Astaxanthin attenuates neuroinflammation contributed to the neuropathic pain and motor dysfunction following compression spinal cord injury

Accepted Manuscript Title: Astaxanthin attenuates neuroinflammation contributed to the neuropathic pain and motor dysfunction following compression spinal cord injury Authors: Sajad Fakhri, Leila Dargahi, Fatemeh Abbaszadeh, Masoumeh Jorjani PII: DOI: Reference:

S0361-9230(18)30628-2 https://doi.org/10.1016/j.brainresbull.2018.09.011 BRB 9516

To appear in:

Brain Research Bulletin

Received date: Revised date: Accepted date:

14-8-2018 11-9-2018 17-9-2018

Please cite this article as: Fakhri S, Dargahi L, Abbaszadeh F, Jorjani M, Astaxanthin attenuates neuroinflammation contributed to the neuropathic pain and motor dysfunction following compression spinal cord injury, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.09.011 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 attenuates neuroinflammation contributed to the neuropathic pain and motor dysfunction following compression spinal cord injury Sajad Fakhria, Leila Dargahib, Fatemeh Abbaszadehc, Masoumeh Jorjania,c* a

Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences,

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Tehran, Iran b

Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

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Neurobiology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

*Corresponding author: Masoumeh Jorjani Department of Pharmacology & Neurobiology Research Center, School of Medicine, Shahid Beheshti University of Medical Sciences, Velenjak, Tehran, Iran, Phone number: +98-2122429768, Fax number: +98-21-22431624, [email protected]

Highlights:

Astaxanthin (AST) improved sensory-motor performance after compression SCI

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AST inhibited glutamate-initiated signaling pathway following compression SCI

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AST decreased post- SCI tissue damage and preserved neurons after SCI

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AST treatment led to down-regulation of NR2B, p-p38MAPK and TNF-α in the spine

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Abstract

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Spinal cord injury (SCI) is a debilitating condition in which inflammatory responses in the secondary phase of injury leads to long lasting sensory-motor dysfunction. The medicinal therapy of SCI complications is still a clinical challenge. Understanding the molecular pathways underlying the progress of damage will

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help to find new therapeutic candidates. Astaxanthin (AST) is a ketocarotenoid which has shown antiinflammatory effects in models of traumatic brain injury. In the present study, we examined its potential in

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the elimination of SCI damage through glutamatergic-phospo p38 mitogen-activated protein kinase (pp38MAPK) signaling pathway. Inflammatory response, histopathological changes and sensory-motor function were also investigated in a severe compression model of SCI in male rats. The results of acetone drop and inclined plane tests indicated the promising role of AST in improving sensory and motor function of SCI rats. AST decreased the expression of n-methyl-d-aspartate receptor subunit 2B (NR2B) and p-p38MAPK as inflammatory signaling mediators as well as tumor necrosis factorα (TNF-α) as an inflammatory cytokine, following compression SCI. The histopathological study 1

culminated in preserved white mater and motor neurons beyond the injury level in rostral and caudal parts. The results show the potential of AST to inhibit glutamate-initiated signaling pathway and inflammatory reactions in the secondary phase of SCI, and suggest it as a promising candidate to enhance functional recovery after spinal injury. Keywords: Astaxanthin; Spinal cord injury; Neuroinflammation; Glutamate Signaling; Sensory-motor

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function; Rat

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1. Introduction

Spinal cord injury (SCI) is a debilitating disorder whose prevalence is progressively increasing and leads

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to sensory-motor and autonomic dysfunction as well as nerve tissue degeneration (Gruener et al., 2018;

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Kong and Gao, 2017; Luo et al., 2015; Sekhon and Fehlings, 2001). There are two phases after SCI. The first phase is related to the direct death of cells resulting from traumatic mechanical injury. It affects the

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local cells and disrupts axon tracts connecting the brain and spinal cord, causing neuronal damage to the spinal grey matter to a larger extent when compared to the white matter. Subsequent to the primary injury,

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a cascade of molecular and cellular processes including inflammation can exacerbate the injury resulted by the primary damage, which is called secondary phase (Beattie, 2004; David et al., 2013; Tator and Fehlings,

