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. 2016 Jan 7;36(8):1257–1268. doi: 10.1007/s10571-015-0323-2

Low-Molecular-Weight Fucoidan Attenuates Mitochondrial Dysfunction and Improves Neurological Outcome After Traumatic Brain Injury in Aged Mice: Involvement of Sirt3

Tao Wang 1,✉,#, Mang Zhu 1,#, Zhong-Zheng He 1
PMCID: PMC11482413  PMID: 26743530

Abstract

Traumatic brain injury (TBI) is a leading cause of death and long-term disability. Fucoidan, a sulfated polysaccharide extracted from brown algae, possesses potent anti-oxidative and anti-inflammatory effects. Considering TBI happens frequently in adults, especially in aged individuals, we herein sought to define the protective effects of low-molecular-weight fucoidan (LMWF) in the aged mice. 16- to 18-month-old mice administered with LMWF (1–50 mg/kg) or vehicle were subjected to TBI using a controlled cortical impact (CCI) model. LMWF at the doses of 10 and 50 mg/kg significantly reduced both cortical and hippocampal lesion volume. This protection was associated with reduced neuronal apoptosis, as evidenced by TUNEL staining. Importantly, LMWF was effective even when administered up to 4 h after TBI. Treatment with LMWF improved long-term neurobehavioral outcomes, including sensorimotor function, and hippocampus-associated spatial learning and memory. In addition, LMWF significantly suppressed protein carbonyl, lipid peroxidation, reactive oxygen species (ROS) generation, as well as mitochondrial dysfunction, which was evidenced by mitochondrial cytochrome c release and collapse of mitochondrial membrane potential (MMP). To evaluate the underlying molecular mechanisms, the expression of sirtuin 3 (Sirt3) was detected by RT-PCR and Western blot. The results showed that TBI significantly increased the expression of Sirt3, which was further elevated by LMWF treatment. Knockdown of Sirt3 using intracerebroventricular injection of small interfering RNA (siRNA) partially prevented the therapeutic effects of LMWF. Collectively, these findings demonstrated that LMWF exerts neuroprotection against TBI in the aged brain, which may be associated with the attenuation of mitochondrial dysfunction through Sirt3 activation.

Keywords: Fucoidan, TBI, Mitochondrial dysfunction, Sirt3, Aging

Introduction

Traumatic brain injury (TBI), the damage to the brain from external mechanical force caused by transportation, falls, assault, or sports, mainly affects young individuals and is the leading cause of mortality and disability around the world (Corrigan et al. 2010; Chen et al. 2011). It is reported that the average mortality rate is estimated to be 21 % by 30 days after TBI in the United States (Greenwald et al. 2003). Survived TBI patients frequently suffer from long-term personality changes and deficits in cognitive and motor performance. To date, many clinical trials evaluating neuroprotective agents have failed in demonstrating clinical efficacy in TBI patients.

Nowadays, it is widely accepted that brain injury after TBI can be classified by its time course into primary injury and secondary injury, which is considered to be the reason why 40 % of TBI patients deteriorate even after being hospitalized (Narayan et al. 2002). Secondary injury occurs in hours or days after the injury as a consequence of systemic or intracranial complications, and causes persistent functional deficits, leading to a loss of decades of productive life with huge costs to the patient and the society (Masel and DeWitt 2010). Importantly, prolonged and supportive neurorehabilitation following the primary injury phase may aid in improving outcome where many agents have been evaluated to ameliorate post-injury disabilities (Napolitano et al. 2005). Thus, finding drugs and prove clinical efficacy targeting secondary injury after TBI is a major challenge ahead of the researchers.

Fucoidan refers to a fucose-containing sulfated polysaccharide derived from brown algae species such as Laminaria and Fucus (Li et al. 2008). It is commercially available as nutritional supplement and has been used in traditional Chinese medicines for many years (Berteau and Mulloy 2003). For the past decade, fucoidan has been extensively studied due to its biological activities, which varies with species, molecular weight, composition, structure, and the route of administration (Fitton 2011). A fraction of low-molecular-weight fucoidan (LMWF) (~7 kDa) is obtained by radical depolymerization of extracts from brown seaweed. Recent evidence suggests that LMWF possess potent protective effects. Luyt et al. showed that LMWF promotes therapeutic revascularization in a rat model of critical hind limb ischemia (Luyt et al. 2003). Chen et al. observed that LMWF protects against renal ischemia via inhibition of the MAPK signaling pathway (Chen et al. 2013). More recently, LMWF was shown to alleviate cardiac dysfunction in rats by reducing oxidative stress and cardiomyocyte apoptosis (Yu et al. 2014). Therefore, we proposed that LMWF might be a possible candidate for treating TBI.

