’t had any significant effect on decrease catalase activity as

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compared to LPS treated group. However, higher concentrations (1, 5 and 10 μM) of

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astaxanthin showed significant (p<0.05, p<0.001) restoration of decrease catalase activity as compared to LPS treated group in a concentration dependent manner (Figure

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3e).

3.2.7. Effect of astaxanthin on LPS-induced reactive oxygen species (ROS):

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NBT assay revealed increase in % ROS production in C6 glial cells by LPS (100 ng/mL) as compared to control cells. This increase in ROS level was significantly decreased by astaxanthin. Lower concentration of astaxanthin (0.1μM) produced significant (p<0.05) decrease in ROS levels in C6 glial cells. However, when concentration of 1, 5 and 10

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μM of astaxanthin were used, there was further significant (p<0.01) reduction of ROS in C6 glial cells as compared to LPS treated cells (Figure 3f). 3.2.8. Immunocytochemistry of GFAP expression:

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Fluorescence intensity of Fluorescein isothiocyanate (FITC) tagged GFAP is important indicator of astrocytic activation. C6 glial cells treated with LPS media showed bright green fluorescence as compared to that in (control) normal media condition. LPS exposed cells showed significant increase (p<0.001) in expression of GFAP as compared to control cells. The treatment of astaxanthin (0.1 and 1μM) did not alter the

U

expression profile of GFAP as compared to LPS group, however, higher concentration

N

of astaxanthin (5 and 10μM) significantly decreased (p<0.01) expression profile of

A

GFAP as compared to LPS treated group (Figure 4).

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3.3. In vivo studies:

3.3.1. Effect of astaxanthin on nerve injury-induced mechanical allodynia:

D

All the CCI-injured animals showed significant decrease in paw withdrawal threshold (in

TE

gm) as compared to their pre-injury (CCI) baseline on 1 week post CCI surgery. Signs

EP

of the development of neuropathic pain were also easily observed like paw licking, autotomy, walking with limp etc. There was no significant difference seen in the

CC

threshold values for normal animals over the period of time. Astaxanthin treated animals showed significant reversal in mechanical allodynia in ipsilateral paws as compared to

A

pre-drug baseline (before drug treatment) as well as compared to CCI group animals, as significant increase (P<0.01) in paw withdrawal threshold was observed at 60 min and 120 min after astaxanthin (5 and 10 mg/kg) administration (Fig. 5a). When the

19

contralateral paw was stimulated, no differences were reported among naive, CCI and CCI with astaxanthin treated groups before and after surgery (Fig.5b). Improve in sensitivity thresholds (P<0.001) was also observed in gabapentin (30

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mg/kg) i.p. treated rats, when tested on the ipsilateral side, with no difference compared with contralateral paws, when tested after 60 min as well as 120 min after astaxanthin treatment.

3.3.2. Effect of astaxanthin on nerve injury-induced thermal hyperalgesia:

The CCI operated animals showed significant decrease in paw withdrawal latency in

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ipsilateral paw when compared to the latency values observed before surgery as well as

N

compared to naïve (uninjured) animals. A significant increase (P<0.001) in paw

A

withdrawal latencies were observed after 60 minutes as well as 120 minutes of

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administration of 30 mg/kg i.p. dose of astaxanthin. However, 10 mg/kg dose of astaxanthin showed significant effect at only (P<0.05) 120 min after the drug

D

administration (Fig.6a). No changes in PWT were observed in contralateral paws of rats

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after nerve injury and drug treatment (Fig.6b). Gabapentin significantly (P<0.001)

EP

increased paw withdrawal latency at 60 min after 30 mg/kg i.p. of drug administration. Thus, astaxanthin showed significant effects comparable to gabapentin at 30 mg/kg

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dose.

4. Discussion

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4.1. Binding of astaxanthin with NMDA receptors: From previous studies, it is proven that glutamatergic receptors play a vital role in conditions reflecting long-term plastic reformations in the central nervous system, for example in neuropathic pain and malfunctioning of the glutamatergic neurotransmission

20

is responsible for causing various neurological diseases. Glutamate stimulates NMDARs, responsible for central sensitization of nociceptive neurons present in spinal cord as well as activation of astrocytes by increasing intracellular Ca 2+ ion

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concentration. Ca2+ influx plays important role in pain signaling by enhancing neurotransmitter release and altering cell membrane excitability. In-silico molecular docking studies revealed that marine natural bioactive astaxanthin perfectly fits into the inhibitory binding pocket of NMDA receptor particularly with NR2B protein which is involved in nociception. There are probable chances that astaxanthin may work via

N

4.2. Astaxanthin and neuroprotective activity:

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NMDA receptor inactivation in attenuation of neuropathic pain.