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1991). The secondary phase starts with the inflammatory responses and is characterized by increased bloodbrain barrier (BBB) permeability, glial and neuronal cell apoptosis, alongside complex neuroinflammatory

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responses (Donnelly and Popovich, 2008; Profyris et al., 2004). The secondary phase lasts for months and years and causes sensory-motor dysfunction. Despite the progress in the preclinical and clinical studies, there is no effective treatment for improving sensory-motor

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dysfunction following SCI; therefore, developing new therapies via targeting the main mechanisms involved in the SCI is of special clinical importance. However, the mechanisms of secondary injury have remained poorly defined, and unlike the acute phase, it may be reversible (Koda et al., 2004; Lerch et al., 2014; Schwab and Bartholdi, 1996). Among the underlying mechanisms, inflammation is the most important (Faden et al., 2016). Subjects with SCI have a higher plasma level of cytokines (e.g. tumor necrosis factor-α), chemokines (e.g. Tumor necrosis factor) and other inflammatory agents (Bank et al., 2

2015). So, trying to find a new strong anti-inflammatory agent can be a key therapeutic goal (Kidd, 2011; Shen et al., 2009). Astaxanthin (AST) is a lipid-soluble keto-carotenoid (Baralic et al., 2015; Nakao et al., 2010), which is found in different microorganisms (Ambati et al., 2014), marine animals, seafood (Higuera-Ciapara et al., 2006), and phytoplanktons (Balietti et al., 2016), and acts as a strong anti-oxidant agent (Pan et al., 2017;

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Xue et al., 2017). It captures radicals in the membrane and scavenges radicals outside and inside the membrane because of the structure of its polyene chain and terminal rings, respectively (Augusti et al., 2012). This polar-nonpolar-polar structure of AST allows for making a precise fit into the same structural part of the cell membrane, permitting AST cross easily through the BBB and affecting different signaling pathways (Higuera-Ciapara et al., 2006; Kidd, 2011). The reactive oxygen species are a primary candidate stimulus for the induction of inflammation (Elmarakby and Sullivan, 2012). Therefore, owing to its

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antioxidant effects, AST is an anti-inflammatory agent and possesses promising effects in different acute

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and chronic neurodegenerative conditions (Speranza et al., 2012; Suzuki et al., 2006). In mechanistic point of view, there are several in-vivo and in-vitro reports clarifying the anti-inflammatory

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effects of AST. In an in-vitro study, AST is shown to reduce the gene expression of inflammatory mediators

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like interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in response to H2O2 induced cytotoxicity in U937 cell line (Suzuki et al., 2006). AST also inhibits cyclooxygenase-1 enzyme (COX-1) and nitric oxide (NO), demonstrating its anti-inflammatory actions (Choi et al., 2008; Nakano et

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al., 2008; Ohgami et al., 2003). AST has also proven to block the nuclear factor kappa B (NF-κB)-dependent

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signaling pathway in endotoxin-induced uveitis in rats (Speranza et al., 2012). It has been also shown that AST increases the anti-oxidant enzymes catalase (CAT), superoxide dismutase (SOD) (Heidari Khoei et

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al., 2018), along with peroxidase and thiobarbituric acid reactive substances in rat plasma and liver (Ranga Rao et al., 2010; Wu et al., 2015). In other studies, it is shown that AST modulates p38/mitogen-activated protein kinase (p38MAPK), activates phosphoinositide 3-kinase (PI3K)/AKT survival pathway, enhances

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Bad phosphorylation, and decreases the activation of cytochrome c and caspase 3 and 9 (Dong et al., 2013; Guo et al., 2015; Wu et al., 2015; Zhang et al., 2014). Elsewhere, it is also shown that AST affects the n-

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methyl-d-aspartate (NMDA) receptor subunit 1 (NR1) signaling pathway to prevent NMDA-triggered retinal damage, reduces the apoptotic death of retinal ganglion cells, decreases lipid peroxidation and oxidative DNA damage, and antagonizes methyl phenylpyridinium-induced oxidative stress (Dong et al., 2013; Nakajima et al., 2008; Ye et al., 2013). In our previous study we also reported the anti-apoptotic and protective effects of AST in a contusion SCI model of rats (Masoudi et al., 2017).

