Astaxanthin improves cognitive performance in mice following mild traumatic brain injury

Astaxanthin improves cognitive performance in mice following mild traumatic brain injury

Accepted Manuscript Research report Astaxanthin improves cognitive performance in mice following mild traumatic brain injury Xinran ji, Dayong Peng, Y...

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Accepted Manuscript Research report Astaxanthin improves cognitive performance in mice following mild traumatic brain injury Xinran ji, Dayong Peng, Yiling Zhang, Jun Zhang, Yuning Wang, Yuan Gao, Ning Lu, Peifu Tang PII: DOI: Reference:

S0006-8993(16)30860-5 http://dx.doi.org/10.1016/j.brainres.2016.12.031 BRES 45235

To appear in:

Brain Research

Received Date: Accepted Date:

14 September 2016 30 December 2016

Please cite this article as: X. ji, D. Peng, Y. Zhang, J. Zhang, Y. Wang, Y. Gao, N. Lu, P. Tang, Astaxanthin improves cognitive performance in mice following mild traumatic brain injury, Brain Research (2016), doi: http://dx.doi.org/ 10.1016/j.brainres.2016.12.031

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Astaxanthin improves cognitive performance in mice following mild traumatic brain injury Running Title: :ATX improves cognitive in TBI in mice Xinran ji1#, M.D. Dayong Peng2#, M.D. Yiling Zhang1,M.D Jun Zhang1, M.D. Yuning Wang1, Yuan Gao1, M.D. Ning Lu 1*, M.D. Peifu Tang1*, M.D 1, The Department of Orthopaedic Surgery. Chinese People's Liberation Army General Hospital(301 Hospital), 28 Fuxing Road, Wukesong, Beijing 100000, China. 2, Department of orthopedics, Shandong Qianfoshan Hospital, Shandong University Jing Shi Road,Jinan,Shandong 250014,P.R.China. # Co-first authors *Co-Corresponding authors: Peifu Tang, Tel: +86 -18510622211 Email: Peifu Tang: [email protected] Ning Lu: [email protected] Address: The Department of Orthopaedic Surgery. The General Hospital of People's Liberation Army (301 Hospital), 28 Fuxing Road, Wukesong, Beijing 100000, China.

Abbreviations: AST, astaxanthin; SAH, subarachnoid hemorrhage; TBI, traumatic brain injury; BDNF, brain-derived neurotrophic factor; GAP-43, growth-associated protein-43; NSS, Neurological Severity Score; CNS, central nervous system; SYP, synaptophysin; ORT, object recognition test; Nrf2, nuclear factor erythroid 2-like 2; PBS, phosphate buffered saline; P.O. per os

Abstract Background: Traumatic brain injury (TBI) produces lasting neurological deficits that plague patients and physicians. To date, there is no effective method to combat the source of this problem. Here, we utilized a mild, closed head TBI model to determine the modulatory effects of a natural dietary compound, astaxanthin (AST). AST is centrally active following oral administration and is neuroprotective in experimental brain ischemia/stroke and subarachnoid hemorrhage (SAH) models. We examined the effects of oral AST on the long-term neurological functional recovery and histological outcomes following moderate TBI in a mice model.. Methods: Male adult ICR mice were divided into 3 groups: (1) Sham + olive oil vehicle treated, (2) TBI + olive oil vehicle treated, and (3) TBI + AST. The olive oil vehicle or AST were administered via oral gavage at scheduled time points. Closed head brain injury was applied using M.A. Flierl weight-drop method. NSS, Rotarod, ORT, and Y-maze were performed to test the behavioral or neurological outcome. The brain sections from the mice were stained with H&E and cresyl-violet to test the injured lesion volume and neuronal loss. Western blot analysis was performed to investigate the mechanisms of neuronal cell survival and neurological function improvement. Results: AST administration improved the sensorimotor performance on the Neurological Severity Score (NSS) and rotarod test and enhanced cognitive function recovery in the object recognition test (ORT) and Y-maze test. Moreover, AST treatment reduced the lesion size and neuronal loss in the cortex compared with the vehicle-treated TBI group. AST also restored the levels of brain-derived neurotropic factor (BDNF), growth-associated protein-43 (GAP-43), synapsin, and synaptophysin (SYP) in the cerebral cortex, which indicates the promotion of neuronal survival and plasticity. Conclusion: To the best of our knowledge, this is the first study to demonstrate the protective role and the underlining mechanism of AST in TBI. Based on these neuroprotective actions and considering its longstanding clinical use, AST should be considered for the clinical treatment of TBI.

Keywords: traumatic brain injury, astaxanthin, NSS, rotarod test, Y-maze, object recognition test, growth-associated protein, synaptic protein

.