A previous study showed that fucoidan was able to effectively prevent mitochondrial damage and thereby attenuated the renal damage in hyperoxaluria (Veena et al. 2008). More recently, a study using renal ischemia–reperfusion injury (IRI) models demonstrated that LMWF may serve as a potential therapeutic agent for acute renal IRI via inhibiting mitochondrial apoptosis (Chen et al. 2013). Sirt3, a member of the sirtuin family that is preferentially localized to mitochondria, has been shown to play important roles in mitochondrial function-associated aging process (McDonnell et al. 2015; Bause and Haigis 2013). In the present study, we investigated the therapeutic effect of LMWF against TBI in an in vivo mouse model, as well as the potential underlying mechanism with focus on Sirt3.

Materials and Methods

Experimental Animals

Normal adult (2–4 months old) and aged (16–18 months old) Male C57BL/6 mice locally bred under a 12/12 h light/dark cycle were used in this experiment. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and approved by the Institutional Animal Care and Use Committee of the Xi’an Jiaotong University.

TBI Model

TBI was produced using a controlled cortical impact (CCI) model in accordance with previously detailed methods (Chen et al. 2011). Briefly, mice were anesthetized using 2 % isoflurane in oxygen and placed in the stereotaxic frame. A 5-mm-diameter craniotomy was performed using a portable drill over the right parietal cortex between bregma and lambda, 1 mm lateral to the midline. The dura mater was kept intact over the cortex. To induce injury, a pneumatic piston impactor device with a 3 mm diameter and rounded tip was used to impact the brain at a depth of 1 mm (velocity 5 m/s). The animal’s core body temperature was maintained at 37 ± 0.5 °C with a thermostatically controlled heating pad during surgery.

Experimental Design

Experiment 1 (Fig. 1): To investigate the potential protective effects of LMWF, 60 mice were randomly divided into five groups: Sham, Vehicle, LMWF 1 mg/kg, LMWF 10 mg/kg, and LMWF 50 mg/kg groups. At 48 h after TBI, 30 mice were killed for detecting brain edema, and 30 mice were killed for measuring lesion volume and TUNEL staining. To determine the toxicity of LMWF in our experimental conditions, 24 mice were randomly divided into four groups: Control, LMWF 1 mg/kg, LMWF 10 mg/kg, and LMWF 50 mg/kg groups. At 48 h after LMWF administration, 24 mice were killed for detecting brain edema.

Fig. 1.

Fig. 1

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LMWF attenuates neuronal damage after TBI in aged mice. a–f Aged mice were intraperitoneally pretreated with LMWF at different doses (1, 10 or 50 mg/kg) 30 min before TBI. The brain water content was assayed at 48 h after TBI (a). The lesion volume in total (b), cortex (c), and hippocampus (d) were measured at 7 days post trauma. TUNEL staining was performed at 48 h after TBI to detect neuronal apoptosis (e and f). Scale bars: 50 μm. g Aged mice were intraperitoneally treated with LMWF at different doses, and the brain water content was measured 48 h later. Data are shown as mean ± SEM of five experiments (n = 6). # p < 0.05 versus Sham. *p < 0.05 versus vehicle

Experiment 2 (Fig. 2): To determine the therapeutic window of LMWF, 60 mice were randomly divided into five groups: Vehicle, LMWF 0 h, LMWF 2 h, LMWF 4 h, and LMWF 6 h groups. At 48 h after TBI, 30 mice were killed for detecting brain edema, and 30 mice were killed for measuring lesion volume and TUNEL staining.

Fig. 2.