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In the present study, we aimed to investigate the potential effects of AST to modulate sensory-motor and histopathological dysfunctions, through modifying the NR2B and phospho-p38MAPK (p-p38MAPK) signaling elements as well as an inflammatory cytokine (TNF-α), in a severe compression model of SCI in rats. 2. Material and methods

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2.1. Experimental animals

Totally, 75 adult male Wistar rats (230-270g, breeding colony of Neuroscience Research Center, Shahid Beheshti University of Medical Sciences) were kept in a room under standard laboratory conditions (temperature 24 ± 2 °C, 12-h light/dark cycle, with fresh food and water ad libitum, 10 days prior to the study). All the experiments were conducted in line with the guidelines and policies of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animal and approved by the institutional animal

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care and use committee at Shahid Beheshti University of Medical Sciences (IR.SBMU.REC.1396,321). We

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attempted to use the minimum number of animals, incur the minimum level of suffering, and used aseptic

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techniques in surgical operations.

Twenty-one rats were randomly divided into three major groups as sham, SCI and AST-treated, consisting

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of 7 rats per each group. The sham group received laminectomy without compression lesion. The SCI group underwent compression lesion and were treated with 5% dimethylsulfoxide (DMSO) as vehicle. The AST-

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treated group (AST) received compression injury and were treated with AST (10 μl of 0.2 mM, Intrathecal

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(i.t.)) just 30 min after injury. All rats in these three groups, were subjected to behavioral testing prior to surgery and weekly on days 7, 14, 21 and 28 post surgery. On day 28, six animals of each group were deeply anesthetized with a mixture of ketamine/xylazine (100/20 mg/kg, i.p.) and sacrificed. Three spines

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of each group were used for western blot and 3 spines for histological analysis. For the molecular and histological assessments on days 7, 14 and 21 post surgery, we assigned 54 rats, divided into the same

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experimental groups including the sham, SCI and AST. On each time points post-surgery (days 7, 14 and 21), 6 rats per group were sacrificed and 3 spines of each group were used for western blot and 3 for

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histological analysis. 2.2. Spinal cord compression injury

For the sham and SCI surgeries, all rats received deep anesthesia with i.p. administration of ketamine/xylazine (80/10 mg/kg). The surgical site was shaved and disinfected with ethanol 70%. Laminectomy was done at the T8-T9 level with a micro rongeur (Fine Science Tools, USA). Subsequently, the extradural clip compression SCI was generated by closing an aneurysm clip (Aesculap, Tuttlingen, 4

Germany) with a 90g calibrated closing force, around the spinal cord for 1 min. After sham or SCI surgery, muscles and skin were sutured, and rats were allowed to recover on a 30°C heating pad, and then received saline (2 ml, twice a day, subcutaneous) and Cefazoline (40 mg/kg twice on the day of surgery, i.p.) to rehydrate and prevent urinary tract infection, respectively. SCI rendered all animals completely paraplegic. Urinary excretion was manually carried out two times per day until gaining bladder-emptying reflex

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recovery.

2.3. Intrathecal drug injection

AST (Sigma, St.Louis, USA) was dissolved in 5% DMSO at the concentration of 0.2mM. We used the modified method of i.t. injection described by Mestre et al (Mestre et al., 1994). In anesthetized rats, 30

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min after SCI surgry, AST or the vehicle (5% DMSO) were injected i.t. with a 25μl Hamilton microsyringe at the volume of 10μl slowly over 10 s into the tissues between the dorsal aspects of L5 and L6. In order to

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prevent outflow of the drug, the needle was held for a few more seconds. The quality of the i.t. injection

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was confirmed by observing a tail flick with no other specific sign of pain at the time of injection.

2.4.1.