A

Oxidative stress has been implicated as a contributing factor to various painful

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symmetrical sensorimotor polyneuropathy. Elevated levels of ROS and MDA and depleted levels of GSH in activated astroglial cells is the main hallmark of which

progressively

D

neuroinflammation

leads

to

neuronal

death

in

various

TE

neurodegenerative disorders (Niranjan et al., 2010). Intracellular ROS triggers various

EP

signaling pathways involving activation of numerous transcription factors and release of pro-inflammatory mediators and subsequent neuronal death.

CC

Another crucial event of oxidative stress is generation of malondialdehyde (MDA) (Nielsen et al., 1997), also been observed in reactive astrogliosis formation (Kamendulis

A

et al., 1999). Significant increase in intracellular levels of ROS, MDA formation and decrease in GSH. SOD and catalase levels were found in C6 cells upon LPS treatment as compared to non-stimulated cells which is similar to the oxidative stress conditions observed in various CNS disorders. Astaxanthin significantly reduced nitrite levels, %

21

ROS generation, and MDA formation and restored the diminished GSH, SOD and catalase levels signifying decline in intracellular oxidative stress enlightening a crucial antioxidant role of this drug in managing the intracellular oxidative stress and

SC RI PT

neuroprotection. Thus, astaxanthin can be beneficial in curing disease involving oxidoinflammatory cascade such as neuropathic pain. 4.3. Astaxanthin and astrocytic activation:

Astrocytes are glial cells that are phagocytic in nature, which play a vital role in development of neurons and establishment of blood brain barrier BBB. Physiological

U

changes that occur after trauma, ischemia, infection etc. lead to activation of microglia

N

and astrocytes. These changes induce hypertrophy, proliferation, increased expression

A

of intermediate filaments, for example Glial Fibrillary Acidic Protein (GFAP) of

M

astrocytes. It is reported that hypertrophy, astrocytic activation and increased GFAP expression can be induced upon LPS treatment in C6 glial cells. Astaxanthin treatment

D

at 5 and 10μM doses was able to reduce LPS induce inflammation by decreasing

TE

astrocytic activation. Activation of astrocytes is often associated with the development

EP

and progression of neuropathic pain, thus astaxanthin induced decrease in astrocytic activation may be the potential mechanism involved in attenuation of neuropathic pain.

CC

4.4. Astaxanthin and neuropathic pain: In-vivo behavioral testing in nerve injured rats was carried out to check the

A

mechanical and thermal pain behavior. Intraperitoneal administration of astaxanthin at 5 mg/kg caused significant reduction in mechanical hyperalgesia whereas 10 mg/kg of astaxanthin significantly attenuated both mechanical and thermal hypersensitivity in nerve injured rats. Astaxanthin showed significant results comparable to standard drug

22

gabapentin (30 mg/kg, ip). Thus, in future, astaxanthin can be looked as an important natural drug for the treatment of neuropathic pain. 5. Conclusion: Findings from the current study suggests that astaxanthin treatment

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significantly attenuated neuropathic pain behavior in nerve injured rats by modulating oxidative-nitrosative stress and attenuating astrocyte activation. Therefore, astaxanthin can be used as a potential alternative in the treatment of chronic neuropathic pain. However, more detailed investigations are required to further probe the in-depth mechanism of action of astaxanthin.

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Funding: This work is supported by Department of Pharmaceuticals, Ministry of Chemical and Fertilizers, Govt of India, National Institute of Pharmaceutical Education

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and Research (NIPER) Ahmedabad, Gandhinagar, Gujarat, India and Department of

A

Science and Technology (DST), Government of India, Early Career Research Grant

D

M

(ECR/2016/001846) awarded to Dr. Vinod Tiwari.

TE

Conflict of interest

No conflicts of interest exist.

EP

Acknowledgement

This work is supported by Department of Pharmaceuticals, Ministry of Chemical and

CC

Fertilizers, Govt of India, National Institute of Pharmaceutical Education and Research (NIPER) Ahmedabad, Gandhinagar, Gujarat, India and Department of Science and

A

Technology

(DST),

Government

of

India,

Early

Career

Research

Grant

(ECR/2016/001846) awarded to Dr. Vinod Tiwari. Authors also want to express their thanks to Director, NIPER Ahmedabad for providing necessary facilities and infrastructure.