Introduction Traumatic brain injury (TBI) is a significant public health problem and a major cause of mortality and morbidity worldwide [1, 2]. Most cases of TBI are not life threatening; however, TBI exerts a severe impact on motor, cognitive, and intellectual functioning, as well as other health problems in affected individuals [3]. Despite intense efforts invested in the identification of therapeutic measures for TBI, the current therapies are limited and far from satisfactory [4]. TBI induces neural cell death and neurological dysfunction via immediate, direct physical forces, as well as long lasting but potentially reversible secondary injury [5]. TBI-induced neuronal loss and its associated cognitive deficits have been demonstrated in rat and mouse models [6, 7]. TBI is a disease with complex actions and dysregulation throughout the brain that results in neuronal loss [8]. The mechanisms of secondary injury involve a complex cascade of changes, including pathobiological, metabolic, and gene expression-related changes, which not only induce neuronal cell death but also activate chronic inflammatory, calcium release, oxidative, and apoptosis signaling cascades that contribute to further neuro-degeneration [1, 9-11]. These multifactorial dysregulations that comprise secondary injuries result in extended neuronal loss and diffuse axonal injury [12, 13]. TBI induced axonal injury is a serious condition and leads to the degradation of neuronal circuitry [12, 13]. The axonal damage induced by secondary injuries may persist following the primary injury. TBI initiated secondary injuries comprise the targets for TBI treatments. If sufficient neurons and axons may be preserved, then the cognitive deficits and prolonged symptoms will be reduced in patients. Astaxanthin (AST), a dietary carotenoid, is protective in various models of disease via anti-inflammation, anti-oxidation [14-18]. However, the potential effects of AST on TBI remain uninvestigated. The current study aimed to investigate the protective effects of AST on TBI-induced neurological deficits and its underlying mechanisms.

2. Results

2.1 Neurological Severity Score (NSS) In the assessment of the NSS, the dosing regimen of AST was examined in mice. The NSS test was previously developed to evaluate post-traumatic functional impairments in mice [19, 20], and it comprises a powerful and useful tool to assess spontaneous or drug-induced recovery following TBI. Ten different tasks are used to evaluate motor ability, balancing, and alertness. One point indicates the failure to perform a task, and 0 indicates success. Scores range from zero in healthy uninjured animals to a maximum of 10 in impaired mice. The NSS at 1 h post-trauma reflects the initial severity of injury. One hour after TBI, all mice regardless of group exhibited NSS values in the range of 6-7, which indicates moderate injury. As shown in Fig. 1, the NSS values decreased with time in all groups as a result of spontaneous recovery; however, the recovery of the AST-treated animals was faster. The group that was treated with a higher dose of AST (75 mg/kg) post-injury exhibited a significantly greater recovery beginning on day 7 compared with the vehicle-treated mice. AST treatment at a lower dose (25 mg/kg) was not sufficient to induce a significant difference between the AST-treated and vehicle-treated injured mice at 7 to 28 days post-injury. We therefore selected the higher dose of AST (75 mg/kg) for our subsequent experiments.

2.2 Rotarod test The Rotarod, which is used to assess sensorimotor coordination and motor learning in rodent models of central nervous system (CNS) disorders [21], was conducted in mice post injury. At 3 days after TBI injury, the AST-treated mice exhibited significantly increased performance scores compared with the vehicle-treated mice (Fig. 2). However, the animals in both experimental groups improved with time. Thus, there was no significant difference between the AST- and vehicle-treated groups from days 7 to 28.

2.3 Object recognition test (ORT) The ORT, which is used to assess the visual memory and nonspatial cognitive

performance of mice [22], was performed to determine the effects of AST treatment. As shown in Fig 3, TBI reduced the preference index in the ORT on days 7 and 28 post-injury; however, AST administration in TBI mice abolished this decrease in the preference index.

2.4 Y-maze The Y maze test was used to assess spatial memory [23]. Similar to the previously described findings, AST prevented the spatial memory deficits following TBI as assessed via the Y-maze. At 7 days post-TBI, the preference index was significantly damaged in the TBI mice compared with the sham operated mice; however, this impairment was significantly inhibited by AST. Moreover, AST maintained its protective effect at 28 days post-injury.

2.5 Cerebral infarct volume The TBI-induced lesion volume was quantified in hematoxylin and eosin (H&E) stained coronal brain sections obtained from the sham, vehicle-treated and AST-treated groups at 7 days after TBI injury using stereological methods [24, 25]. AST treatment significantly reduced the TBI-induced lesion volumes compared with the vehicle-treated group (Figure 3B). Representative images from each group are shown in Figure 3A.