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2.4. Behavioral assessments Acetone drop test

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The rats were placed in acrylic cages on top of a wire mesh grid to allow access to the paws. Following a 45-min acclimatization time, a cold stimulation reaction was tested on the plantar surface of the hind paw

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by applying about 100 μl acetone from a distance of 2 cm. A response was recorded if the rat withdrew its hind paw, which were classified as: no reaction, startle response without paw withdrawal, brief withdrawal

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of the paw, prolonged withdrawal (5-30s), prolonged and repetitive withdrawal (30s) with flinching, and licking which scored 0,1,2,3, 4, and 5, respectively (Kauppila, 2000). This acetone test has been described as constituting cold, chemical, and possibly mechanical stimulation (Dowdall et al., 2005). An observer

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tested cold allodynia in a blinded manner.

Inclined plane test

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2.4.2.

We evaluated motor performance in rats using an adjustable 60 × 40 cm wood plane which could be inclined at an angle of 0° (horizontal) to 60° inclined plane (Samini et al., 2013). The maximum angle on the inclined plane at which each rat could stay stable for 5 sec was evaluated by two observers who were blinded to the animal groups, and the average angle was recorded.

2.5. Western blot analysis 5

Western blot was done based on our previously reported protocols on the days when molecular assessments were made and the epicentral segments of the injured spinal cord were dissected (Naseri et al., 2013). Briefly, the tissues were removed and homogenized with lysis buffer containing NaCl 150 mM, Triton X100 0.1%, Sodium dodecyl sulfate (SDS) 0.1%, Tris–HCL 50 mM, EDTA 1 mM, sodium deoxycholate 0.25%, and protease inhibitor cocktail 1% for 10-15 min on ice. The homogenates were centrifuged at 4 °C

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for 45 min at 12000 rpm. After collecting the supernatants, the protein concentration was measured using Bradford assay. For western blotting, the proteins were fractioned on SDS-polyacrylamide gel electrophoresis, followed by transference onto polyvinylidene difluoride membrane. The membranes were blocked with non-fat dry milk 2% (Amersham, ECL Advance TM) for 75 min and then were incubated with polyclonal anti-NR2B (1:10000 v/v, Abcam Co.), polyclonal anti-p-p38MAPK (1:1000 v/v, Cell signaling Co.), polyclonal anti-TNF-α (1:500 v/v, Abcam Co.), and anti-β-actin (1:1000 v/v, Cell signaling Co.) antibodies overnight at 4 °C. Then, the membranes were incubated with a secondary antibody (1/8000,

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Cell signaling Co.) at room temperature for 2 h. Afterward, the membrane was washed three times with

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Tris-buffered saline with Tween 80.

The membranes were developed with a western ECL substrate chemiluminescent detection reagent (ECL

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solution) and then were exposed to x-ray films. Image J software (NIH) was used to quantify the intensity

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of specific bands. Finally, the ratios of NR2B and p-p38MAPK as well as TNF-α to β-actin were reported.

2.6. Histological analysis

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The rats were deeply anesthetized with a mixture of ketamine and xylazine (100 and 20 mg/kg, i.p.).

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Perfusion of 200 ml of cold 0.1 M phosphate-buffered saline (PBS) and then 200 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH = 7.4) was performed transcardially. About 1.5 cm

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of spinal cord sections on the compression site were removed and stored in 4% PFA at 4°C overnight. The removed spinal cord was processed in a tissue processor (Didban sabz DS2080/H; Iran) and embedded in paraffin. Three sections rostral (-1800 µm) and three sections caudal (+1800 µm) to the lesion center per

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rat, were stained with Cresyl violet and Luxol fast blue (Masoudi et al., 2017). The stained sections were dehydrated in alcohol and xylene, coverslipped, visualized under the light

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microscope Nikon E600 equipped with an optical camera, and was photographed at 10 X and 40 X magnifications. Image J software was used to quantify the spared myelin and count the average number of ventral horn motor neurons. The percentage of spared myelin was calculated by dividing the area of tissue displaying normal myelin to the total area of spinal cord sections. The number of survived motor neurons was counted on two ventral horns of gray matter and then the mean was calculated.

2.7. Statistical analysis 6

The data are expressed as mean ± standard error of the mean (SEM) and analyzed using the GraphPad Prism Program, Version 6.0 (GraphPad Software, version 6.0). Two-way repeated measures analysis of variance (ANOVA) with Bonferroni post-hoc analysis was used to compare three groups across all measuring times. In all calculations, a difference with p < 0.05 was regarded as significant.