Fig. 2

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Therapeutic window of LMWF against TBI. Aged mice were intraperitoneally treated with a bolus of LMWF (50 mg/kg) at 0, 2, 4, or 6 h after TBI. The brain water content was assayed at 48 h after TBI (a). The lesion volume in cortex (b), hippocampus (c), and total (d) were measured at 7 days post trauma. TUNEL staining was performed at 48 h after TBI to detect neuronal apoptosis (e). Data are shown as mean ± SEM of five experiments (n = 6). *p < 0.05 versus vehicle

Experiment 3 (Fig. 3): To assess the effects of LMWF on neurological function, 36 mice were randomly divided into three groups: Sham, Vehicle, and LMWF. Eighteen mice were used to perform Beam-walking task at 2, 7, 14, and 21 days, and then killed for measuring cortical lesion volume at 30 days after TBI; another 18 mice were used to perform Barnes maze task from 19 to 23 days, and killed for measuring hippocampal lesion volume at 30 days after TBI.

Fig. 3.

Fig. 3

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LMWF provides long-term protection from neuronal damage and neurological defects after TBI in aged mice. Aged mice were intraperitoneally pretreated with 50 mg/kg LMWF at 30 min before TBI. Histopathological quantification of cortical lesion area in brain sections is shown (a), and cortical lesion areas were calculated at 30 days post trauma (b). Beam-walking test was performed at 2, 7, 14, and 21 days post trauma (c), and correlation between cortical damage and motor deficits is shown (d). The hippocampal lesion volume was calculated at 30 days post trauma (e). Barnes maze acquisition was performed at 19–23 days post trauma (f), and the results were calculated by measuring the area under the curve (g). The correlation between hippocampal damage and memory deficits is shown (h). Data are shown as mean ± SEM of five experiments (n = 6). *p < 0.05 versus vehicle

Experiment 4 (Fig. 4): To detect the effects of LMWF on oxidative stress and mitochondrial dysfunction, 24 mice were randomly divided into four groups: Sham, LMWF, Vehicle, and TBI + LMWF groups. At 48 h after TBI, mice were killed for biochemical analysis.

Fig. 4.

Fig. 4

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LMWF inhibits oxidative stress and mitochondrial dysfunction after TBI in aged mice. Aged mice were intraperitoneally pretreated with 50 mg/kg LMWF at 30 min before TBI. The brain tissue samples were obtained at 48 h after TBI. The protein carbonyl concentration was assayed to assess protein oxidation (a). The MDA (b) and 4-HNE (c) levels were measured to determine lipid peroxidation. The activities of endogenous oxidant enzymes (d, e, and f) were measured. The mitochondrial cytochrome c (g), cytosolic cytochrome c (h), MMP (i), and ROS generation (j) were examined, respectively. Data are shown as mean ± SEM of five experiments (n = 6). *p < 0.05 versus vehicle

Experiment 5 (Fig. 5): To investigate the expression of Sirt3 in adult and aged mice, 6 adult and 6 aged mice were killed for RT-PCR and Western blot analysis. To investigate the effect of TBI on Sirt3 expression, 36 mice were used (6 in Sham group and 30 in TBI groups). Mice were killed at different time points (1, 3, 6, 12, and 24 h after TBI) for RT-PCR and Western blot analysis. To investigate the effects of LMWF on Sirt3 expression, 24 mice were randomly divided into four groups: Sham, TBI, Vehicle, and TBI + LMWF groups. Mice were killed at 24 h after TBI for RT-PCR and Western blot analysis.

Fig. 5.

Fig. 5

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LMWF increases the expression of Sirt3 after TBI in aged mice. a–b The expression of Sirt3 at mRNA (a) and protein (b) levels in normal adult and aged mice were measured by RT-PCR and Western blot, respectively. c–d Aged mice were injured by TBI, and the expression of Sirt3 mRNA (c) and protein (d) were detected at different time points (1, 3, 6, 12, and 24 h after TBI). (e–f) Aged mice were intraperitoneally pretreated with 50 mg/kg LMWF at 30 min before TBI, and the expression of Sirt3 mRNA (e) and protein (f) were assayed at 12 h after TBI. Data are shown as mean ± SEM of five experiments (n = 6). *p < 0.05 versus Sham. *p < 0.05 versus TBI

Experiment 6 (Fig. 6): To investigate the effects of siRNA transfection, 18 mice were randomly divided into three groups: Control, Si-Control, and Si-Sirt3 groups. At 72 h after transfection, mice were killed for RT-PCR and Western blot analysis. To determine the involvement of Sirt3 in LMWF-induced protection, 90 mice were randomly divided into five froups: Sham, Vehice, LMWF, LMWF + Si-Control, and LMWF + Si-Sirt3 groups. At 48 h after TBI, 30 mice were killed for detecting brain edema, and 30 mice were killed for measuring lesion volume, and the other 30 mice were used for neurological function assessment.