2.6 Neuron loss The TBI-induced neuronal loss in the cortex was quantified in Nissl stained brain sections obtained from the sham-injured, vehicle TBI, and AST TBI groups 7 days after injury using stereological methods [24]. A significant effect of treatment was identified in the cortex neuronal densities, and a significant difference in the neuronal densities was identified between the sham-injured and vehicle TBI groups. Moreover, significantly increased neuronal densities were identified in the cortex of the AST-treated group compared with the vehicle TBI group.

2.7. Western blot analysis Neurotropic and synaptic proteins from cortical segments of the injured hemisphere, which have been linked to the neurological score [26], were detected via western blot analysis. The protein levels were calculated as the relative optical density of the examined protein divided by the beta-Tubulin level within the same lane, and the mean value for each treatment group was calculated and compared with the sham group. This approach enabled us to determine the effects of AST treatment on the levels of synapsin I, synaptophysin (SYP), GAP-43 and brain-derived neurotrophic factor (BDNF) following TBI. As shown in Fig. 7, the synapsin I, SYP, GAP-43, and BDNF levels were significantly decreased after injury, whereas AST administration increased the levels of these proteins (Fig. 7).

3. Discussion The present study assessed the effects of AST on the extent and rate of recovery of motor and cognitive behavioral tasks following a focal lesion induced by TBI in mice. The cerebral infarct volume, neuronal loss, neurotropic proteins, and markers of neural plasticity were also investigated. AST is a natural compound in algae, crustaceans, shellfish, and various plants [27], and it exhibits anti-inflammatory, anti-oxidative, anti-cancer and immunomodulatory properties [28-32]. AST was approved by the United States Food and Drug Administration as a feed additive in 1987 and a dietary supplement in 1999 [33]. It also exhibits a neuroprotective property [17, 18, 34]. AST is beneficial in ischemic brain injury and SAH in vivo, as well as protective against neurotoxicity in neural and retinal ganglion cells [15, 35, 36]. However, most previous studies have primarily focused on the protective property of AST during the acute stage. In contrast, there is limited knowledge regarding its long-term effects on CNS injury. We used a well-established TBI model in mice to determine the effects of AST on neurological deficits following TBI because long-term motor and cognitive impairments have previously been demonstrated [37]. Previous studies have indicated that AST is highly effective when delivered via intraperitoneal injection. Zhang et al. utilized a P.O.

administration of AST (75mg/kg) following SAH and identified improvements in the neurological deficits similar to the intraperitoneal administration route [28, 35]. Thus, AST penetrates into the brain to activate protective pathways and leads to neurological recovery. Following TBI, the blood brain barrier is compromised, and an increase in Evans blue permeability has been reported from 4 h until 7 days post-injury [38]. Moreover, P.O. administration is the most suitable route for long term, repeated treatments. Thus, we utilized P.O. delivery in this study. The assessment of neurological function via the NSS demonstrated that the post-treatment of two doses once daily from 30 min after TBI is beneficial. Moreover, the administration of 75 mg/kg AST post-injury resulted in faster and better recovery, whereas the administration of 25 mg/kg AST exhibited minimal differences compared with the vehicle-treated injured mice at 7 to 28 days post injury. Thus, we selected the higher dose of AST to treat secondary injuries following TBI in mice. Furthermore, at 3 days post-injury, the AST-treated mice performed better than the vehicle-treated mice in the Rotarod test. However, as a result of spontaneous recovery following TBI, the significant difference between the groups was lost over time. The ORT is a well-validated method to assess memory deficits in rodents. In general, mice are able to distinguish between familiar and novel objects and spend more time exploring a novel object compared with a familiar object. The failure to show a preference for the novel object is interpreted as an impairment in either the storage or retrieval of the memory of the previously presented object [39]. The Y-maze test measures the ability to memorize spatial cues and the innate interest in exploring new environments [40]. The mice treated with AST exhibited a significantly better performance in both the ORT and Y-maze test compared with the vehicle-treated mice. In the ORT, the preference for the novel object was superior in the AST-treated animals 7 days after TBI induction, and this difference remained at 28 days. We used the same mice to investigate the ORT at early (7 days) and late (28 days) time points post-injury to ensure that, given the time frame of 3 weeks apart, we assessed memory recognition rather than long-term memory. Similarly, in the Y-maze task, the number of entries into the new arm was significantly reduced 7 and 28 days after TBI, and AST