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3. Results 3.1. AST treatment improves sensory-motor dysfunction following SCI

The inclined plane test was used to assess the locomotor recovery after SCI. As demonstrated in Fig. 1, the rats in the laminectomy sham group continued their normal angle stay on inclined plane apparatus, suggesting that the surgery per se did not cause any functional impairments. SCI caused a significant reduction in the angle stays. In other words, the spinal cord injured rats indicated a decrease in the mean angle of stay on the inclined-plane apparatus as compared to the sham. Interestingly, the rats treated with

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AST experienced a significantly faster recovery during the weeks after SCI, such that from week one there

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was a significant difference between SCI and AST group. In the sham group, rats continued their normal cold hypersensitivity in a 4-week follow-up. SCI rats developed a significant cold hypersensitivity in

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comparison to the sham group, as shown by the mean acetone test score. AST caused improvements in the

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rats' response to cold stimulation compared to the SCI group to such an extent that this threshold returned to almost normal level.

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3.2. AST treatment reduced neuroinflammatory signaling elements following SCI

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To check the inflammatory response in the severe compression SCI, the expression levels of NR2B, p-p38 MAPK (threonine 180 and tyrosine 182 phosphorylated p38MAPK) and TNF-α were evaluated in sham, SCI and AST treated groups on days 7, 14, 21, and 28. We further examined spinal changes of

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housekeeping protein β-actin at the same four-time points and across the three groups. At each time point, the ratios of NR2B/β-actin and p-p38MAPK/β-actin as inflammatory signaling mediators as well as TNF-

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α/β-actin as an inflammatory cytokine were compared between the three experimental groups. SCI rats revealed a significant increase in NR2B/β-actin, p-p38MAPK/β-actin, and TNF-α/β-actin. Interestingly, on treatment with AST, NR2B/β-actin, p-p38 MAPK/β-actin, and TNF-α/β-actin were significantly reduced

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when compared to the SCI group (Fig. 2).

3.3. AST treatment preserved myelinated white matter and neurons following SCI SCI caused a signficant demyelination and a faint myelin staining in the dorsal white matter and ventrolateral parts of rostral side from day 14 following injury and continued to day 28, along with caudal on day 14 following injury. The operated sections of the sham group were undamaged in structure and 7

showed normal white matter myelin staining all over the ventral and dorsal sides of both rostral and caudal parts (Fig. 3). AST treatment significantly preserved the normal staining of white matter and lowered the myelin loss of rostral part of the spinal cord on day 28 and caudal part on days 21 and 28. Counting the number of survived motor neurons in the ventral horn of gray matter on the same time points after injury proved the same

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results. As displayed in Fig. 4, although compression SCI caused a significant drop in the number of motor neurons compared to the sham group, AST improved the survival of neurons.

4. Discussion

Our results suggest that AST treatment produces a remarkable effect against post-SCI sensorimotor

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dysfunction. The downregulation of NR2B, p-p38MAPK, and TNF-α, as cytotoxic and inflammatory

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signaling elements, can be introduced as the underlying mechanism through which AST limits SCI-induced demyelination and tissue damage, pain-like behavior and motor dysfunction.

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Complex pathophysiological mechanisms (Kwon et al., 2011a), high morbidity, and serious complications

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of SCI have raised the needs for multi-target treatments attenuating neuronal hyperexcitability, apoptosis, neuroinflammation, neuroplasticity, and neurodegeneration processes (Bradbury and McMahon, 2006; Kwon et al., 2011b). We previously reported anti-apoptotic effects of AST in a contusion model of SCI

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(Masoudi et al., 2017), and in the present study, in order to introduce AST as a multi-target drug, examined

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its anti-neuroinflammatory effects in a clip compression SCI model. Compression, contusion, and hemisection are studied in rodent models of SCI for simulating the clinical situation in terms of sensory-motor dysfunctions and injury mechanisms (Borbély et al., 2017; Nakae et al.,

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2011; Šedý et al., 2008). Clip compression model is preferred since it provides a precise control over injury to evaluate SCI-induced chronic pain and motor dysfunctionality (Cheriyan et al., 2014).