Fig. 6.

Fig. 6

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Involvement of Sirt3 in LMWF-induced neuroprotection against TBI in aged mice. a–b Aged mice were intracerebroventricularly injected of Sirt3-targeted siRNA (Si-Sirt3) or control siRNA (Si-Control), and the expression of Sirt3 mRNA (a) and protein (b) were measured by RT-PCR and Western blot, respectively. c–f Aged mice were treated with siRNAs and 50 mg/kg LMWF and injured by TBI. The brain water content was assayed at 48 h after TBI (c). The total lesion volume was measured at 7 days post trauma (d). Beam-walking test was performed at 2, 7, 14, and 21 days post trauma (e), and Barnes maze acquisition was performed at 19–23 days post trauma (f). Data are shown as mean ± SEM of five experiments (n = 6). # p < 0.05 versus Sham. & p < 0.05 versus vehicle. *p < 0.05 versus Si-control

Measurement of Brain Edema

Brain edema was determined with the wet–dry method. Briefly, mice were killed by decapitation under deep anesthesia, and the brain was quickly removed. Tissue samples from injured hemispheres were dissected and weighed immediately to get wet weight. Dry weight was determined after heating the tissue for 48 h at 100 °C. Brain water content was then calculated using the following formula:  % H2O = (1 − dry weight/wet weight) × 100 %.

Quantitative Assessment of Lesion Volume

At 7 or 30 days after TBI, mice were sacrificed and brains were processed for assessment of brain lesion volumes using the stereology technique as described (Hadass et al. 2013). Coronal sections were serially cut through the brains at 40 μm thickness with a vibratome, and 120–150 tissue sections from each brain were sequentially collected into 24-well plates. Every the fifth section was mounted on poly-l-lysine-coated glass slides and stained with cresyl violet to quantify brain lesion volumes. In order to calculate lesion volumes, areas of the contralateral and the lesioned cortex or hippocampus were measured in photomicrographs of each coronal section to obtain values in mm2. The area of the lesioned cortex was subtracted from that of the contralateral cortex. Sections were then assigned a position along the rostro-caudal axis of the brain based on the anterior–posterior axis of the brain coordinate to Bregma and the difference plotted on the y-axis versus the anterior–posterior coordinates of the section on the x-axis. A second degree polynomial was generated in MS Excel to best fit the data points in order to visualize data trends. The area of the lesioned cortex or hippocampus was subtracted from that of the contralateral cortex or hippocampus. The lesion area of each section was multiplied by a step size of 200 mm (the distance between adjacent sections), and these values summed together in order to yield the lesion volume (in mm3) for each brain.

TUNEL Staining

Neuronal apoptosis was measured by TUNEL staining, a method used to observe DNA strand breaks in nuclei. In brief, sections of 4 μm thick were cut and mounted on poly-l-lysine-coated slides, and treated with proteinase K solution (20 μg/ml) for 10 min at room temperature to permeabilize tissues. TUNEL staining was performed by labeling with fluorescein TUNEL reagent mixture for 60 min at 37 °C according to the manufacturer’s suggested protocol, and examined under a fluorescence microscopy. The number of TUNEL-positive cells in each section in 10 microscopic fields (at × 600 magnification) was counted by an investigator blinded to the grouping.

Beam-Walking Task

Evaluation trials were conducted at 2, 7, 14, and 21 days post trauma to assess motor function. The apparatus consisted of a horizontal wooden beam, 6 mm wide, 90 cm long, and elevated to 48 cm above the ground. Each mouse was placed on one end of the beam and allowed to walk across it towards the opposite end into a dark goal box. The performance of animals was video recorded, and the number of foot faults for the right hind limb was counted during the first 50 steps. Experiments and analyses were performed by observers blinded to the grouping.