treatment increased the percentage of entries into the new arm. The results of both memory tests indicate that AST has beneficial effects on recovery following TBI. The present work was also performed to clarify the variable histological outcomes to determine the protective potential of AST at 7 days after TBI injury. We assessed the TBI-induced lesion volume and neuronal loss in these animals using unbiased, quantitative stereological analyses [24, 25]. A previous study demonstrated that neuronal cell loss in the cortex was relatively modest early after injury, which was followed by significant and progressive neuronal cell death at a later time point [41]. Thus, the investigation of late time points is important to obtain an accurate assessment of neuronal loss. The administration of AST, initiated 1 h after TBI, significantly reduced the lesion volume and attenuated neuronal degeneration in the cortex at 7 days post-injury, which may be responsible for the recovery of sensorimotor function. Thus, the reduction of the cortical lesion size and neural cell loss may be responsible for the attenuation of the sensorimotor deficits in the AST-treated TBI mice in the NSS test. The mechanisms of neuronal cell survival and neurological function improvement at later time points after TBI may involve various endogenous growth factors, as well as neuronal plasticity. Quantitative Western blotting was used to determine the levels of BDNF and the synaptic associated protein synapsin, GAP-43, and SYP in the cortex following TBI. BDNF promotes the growth and survival of brain cells and functions as a communication link between them. Synapsins are synaptic vesicle-associated proteins that regulate synaptic vesicle exocytosis and are involved in synaptogenesis. GAP-43 is a marker for axonal growth during development, as well as axonal remodeling and regeneration in adults. SYP is a protein that is involved in the formation and cycling of the synaptic vesicle, which is highly concentrated in the axonal terminals in neurons. TBI decreased the overall BDNF, synapsin, GAP-43, and SYP levels in the cortex, whereas AST partially restored these levels. The effects of AST on the synaptic protein levels in the injured brain help explain its effects in improving performance in the NSS, ORT, and Y-maze tasks. The pathological cascade in the brain following injury involves numerous pathways that trigger neuronal cell

death [42]. Oxidative stress and inflammation are among the early events that have been reported in models of TBI and are implicated in the secondary injury process [8, 38]. Studies have demonstrated that AST has anti-oxidative and anti-inflammatory properties [28, 34], and it activates protective signaling pathways, such as the Akt and ERK pathways [17, 18]. The current study demonstrated that AST has a protective property by simulating neurotrophic factors and synaptic associated proteins to promote neural cell and axonal neurite survival. Taken together, our findings support the hypothesis that AST has a therapeutic potential in TBI-induced neurological disorders. AST is also an attractive candidate for clinical translation because of its history of safe use. Nevertheless, there are several study limitations that should be considered in the interpretation of our results. First, the experimental results are exciting; however, these findings are based on mice, and AST may not have the same effect in humans. Second, although AST was obtained from a dietary organism, its toxicity must be investigated, especially for longer usage. Therefore, additional studies are necessary to address these issues.

4. Conclusions The current findings demonstrate that AST administration after TBI attenuated TBI-induced sensorimotor and cognitive function deficits. Using quantitative stereological methods, AST treatment was associated with a reduced cortical lesion volume, neuronal cell loss, and neurodegeneration in the cortex, which may occur via the simulation of neuro-trophic factors and the promotion of synaptic survival. Thus, AST comprises an attractive candidate for the treatment of TBI.

5. Experimental procedures 5.1 Animals Male ICR mice (weight: 28-32 g) (the Laboratory Animal Center of the Academy of Military Medical Sciences, Beijing) were bred and raised in our vivarium and were housed 10 mice per cage under a constant 12 h light/dark cycle. The mice were

maintained at 23 C ± 1°C. Food and water were supplied ad libitum. All experiments were performed in accordance with the Animal Care and Use Committee of the Medical School of Chinese People’s Liberation Army and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health (NIH).

5.2 Experimental design AST (Santa Cruz Biotechnology, Dallas, TX, USA, 97% pure) was diluted in olive oil (1 mL/kg) prior to use. AST or an equal volume of olive oil was administered 30 min after the surgery via oral gavage. The mice were divided into 3 groups: (1) Sham + olive oil vehicle treated, (2) TBI + olive oil vehicle treated, and (3) TBI + AST. The olive oil vehicle or AST (at 25 or 75 mg/kg) were administered via oral gavage beginning 30 min post trauma and involved six additional daily oral gavages. The administration was based on the effective doses reported in rodent models of cerebral ischemia and subarachnoid hemorrhage. The behavioral tests included the NSS, Rotarod, ORT, and Y-maze, and the tests were performed one after the other.

5.3 Mouse TBI model The M.A. Flierl weight-drop model was used to induce TBI with modifications [20] in the mice in the present study. Briefly, the mouse was anesthetized with isoflurane, and a 1.5-cm midline longitudinal scalp incision was made to expose the skull. The mouse was subsequently placed onto the platform directly under the weight-drop device. Following the identification of the left anterior frontal area (1.5 mm lateral to the midline on the mid-coronal plane) as the impact area, the weight was released and dropped onto the skull. Based on the previously published protocol [43], the weight was 200 gram, and the height of the weight-drop was 2.5 cm. All procedures were identical in the sham-injured mice with the exception of the weight drop injury. Following the onset of TBI, the scalp wound was sutured, and the mice were returned to their cages. Approximately 8% mortality occurred following the TBI operation.