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Acetone drop test and inclined plane test following compression SCI in this study indicated symptoms of neuropathic pain and motor dysfunction. A single dose AST treatment ameliorated pain-like behavior as

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well as motor dysfunction. The beneficial effect of AST on behavioral parameters was manifested from day 7 and existed even 28 days after SCI, indicating that the effect provided by AST is long lasting and not transient. Regarding the anti-nocifensive effects of AST, it has been shown that AST mitigates mechanical and thermal hyperalgesia in a carrageenan-induced inflammation and pain model in mice with an efficacy comparable to indomethacin (Kuedo et al., 2016). In other studies on chronic constriction injury model of neuropathic pain, AST is also shown to have therapeutic effects on thermal and mechanical hyperalgesia (Jiang et al., 2018; Sharma et al., 2018). 8

Glutamate-induced neurotoxicity is repeatedly shown to contribute with the progress of neurodegenerative diseases like SCI (Lancelot and Beal, 1998; Sheldon and Robinson, 2007). Malfunctioning of the glutamatergic neurotransmission is also involved in different neurological diseases including neuropathic pain. Glutamate stimulates NMDARs and by increasing intracellular concentration of calcium ions affects neurotransmitter release, alters the cell membrane excitability and plays a crucial role in the central

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sensitization of nociceptive neurons and also activation of astrocytes (Sharma et al., 2018). Glutamate excitotoxicity is responsible for neuronal apoptosis via modulating the expression of B-cell lymphoma 2 (Bcl-2) family of proteins (Hu et al., 2011; Schelman et al., 2004) and the activation of caspase 3 (Bachis et al., 2001; Brecht et al., 2001; Du et al., 1997). Although glutamate is a central player in neurotoxic events, it has been reported that the release and uptake of other neurotransmitters like noradrenaline is also imbalanced in SCI, an event which may be secondary to glutamate overconcentration or even independent to glutamate, leading to neural toxicity (Borbély et al., 2017; Végh et al., 2017). Given the role of glutamate

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signaling in the pathogenesis of SCI, we measured the expression level of NR2B subunit of NMDARs in

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SCI rats, and interestingly showed that treatment with AST attenuates the SCI-induced upregulation of NR2B. In this regard, it has been shown that NR2B subunit is enriched in extra-synaptic NMDARs which

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are central players in excitotoxicity-induced neurodegeneration and their blockade are proved to provide

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neuroprotection (Kiss et al., 2012; Vizi et al., 2013). Accordingly, it has been reported that AST pretreatment significantly suppresses glutamate-induced elevation of intracellular Ca2+ concentration (Araújo et al., 2010; Rami et al., 2000; Ray et al., 2000; Zhang and Bhavnani, 2006), and protects neuroblastoma

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SH-SY5Y cells against glutamate-induced apoptosis (Lin et al., 2017). AST may also work directly via

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NMDA receptor inactivation to attenuate neuropathic pain, since in-silico molecular docking studies have revealed that AST particulary fits into the NR2B subunit which is involved in nociception (Sharma et al.,

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2018).

Upregulation of p38MAPKs pathway following injury and excitotoxicty (Hui et al., 2014; Jiang et al., 2000; Stanciu et al., 2000) is shown to play a key role in post-SCI neuropathic pain (Choi et al., 2012) as well

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as apoptosis and cell death (Evans et al., 2015). Herein, we showed that the active phosphorylated form of p38MAPK increases following SCI, and AST treatment decreases it to almost normal levels. These results are in line with other findings indicating that activated p38MAPK mediate changes in nociceptive reactivity

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in various models of peripheral injury (Kim et al., 2002; Song et al., 2005; Zhang et al., 2005). And, also in line with studies revealing that AST inhibits acetaldehyde- (Yan et al., 2016) and 6-hydroxydopamine(Ikeda et al., 2008; Shen et al., 2009) induced p38MAPK activation in a dose-dependent manner (Yan et al., 2016). Attenuation of p38MAPK activation by AST treatment in the context of SCI may therefore underlie its anti- allodynia and neuroprotective effect.