Barnes Maze Task

The apparatus consisted of a circular platform (75 cm in diameter) elevated 56 cm above the floor with 20 holes (each 5 cm in diameter) evenly spaced around the perimeter. The platform was surrounded by a black wall with four visual cues (a triangle, square, circle, and cross) inside the wall. Black fabric curtains covered the floor beneath the maze apparatus and hung 150 cm high from floor level to ensure that the mice were using the visual cues provided in the maze, instead of distal cues within the testing room. Each mouse was assigned an escape hole number and the escape box location remained constant for any individual mouse across test trials. Behavioral testing consisted of two shaping trials on day 19 post-trauma, followed by 10 evaluation trials for five days with a 30-min inter-trial interval. Each day, the animals were transferred from their cage room to the testing room 30 min prior to the start of testing; a trial began by placing the mouse under a black starting box positioned in the center of the platform. After 1 min, the box was lifted and the mouse had a maximum of 5 min to find and enter the escape box. Latency (time it took for the mouse to find the escape box) and total errors (nose-pokes into non-escape holes) were recorded. If the mouse did not enter the escape box within 5 min, it was gently guided there.

Measurement of Protein Carbonyl and Lipid Peroxidation

After various treatments, the ipsilateral cortical tissues were dissected from the mice and homogenized in chilled PBS, and then centrifuged at 10,000g at 4 °C for 10 min. The supernatants were collected, and the protein content was determined using a BCA protein assay kit. The levels of protein carbonyl, MDA, and 4-HNE were detected by commercial kits according to the manufacturer’s suggested protocol (Nanjing Jiancheng Bioengineering Institute, China).

Determination of Antioxidant Enzyme Activity

The brain tissue samples obtained above were also used for detecting the antioxidant enzymatic activities. The enzyme activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in tissue homogenates were measured according to the technical manual of the detection kits (Cayman Chemical, USA). The activities were expressed as the percentage of sham.

Measurement of Cytochrome c Release

Cytochrome c release was assessed after subcellular fraction preparation. The brain tissue homogenates were centrifuged for 10 min at 750g at 4 °C, and the pellets containing the nuclei and unbroken cells were discarded. The supernatant was then centrifuged at 15 000×g for 15 min. The resulting supernatant was removed and used as the cytosolic fraction. The pellet fraction containing mitochondria was further incubated with PBS containing 0.5 % Trition X-100 for 10 min at 4 °C. After centrifugation at 16 000×g for 10 min, the supernatant was collected as mitochondrial fraction. The levels of cytochrome c in cytosolic and mitochondrial fractions were measured using the Quantikine M Cytochrome c Immunoassay kit obtained from R&D Systems (R&D, Minneapolis, MN, USA). Data were expressed as percentage of sham.

Determination of ROS Generation and MMP

The injured brain tissues from cortex and hippocampus were dissected at 24 h after TBI. The purified mitochondria were stained with JC-1 to measure MMP. Mitochondria samples (0.5 mg/ml, 1 ml) were incubated with 19 ml JC-1 staining buffer according to the manufacture’s instruction (Sigma, CA, USA). At the end of the experiments, valinomycin was added as a negative control. Fluorescence intensity was determined at 37 °C in a fluorescence spectrophotometer. The ratio of aggregates (red in the web version, 590 nm) to monomer (green in the web version, 525 nm) was calculated as an indicator of MMP. Mitochondrial ROS production was measured using the indicator dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen, USA) as described earlier (Ye et al. 2011).

Real-Time RT-PCR

Total RNA was isolated using Trizol reagent (Invitrogen, CA, USA). After the equalization of the RNA quantity in each group, the mRNA levels were quantitated using a Bio-Rad iQ5 Gradient Real-Time PCR system (Bio-Rad Laboratories, CA, USA), and GAPDH was used as an endogenous control. Primers for all Real-Time PCR experiments were listed as follow: Sirt3: forward: 5′-TAC TTC CTT CGG CTG CTT CA-3′, reverse: 5′-AAG GCG AAA TCA GCC ACA-3′; GAPDH: forward: 5′-AAG GTG AAG GTC GGA GTC AA-3′, reverse: 5′-AAT GAA GGG GTC ATT GAT GG-3′. Samples were tested in triplicates and data from five independent experiments were used for analysis.