5.4 Behavioral tests NSS The animals were weighed and examined using a modified NSS at 1 h after TBI and once a week thereafter, until the end of the experiment. This evaluation was performed by an observer who was blind to the treatment groups. This scoring system was developed to evaluate post-traumatic functional impairment in mice [19], and it is a powerful and useful tool to identify spontaneous or drug-induced recovery after TBI. Ten different tasks are used to evaluate motor ability, balancing, and alertness. One point indicates failure to perform a task, and 0 indicates success. The scores range from zero in healthy uninjured animals to a maximum of 10 in TBI mice, which indicates severe neurological dysfunction, with failure in all tasks. The NSS at 1 h post-trauma reflects the initial severity of the injury. Immediately after the initial NSS evaluation (1 h post-TBI), the mice were assigned to one of two treatment groups, which were evenly distributed to a TBI homogenous groups.

Rotarod The rotarod is used to assess sensorimotor coordination and motor learning in rodent models of CNS disorders [44]. The task involves both forelimb and hindlimb coordination. The mice were placed on a rotating rod with a constant rotation. The latency to fall was recorded, in which the animals fell safely 30 cm below the rotating rod. During the preinjury training phase, the subjects learned to balance on the rod, which was constantly rotating at 10 revolutions per minute (rpm), for the duration of 5 min in five consecutive trials. This time comprised the baseline performance for each mouse. Twenty-four hours later, TBI was induced, and the test trial was performed at the following post-injury time intervals: 3, 7, 14, 21, and 28 days, during which the mice were placed on the rod for 3 consecutive trials. The mean latency to fall was recorded. If a mouse did not fall from the rod within 5 min, it was removed by the experimenter. The latency to fall was recorded. For each mouse, the average performance time was divided by 300 s to express the results as the percent time spent on the Rotarod compared with the preinjury control.

ORT The ORT was performed as previously described [37]. This test is used to assess recognition memory and is based on the tendency of rodents to discriminate a familiar object from a novel object. The open field comprised a 59 cm arena, which was surrounded by 20 cm black Plexiglas walls. The floor of the arena was also black and divided into 37 identical squares via white gridlines. Each mouse was placed in an empty arena for 5 min of habituation. After 24 h, the mice were placed into the arena with two identical objects, A and B (e.g., bottles), positioned 40 cm from each other and 10 cm from the walls for 5 min. On the subsequent day (day 3), the mice were placed into the arena again with object A (the same as on the second day) and object C for 5 min. The arena and the objects were cleaned with 70% ethanol between each trial. Exploration of an object was defined as rearing on the object or sniffing it at a distance of less than 2 cm and/or touching it with the nose. The discrimination of visual novelty was assessed using a preference index: (time exploring the ‘new’ object - time exploring the ‘old’ object)/(total time exploring the objects).

Y maze The Y maze test was used to assess spatial memory [6]. The maze was composed of black Plexiglas and consisted of three identical arms that created a Y-shaped maze. One arm was defined as the “start arm”. The mice were placed in the start arm during the first trial, which lasted 5 min; one of the two remaining arms was randomly designed the “novel arm”, which remained closed during the first trial. After this session, the mouse was returned to his home cage for 2 min of habituation. In the second trial, which lasted 2 min, the mice were again placed in the start arm with all arms open. Between each trial and between each mouse, the maze was cleaned with a 70% ethanol solution. The time that the mice spent in each of the three arms was measured, and the difference between the time spent in the novel and old arms was used to assess memory. The discrimination of spatial novelty was calculated using a preference index: (time in new arm - time in old arm)/(time in both arms).

6. Lesion volume and neuronal loss The mice were anesthetized and transcardially perfused with saline and 10% buffered formalin phosphate solution at 7 days after TBI. The brains were removed, post-fixed in paraformaldehyde, and subsequently embedded in paraffin for further analysis. The sections were stain with H&E, dehydrated, and mounted for analysis. The lesion volume was estimated based on Cavalieri’s method of unbiased stereology using Stereologer 2000 program software (Systems Planning and Analysis, Alexandria, VA, USA) as previously described [45]. Stereoinvestigator software (MBF Biosciences, Williston, VT, USA) was used to count the total number of surviving neurons in the cortex via the optical fractionator method of unbiased stereology. The sampled region was demarcated in the injured hemisphere, and the cresyl-violet neuronal cell bodies were counted. The cortical volume was measured using a Cavalieri estimator method. The estimated number of surviving neurons in each field was divided by the volume of the region of interest to obtain the cellular density expressed in cells/mm3.