23

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References:

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Figure Legends: Figure 1. In-silico molecular docking studies with NR2A; Interaction of astaxanthin with

SC RI PT

NR2A receptor protein, 3D and 2D representations.

Figure 2: Effect of astaxanthin on (A) cytotoxicity (expressed as %cell viability) (B) LPS induced morphological changes in C6 glial cells; LPS Dose= 100ng/ml (24hrs). The data are expressed as the mean ± SEM and analyzed for statistical significance using one way ANOVA with a post hoc Bonferroni test for multiple comparison, (n=6). Cell

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morphology was observed under inverted fluorescence microscope at 100X

A

N

magnification.

M

Figure 3: Effect of astaxanthin on LPS-induced (A) Nitrite levels: increase in nitrite levels in the C6 cells (expressed as μM/mg protein). (B) Glutathione levels: decrease in

D

glutathione levels in the C6 cells (expressed as μM/mg protein). (C) Malonaldehyde

TE

(MDA) levels: increase in malonaldehyde (MDA) levels in the C6 cells (expressed as

EP

μM/mg protein). (D) SOD activity: decrease in SOD activity (expressed as units/mg protein). (E) Catalase activity: decrease in catalase activity in the C6 cells (expressed

CC

as kU/mg protein). (F) ROS levels: increase in the C6 cells (expressed as % ROS production). Values are expressed as Mean ± SEM (n=3). # represents significance

A

compared to LPS-induced group (# p<0.05, ## p<0.01 and ### p<0.001). * represents significance compared to control group (*** p<0.001).

27

Figure 4: Effect of astaxanthin on LPS induced astrocyte activation in C6 glial cells by immunocytochemistry; (A) GFAP expression was visualized by fluorescent labelling using FITC (green), nuclei were counterstained using DAPI (blue). Increased

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expression of GFAP was found in LPS stimulated group as the intensity of green fluorescence was found to be high. Astaxanthin treatment significantly and dose dependently reduced LPS-induced increased GFAP expression. Images were taken using inverted fluorescence microscope at 100x magnification. (B) The intensity of

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GFAP is expressed as Mean ± SEM (n=3). ***p<0.001 vs control; ##p<0.01 vs LPS.

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Figure 5. Effect of astaxanthin on mechanical allodynia of (A) ipsilateral and (B)

A

contralateral paws of nerve injured rats. Results are expressed as mean ± SEM and

M

analyzed for statistical significance using two way ANOVA with a post hoc Bonferroni test for multiple comparison, n=5; ***p<0.001 all groups vs Pre-CCI; ##p<0.01

D

astaxanthin (5 and 10 mg/kg) 60 mins after administration vs pre drug; ###p<0.001

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astaxanthin (5 and 10 mg/kg) 120 mins after administration vs pre drug; ###p<0.001

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gabapentin (30 mg/kg) 60 and 120 mins after administration vs pre drug,AST: astaxanthin, GP: gabapentin, CCI: chronic constriction injury, PWT: paw withdrawal

CC

threshold.

A

Figure 6. Effect of astaxanthin on thermal hyperalgesia of (A) ipsilateral and (B) contralateral paws of nerve injured rats. Results are expressed as mean ± SEM and analyzed for statistical significance using two way ANOVA with a post hoc Bonferroni test for multiple comparison, n=5; ***p<0.001 all groups vs Pre-CCI; #p<0.05

28

astaxanthin (10 mg/kg) 120 mins after administration vs pre drug; ###p<0.001 gabapentin (30 mg/kg) 60 and 120 mins after administration vs pre drug .AST: astaxanthin, GP: gabapentin, CCI: chronic constriction injury, PWL: Paw withdrawal

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A

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latency.

29

30

D

TE

EP

CC

A

SC RI PT

U

N

A

M

31

D

TE

EP

CC

A

SC RI PT

U

N

A

M

32

D

TE

EP

CC

A

SC RI PT

U

N

A

M

D

TE

EP

CC

A

SC RI PT

U

N

A

M

Fig 4

33

34

D

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EP

CC

A

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U

N

A

M

35

D

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CC

A

SC RI PT

U

N

A

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36

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Table 1: Docking results of the probable targets with astaxanthin and ketamine obtained by molecular docking (glide version 7.0) in the Schrödinger Suite.

PDB-ID

Ligand

G-score

NR2-B NR2-B

5EWJ 5EWJ

Ketamine Astaxanthi n

-6.67 -5.332

D-score

U

-6.645 -5.332

N A M D TE EP CC A

37

Epik state penalty 0.02 0.00

SC RI PT

Protein