Intracerebroventricular Injection of siRNA

The siRNA knockdown technique was used to reduce Sirt3 expression in the brain as previously reported (Zheng et al. 2015). The specific siRNA-targeted Sirt3 (Si-Sirt3, sc-61556) and control siRNA (Si-Control, sc-37007), which should not knock down any known proteins, were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The mice were intracerebroventricularly injected with Si-Sirt3 or Si-Control at 2 days before TBI. The coordinates for the right lateral ventricle were 0.5 mm posterior to, 0.8 mm lateral to, and 2.5 mm below bregma. The siRNA complexes were prepared according to the in vivo siRNA transfection protocol for brain delivery outlined in the PolyPlus Transfection reagent instructions (PolyPlus Transfection, IIIkirch, France). Each mouse was administered 2 μg of siRNA in a 4-μl mixture.

Western Blot Analysis

The homogenates obtained above were also used to Western blot analysis. Forty μg of protein was resolved on 10 % SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5 % non-fat milk and incubated with the Sirt3 or β-actin primary antibodies. Membranes were then washed and incubated for 1 h at room temperature with secondary antibodies. The ImageJ software was used to quantify the optical density of each band.

Statistical Analysis

Statistical analysis was performed using SPSS 16.0, a statistical software package. Statistical evaluation of the data was performed by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons or unpaired t test (two groups). A value of p < 0.05 was considered statistically significant.

Results

LMWF Attenuates Neuronal Damage After TBI in Aged Mice

To investigate the potential neuroprotective effect of LMWF, aged mice were pretreated with LMWF at different doses (1, 10, or 50 mg/kg) 30 min before TBI. The results showed that LMWF significantly decreased the brain edema, as measured by brain water content, after TBI in a dose-dependent manner (Fig. 1a). Next, we conducted histological assessment of lesion volume to evaluate the effect of LWMF on brain injury. All brains of TBI-injured mice showed severe contusion injuries with substantial tissue loss in the cortex (Fig. 1c) and hippocampal region (Fig. 1d), which were all partially prevented by treatment with LMWF at 10 and 50 mg/kg, but not 1 mg/kg (Fig. 1b–d). We also detected apoptosis by TUNEL staining in brain sections (Fig. 1e), and the results showed that LMWF at 10 mg/kg and 50 mg/kg, but not 1 mg/kg, significantly reduced TBI-induced apoptosis in aged mice (Fig. 1f). Because LMWF in all doses mentioned before did not show toxicity in aged mice, as measured by brain water content (Fig. 1g), 50 mg/kg LMWF was used in the following experiments.

To determine the time window of LMWF-induced protective effects, aged mice were intraperitoneally treated with a bolus of LMWF (50 mg/kg) at 0 h, 2 h, 4 h, or 6 h after TBI. The results of brain water content (Fig. 2a), brain lesion volume (Fig. 2b–d), and TUNEL staining (Fig. 2e) indicated that the administration of LMWF from 0 h to 4 h after TBI induced a gradually reduced protection, while there was no significant protection if LMWF was administered at 6 h after the initiation of TBI.

LMWF Provides Long-Term Protection from Neuronal Damage and Neurological Deficits After TBI in Aged Mice

To assess the long-term effects of LMWF treatment, we assessed the histological differences 30 days post trauma. A second degree polynomial was generated to fit the data points for each group (Fig. 3a), which indicates a difference in lesion area between vehicle and LMWF-treated mice. We found that LMWF-treated mice exhibited cortical lesion volumes that were significantly smaller by approximately 25 % than those of vehicle-treated mice (Fig. 3b). We also used a beam-walking task in order to test fine motor coordination after TBI, and the results showed that LWMF treatment significantly accelerated motor function recovery after TBI (Fig. 3c). Moreover, quantitative data of the 30-day cortical lesion volume and 7-day beam-walking foot faults were well correlated (Fig. 3d).