7. Western blot analysis The brain tissues were harvested at 7 days after TBI, homogenized, and centrifuged at 14,000 g for 15 min at 4°C. The protein concentration in the supernatant was determined using the Bradford method. Following the addition of SDS sample buffer, the supernatants were boiled for 10 min at 100°C. Thirty µg of each protein were loaded onto 10% SDS-PAGE and subsequently electrotransferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk for 2 h at room temperature and was incubated with primary antibodies (Synapsin Ia/b (Santa Cruz Biotechnology, Dallas, TX, USA), GAP-43 (Santa Cruz Biotechnology, Dallas, TX, USA), BDNF (Santa Cruz Biotechnology, Dallas, TX, USA), β-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA); and Synaptophysin antibody (Abcom company, Cambridge, MA, USA) at 4 °C overnight. The membrane was subsequently washed for 5 min three times in TBST, followed by incubation with the appropriate HRP-conjugated secondary antibody (diluted 1:5000 in TBST) for 2 h

at room temperature. The blotted protein bands were visualized via enhanced chemiluminescence (ECL, Thermo Scientific, USA) and subsequently exposed to X-ray film. The relative changes in the protein expressions were estimated from the mean pixel density, measured via densitometry, using ImageJ 1.48 (National Institutes of Health) and then normalized to β-Tubulin. The data were calculated as the target protein expression/β-Tubulin expression ratios.

8. Statistical Analysis All behavioral data are represented as means ± standard errors of the means (SEMs) and were analyzed with SPSS 17 software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was performed to compare all groups, followed by least squared difference (LSD) post hoc tests. ANOVA was used to analyze the results of the Y maze test, elevated plus maze test, and ORT. Biochemical analyses are presented as means ± SEMs and were analyzed via One-way ANOVA, followed by the Student-Newman-Keuls Multiple Comparisons Test.

References

1.

Loane, D.J., B.A. Stoica, and A.I. Faden, Neuroprotection for traumatic brain injury. Handb Clin Neurol, 2015. 127: p. 343-66.

2.

Parchani, A., et al., Traumatic subarachnoid hemorrhage due to motor vehicle crash versus fall from height: a 4-year epidemiologic study. World Neurosurg, 2014. 82(5): p. e639-44.

3.

Ozen, L.J. and M.A. Fernandes, Slowing down after a mild traumatic brain injury: a strategy to improve cognitive task performance? Arch Clin Neuropsychol, 2012. 27(1): p. 85-100.

4.

Dikmen, S.S., et al., Cognitive outcome following traumatic brain injury. J Head Trauma Rehabil, 2009. 24(6): p. 430-8.

5.

Bramlett, H.M. and W.D. Dietrich, Progressive damage after brain and spinal cord injury:

6.

Rachmany, L., et al., Cognitive impairments accompanying rodent mild traumatic brain injury

pathomechanisms and treatment strategies. Prog Brain Res, 2007. 161: p. 125-41. involve

p53-dependent

neuronal

cell

death

and

are

ameliorated

by

the

tetrahydrobenzothiazole PFT-alpha. PLoS One, 2013. 8(11): p. e79837. 7.

Stoica, B.A., et al., PARP-1 inhibition attenuates neuronal loss, microglia activation and neurological deficits after traumatic brain injury. J Neurotrauma, 2014. 31(8): p. 758-72.

8.

Werner, C. and K. Engelhard, Pathophysiology of traumatic brain injury. Br J Anaesth, 2007.

99(1): p. 4-9. 9.

Algattas, H. and J.H. Huang, Traumatic Brain Injury pathophysiology and treatments: early, intermediate, and late phases post-injury. Int J Mol Sci, 2014. 15(1): p. 309-41.

10.

Fernandez-Gajardo, R., et al., Novel therapeutic strategies for traumatic brain injury: acute

11.

Lozano, D., et al., Neuroinflammatory responses to traumatic brain injury: etiology, clinical

antioxidant reinforcement. CNS Drugs, 2014. 28(3): p. 229-48. consequences, and therapeutic opportunities. Neuropsychiatr Dis Treat, 2015. 11: p. 97-106. 12.

Johnson, V.E., W. Stewart, and D.H. Smith, Axonal pathology in traumatic brain injury. Exp Neurol, 2013. 246: p. 35-43.

13.

Siedler, D.G., et al., Diffuse axonal injury in brain trauma: insights from alterations in

14.

Tripathi, D.N. and G.B. Jena, Astaxanthin intervention ameliorates cyclophosphamide-induced

neurofilaments. Front Cell Neurosci, 2014. 8: p. 429. oxidative stress, DNA damage and early hepatocarcinogenesis in rat: role of Nrf2, p53, p38 and phase-II enzymes. Mutat Res, 2010. 696(1): p. 69-80. 15.

Shen, H., et al., Astaxanthin reduces ischemic brain injury in adult rats. Faseb j, 2009. 23(6): p. 1958-68.