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Measuring TNF-α as a player of neuroinflammtion, we showed that AST treatment reduces TNF-α in spinal cord tissue after compression SCI, though not as efficacious as reducing NR2B and p-p38MAPK. The transient ant-inflammatory effect of AST observed herein can be attributed to applying the treatment at only a single dose, however, there are studies showing that AST plays an immunomodulatory role via down regulation of IL-6, TNF-α and NF-κB expression in the brain and prevents from potassium channel blocker-

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induced neuroinflammatory responses (Sifi et al., 2016). At the level of histopathological outcomes, we showed herein that demyelination and neuron loss can be tracked in rostral and caudal area of injured spinal cord as also reported in other studies ( Kakulas et al., 1998; Hook et al., 2017; O’Shea et al., 2017). We further proved that AST treatment protects form compression SCI-induced degeneration which is consistent with our pervious report in a contusion model of SCI (Masoudi et al., 2017)

In conclusion, the present study suggests NR2B and p-p38MAPK as signaling mediators along with TNF-

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α as an inflammatory cytokine underlying the development of below-level cold allodynia and motor

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dysfunctionality following SCI. AST treatment attenuates post SCI tissue damage, neuropathic pain, and finally motor dysfunction possibly by down-regulation of NR2B, p-p38MAPK and TNF-α in the spinal

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dysfunctions for further translational studies.

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cord. So, AST can be considered as a new candidate to protect against SCI-induced sensorimotor

Conflicts of interest

Acknowledgment

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The authors state that there are no conflicts of interest.

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The current research was performed as part of a Ph.D. thesis project of Sajad Fakhri and was supported by the Neuroscience Research Center (Grant No. S-N-56-1397) of Shahid Beheshti University of Medical

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Sciences.

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Figure legends:

Fig 1: Pain-related and motor performance behaviors in rats undergoing clip compression SCI and AST treatment. Aceton test (A), and inclined plane test (B). Data are presented as mean ± SEM (n=7 per +++

p < 0.001 vs. SCI group. Repeated measures two-way

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group). ***p < 0.001 vs. sham, and ++p < 0.01,

analysis of variance with Bonferroni post-hoc was used. SCI, Spinal cord injury; AST, Astaxanthin (10 μl of 0.2 mM, intrathecal, 30 min post-injury), DMSO, dimethylsulfoxide.

Fig 2: Effects of AST on the NR2B, p-p38MAPK, and TNF-α expression in rats undergoing clip compression SCI and AST treatment. Data are presented as mean ± SEM (n=3 per group). *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham, and +p < 0.05,

++

p < 0.001, +++p < 0.001 vs. SCI group. Repeated

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measures two-way analysis of variance with Bonferroni post-hoc was used. SCI, Spinal cord injury; AST,

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Astaxanthin (10 μl of 0.2 mM, intrathecal, 30 min post-injury); DMSO, dimethylsulfoxide; NR2B, nmethyl-d-aspartate receptor subunit 2B; p-p38MAPK, phospo p38 mitogen-activated protein kinase; TNF-

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α, tumor necrosis factor-α.

Fig 3: Luxol fast blue staining of spinal cord rostral and caudal paraffin transverse sections in rats undergoing clip compression SCI and AST treatment. Rostral (A, B) and caudal (C, D). Data are

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presented as mean ± SEM (n=3 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. sham, and +p <

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0.001, +++p < 0.001 vs. SCI group. Repeated measures two-way analysis of variance with Bonferroni posthoc was used. SCI, Spinal cord injury; AST, Astaxanthin (10 μl of 0.2 mM, intrathecal, 30 min post-injury);

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DMSO, dimethylsulfoxide.

Fig 4: Cresyl violet staining of spinal cord rostral and caudal paraffin transverse sections in rats

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undergoing clip compression SCI and AST treatment. Rostral (A, B) and caudal (C, D). Data are presented as mean ± SEM (n=3 per group). *p < 0.05, **p < 0.01 and ***p < 0.001 vs. sham, and +p < 0.05 and ++p < 0.05 vs. SCI group. Repeated measures two-way analysis of variance with Bonferroni post-hoc

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was used. SCI, SCI, Spinal cord injury; AST, Astaxanthin (10 μl of 0.2 mM, intrathecal, 30 min postinjury); DMSO, dimethylsulfoxide.

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