In addition, we examined the long-term effects of LMWF on the ability to reduce the lesion volumes of the hippocampus, which is required for spatial learning and memory processing. As shown in Fig. 3e, LMWF-treated mice had hippocampal lesion volumes (30 days after TBI) that were significant smaller by approximately 33 % than those of vehicle-treated mice. A 5-day Barnes maze task, which started at 19 days post trauma, was used to evaluate spatial learning and memory in aged mice. Measurement of latency revealed a significant impairment in spatial learning after TBI, which was attenuated by LMWF treatment from 20 to 23 days after the insult (Fig. 3f). For analysis of memory acquisition in the maze, the latency area under the curve (AUC) from trial one to trial ten (5 days) was calculated for each animal (Fig. 3g). As shown in Fig. 3h, quantitative data for the 30-day hippocampal lesion volume and latency AUC show significant correlation.

LMWF Inhibits Oxidative Stress and Mitochondrial Dysfunction After TBI in Aged Mice

Oxidative protein damage was identified by the assessment of protein carbonyl. The results showed that TBI induced a 3-fold increase in carbonyl levels, which were significantly alleviated by LMWF treatment (Fig. 4a). We also estimated lipid peroxidation by measuring the MDA (Fig. 4b) and 4-HNE (Fig. 4c) formation. MDA and 4-HNE levels in mice subjected to TBI showed significant increases when compared with sham mice. Administration of LMWF markedly reduced MDA and 4-HNE generation in cortical and hippocampal areas. To probe whether LMWF could affect the endogenous antioxidant system, the enzyme activities of CAT (Fig. 4d), SOD (Fig. 4e), and GPx (Fig. 4f) were measured. The results showed that TBI-induced decrease in these enzymes activities were preserved by LMWF treatment.

To investigate the effect of LMWF treatment on mitochondrial dysfunction after TBI, we detected the content of cytochrome c in mitochondrial (Fig. 4g) and cytosolic (Fig. 4h) fractions. The results showed that LMWF attenuated cytochrome c release in cortical and hippocampal areas, as evidenced by increased cytosolic cytochrome c and decreased mitochondrial cytochrome c content in LMWF-treated mice when compared with vehicle-treated animals. Furthermore, LMWF also significantly hyperpolarized MMP in injured cortex and hippocampus, while MMP was not different in sham animals with or without LMWF treatment (Fig. 4i). To determine the effect of LMWF on free radical release, we measured the production of hydrogen peroxide using the fluorescent probe H2DCFDA. In agreement with elevation of MMP levels, pretreatment with LMWF significantly reduced the increased ROS production in both injured cortex and hippocampus (Fig. 4j).

LMWF Increases the Expression of Sirt3 After TBI in Aged Mice

To investigate the potential molecular mechanisms underlying LMWF-induced protection, we detected the expression of Sirt3 by RT-PCR and Western blot. Sirt3 was shown to be involved in aging-associated degeneration (Brown et al. 2013). Thus, we first detected the expression of Sirt3 in normal adult and aged mice. The results showed that both Sirt3 mRNA (Fig. 5a) and protein (Fig. 5b) were down-regulated in aged mice. The Sirt3 mRNA level was relatively low in the sham animal, markedly increased from 1-h post-trauma, peaked at 3 h, and then gradually decreased (Fig. 5c). Consistently, TBI also significantly increased the expression of Sirt3 protein at 3, 6, and 12 h after trauma (Fig. 5d). To determine the effect of LMWF on the expression of Sirt3 in our experiments, aged mice were intraperitoneally pretreated with 50 mg/kg LMWF at 30 min before TBI. The results of RT-PCR showed that LMWF markedly increased the expression of Sirt3 mRNA in both sham and TBI-injured mice (Fig. 5e). As shown in Fig. 5f, a similar result in the expression of Sirt3 protein was also observed.

Involvement of Sirt3 in LMWF-Induced Neuroprotection Against TBI in Aged Mice

We used the siRNA knockdown approach to further confirm the involvement of Sirt3 in LMWF-induced protection against TBI. Intracerebroventricular (ICV) injection of siRNAs has been reported to decrease target gene expression in in vivo models. Si-Sirt3 or Si-Control was administered via ICV injection 2 days prior to TBI. As expected, compared to Si-Control, Si-Sirt3 effectively attenuated the expression of Sirt3 at both mRNA (Fig. 6a) and protein (Fig. 6b) levels. Knockdown of Sirt3 partially prevented the LMWF-induced protection against TBI-induced brain edema as measured by brain water content (Fig. 6c). The mice treated with LMWF and Si-Sirt3 displayed lesion volume that was larger than that of the mice treated with LMWF and Si-Control (Fig. 6d). In addition, the LMWF-induced preservation on neurological dysfunction after TBI, as measured by beam-walking task (Fig. 6e) and Barnes maze task (Fig. 6f), was partially reversed by Si-Sirt3 injection as compared with Si-Control-injected animals.