16.

Kidd, P.M., Integrated brain restoration after ischemic stroke--medical management, risk factors, nutrients, and other interventions for managing inflammation and enhancing brain plasticity. Altern Med Rev, 2009. 14(1): p. 14-35.

17.

Wang, H.Q., et al., Astaxanthin upregulates heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells. Brain Res, 2010. 1360: p. 159-67.

18.

Zhang, X.S., et al., Astaxanthin alleviates early brain injury following subarachnoid hemorrhage in rats: possible involvement of Akt/bad signaling. Mar Drugs, 2014. 12(8): p. 4291-310.

19.

Beni-Adani, L., et al., A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice. J Pharmacol Exp Ther, 2001. 296(1): p. 57-63.

20.

Flierl, M.A., et al., Mouse closed head injury model induced by a weight-drop device. Nat Protoc, 2009. 4(9): p. 1328-37.

21.

Hamm, R.J., et al., The rotarod test: an evaluation of its effectiveness in assessing motor

22.

Thau-Zuchman, O., et al., Subacute treatment with vascular endothelial growth factor after

deficits following traumatic brain injury. J Neurotrauma, 1994. 11(2): p. 187-96. traumatic brain injury increases angiogenesis and gliogenesis. Neuroscience, 2012. 202: p. 334-41. 23.

Conrad, C.D., et al., Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment. Behav Neurosci, 1996. 110(6): p. 1321-34.

24.

Kabadi, S.V., et al., Selective CDK inhibitor limits neuroinflammation and progressive neurodegeneration after brain trauma. J Cereb Blood Flow Metab, 2012. 32(1): p. 137-49.

25.

Zhao, Z., et al., Neuroprotective effects of geranylgeranylacetone in experimental traumatic

26.

Weng, S.M., et al., Synaptic plasticity deficits in an experimental model of rett syndrome:

brain injury. J Cereb Blood Flow Metab, 2013. 33(12): p. 1897-908. long-term potentiation saturation and its pharmacological reversal. Neuroscience, 2011. 180: p. 314-21.

27.

Lin, T.Y., C.W. Lu, and S.J. Wang, Astaxanthin inhibits glutamate release in rat cerebral cortex nerve terminals via suppression of voltage-dependent Ca(2+) entry and mitogen-activated protein kinase signaling pathway. J Agric Food Chem, 2010. 58(14): p. 8271-8.

28.

Zhang, X.S., et al., Astaxanthin offers neuroprotection and reduces neuroinflammation in experimental subarachnoid hemorrhage. J Surg Res, 2014. 192(1): p. 206-13.

29.

Ambati, R.R., et al., Astaxanthin: sources, extraction, stability, biological activities and its

30.

Wang, M., et al., Astaxanthin ameliorates lung fibrosis in vivo and in vitro by preventing

commercial applications--a review. Mar Drugs, 2014. 12(1): p. 128-52. transdifferentiation, inhibiting proliferation, and promoting apoptosis of activated cells. Food Chem Toxicol, 2013. 56: p. 450-8. 31.

Fassett, R.G. and J.S. Coombes, Astaxanthin, oxidative stress, inflammation and cardiovascular disease. Future Cardiol, 2009. 5(4): p. 333-42.

32.

Chew, B.P. and J.S. Park, Carotenoid action on the immune response. J Nutr, 2004. 134(1): p. 257S-261S.

33.

Guerin, M., M.E. Huntley, and M. Olaizola, Haematococcus astaxanthin: applications for

34.

Wu, Q., et al., Astaxanthin activates nuclear factor erythroid-related factor 2 and the

human health and nutrition. Trends Biotechnol, 2003. 21(5): p. 210-6. antioxidant responsive element (Nrf2-ARE) pathway in the brain after subarachnoid hemorrhage in rats and attenuates early brain injury. Mar Drugs, 2014. 12(12): p. 6125-41. 35.

Zhang, X.S., et al., Amelioration of oxidative stress and protection against early brain injury by astaxanthin after experimental subarachnoid hemorrhage. J Neurosurg, 2014. 121(1): p. 42-54.

36.

Lu, Y.P., et al., Neuroprotective effect of astaxanthin on H(2)O(2)-induced neurotoxicity in vitro and on focal cerebral ischemia in vivo. Brain Res, 2010. 1360: p. 40-8.

37.

Tsenter, J., et al., Dynamic changes in the recovery after traumatic brain injury in mice: effect of injury severity on T2-weighted MRI abnormalities, and motor and cognitive functions. J Neurotrauma, 2008. 25(4): p. 324-33.

38.

Chen, Y., et al., An experimental model of closed head injury in mice: pathophysiology, histopathology, and cognitive deficits. J Neurotrauma, 1996. 13(10): p. 557-68.

39.

Ennaceur, A., et al., Detailed analysis of the behavior of Lister and Wistar rats in anxiety,

40.

Dellu, F., et al., A two-trial memory task with automated recording: study in young and aged

object recognition and object location tasks. Behav Brain Res, 2005. 159(2): p. 247-66. rats. Brain Res, 1992. 588(1): p. 132-9. 41.

Byrnes, K.R., et al., Metabotropic glutamate receptor 5 activation inhibits microglial associated inflammation and neurotoxicity. Glia, 2009. 57(5): p. 550-60.

42.

Leker, R.R., E. Shohami, and S. Constantini, Experimental models of head trauma. Acta

43.

Kuzelova, K., et al., Group I PAK inhibitor IPA-3 induces cell death and affects cell adhesivity

Neurochir Suppl, 2002. 83: p. 49-54. to fibronectin in human hematopoietic cells. PLoS One, 2014. 9(3): p. e92560. 44.

Laurer, H.L., et al., Mild head injury increasing the brain's vulnerability to a second concussive impact. J Neurosurg, 2001. 95(5): p. 859-70.

45.

Loane, D.J., et al., Amyloid precursor protein secretases as therapeutic targets for traumatic brain injury. Nat Med, 2009. 15(4): p. 377-9.

Figure legends Fig. 1. Effects of two dosage treatment regimens with AST on the Neurological Severity Score (NSS) values of mice for up to 28 days following TBI. Mice that received 75 mg/kg/day of AST exhibited the lowest NSS values, which indicates an increased rate of recovery (2-way ANOVA: *p<0.05 and **p<0.01 vs. the vehicle-treated group, n=10 per group).

Fig. 2. Average percent time (out of 5 min) that mice remained on the Rotarod for up to 28 days following the induction of TBI. A significant improvement in the performance of the AST-treated mice was identified 3 days after injury (t-test: *p=0.02 vs. the vehicle-treated group, n=10 per group).

Fig. 3. Recognition memory was assessed using the object recognition test (ORT) by calculating the relative time that the mice spent near a novel object compared with an old, familiar object. The significant decrease in the preference index in the TBI mice was significantly prevented by the administration of AST at both 7 and 28 days post-injury. One way ANOVA indicated a significant effect of group at both 7 and 28 days (LSD post hoc test, n=10 per group).

Fig 4. Spatial memory was assessed in the Y-maze by calculating the relative time that the mice spent in a novel arm compared with an old, familiar arm. The significant decrease in the preference index in the TBI mice was significantly prevented by the administration of AST. One-way ANOVA indicated a significant effect of group at both 7 and 28 days (LSD post hoc test, n=10 per group).

Fig. 5. AST treatment reduced the lesion size in the cortex following TBI. The lesion volume was quantified using the Cavalieri method. An unbiased stereological assessment of the lesion volume was performed at 7 days after TBI using H&E stained brain sections. (Fig 5-A) Representative images from each group are shown.

(Fig 5-B) A significant reduction in the lesion volume was identified in the AST-treated group (*P<0.05) compared with the vehicle-treated group. Analysis via one-way analysis of variance (ANOVA) followed by LSD post hoc test, n=7 per group.

Fig. 6. AST treatment attenuates neuronal cell loss in the cortex following traumatic brain injury. A stereological assessment of neuronal cells on post-injury day 7 was performed using Nissl stained cortical sections(Fig 6-A). Significant differences in the neuronal density in the cortex were identified between the sham-injured and vehicle TBI groups (**P<0.01). AST treatment significantly increased the neuronal density in the cortex (#P<0.05) compared with the vehicle TBI group. Analysis by one-way analysis of variance (ANOVA), followed by LSD, n=7 per group(Fig 6-B).

Fig. 7. Effects of AST on the levels of axonal and synaptic proteins (synapsin, GAP43, and synaptophysin) and brain-derived neurotropic factor (BDNF). Assays were performed 7 days after TBI on cortical tissues(Fig 7-A). Significant reductions in the expression of synapsin, GAP43, synaptophysin, and BDNF were identified in the cortex of the vehicle groups compared with the sham-injured group (**P<0.01). AST treatment significantly reversed the decrease in proteins compared with the vehicle TBI group (#P<0.05). Analysis by one-way analysis of variance (ANOVA), followed by LSD, n=7 per group(Fig 7-B).

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Highlights 1, AST administration improved the sensorimotor performance on the Neurological Severity Score (NSS) and rotarod test and enhanced cognitive function recovery. 2, AST treatment reduced the lesion size and neuronal loss in the cortex compared with the vehicle-treated TBI group. 3, AST also restored the levels of brain-derived neurotropic factor (BDNF), growth-associated protein-43 (GAP-43), synapsin, and synaptophysin (SYP) in the cerebral cortex.