Discussion

Because brain damage due to the primary insult of TBI is difficult to avoid, treatments targeting the secondary injury-associated biochemical events, such as inflammation, neurotoxicity, ischemia, and edema in the brain, could provide opportunities for the therapeutic intervention (Thal and Neuhaus 2014; Rodriguez-Rodriguez et al. 2014). The current study demonstrated for the first time that LMWF protects aged mice against TBI, as evidenced by reduced brain edema, decreased lesion volume, and attenuated neurological deficit. The protection afforded by LMWF is presented in moderate doses (10–50 mg/kg) and sustained for at least 30 days. Importantly, LMWF is still effective even if the administration was delayed to 4 h after the insult. In addition, LMWF treatment markedly reduced oxidative stress, inhibited mitochondrial dysfunction, and preserved antioxidant defense activities in TBI-injured brains.

In our experiments, the results displayed more information, and because of that the sub-analysis of total lesion volume compared with cortical and hippocampal lesion volume was performed. The spatial pattern of the contusion suggests that LMWF is able to salvage jeopardized tissue in both cortical and hippocampal areas. The hippocampus plays important roles in the consolidation of memory and spatial navigation. TBI patients with extensive hippocampal damage may experience anterograde amnesia—the inability to form or retain new memories (Barker-Collo and Feigin 2008). In consistent with reduced lesion volume in both cortical and hippocampal area, LMWF-treated mice showed improved performance in beam-walking and Barnes maze task, indicating the preserved memory and motor function after LMWF treatment. In addition, we measured the effect of LMWF on TBI-induced lesion volume on 7 and 30 days post trauma, the results of which demonstrated the improved long-term neurological recovery in aged mice. It is well known that a large percentage of people killed by TBI do not die at the moment of injury but rather days to weeks after the event. The lethal secondary injury occurs in hours or days, and lasts for even months after the primary injury (Hofman et al. 2015; Mioni et al. 2014). Thus, ours present results strongly supported that LMWF could be an ideal therapeutic agent targeting secondary injury after TBI.

Another major observation made in the present study was the therapeutic time window of LMWF in the TBI model. To date, most of the neuroprotective agents tested in preclinical experiments were administrated prior to TBI, which was found to be problematic in clinical trials because of the difficulty in obtaining informed consent (Menon 2009). This might be an explanation of the fact that no clinical trial evaluating neuroprotective compounds has succeeded in demonstrating clinical efficacy in TBI-injured patients in the past decades (Marklund and Hillered 2011). Our results showed that the protective effects of LMWF were also present when the compound was given 4 h after TBI, indicating that it is effective in mice in a time window that is more relevant to clinical practice. Biochemical events following the primary injury after TBI are complex depending on the extent of brain damage. In most instances, secondary injury is triggered by onset of a cascade of events that damages the BBB and is followed by inflammation, neurotoxicity, edema, and oxidative stress in the brain (Hadass et al. 2013). Our results showed that LMWF significantly attenuated mitochondrial dysfunction, oxidative stress, and neuronal apoptosis at 48 h after TBI, and reduced contusion volumes at 7 and 30 days later. More interestingly, there was a sustained effect of LMWF treatment, as we observed profound amelioration of fine motor coordination related to cortical lesion and hippocampus-associated spatial learning and memory. These results are important in explaining the improved neurobehavioral outcomes in sensorimotor and cognitive function in mice subjected to TBI.

In addition, as compared to many other neuroprotective agents, LMWF represents unique advantages. LMWF was shown to exert low toxicity, and our results also demonstrated that LMWF did not induce brain edema at the dose of 50 mg/kg. Previous studies suggested that fucoidan was effective in vivo upon oral, intraperitoneal, or intravenous administration (Kwak 2014). Importantly, fucoidan has been demonstrated to exert antioxidant and anti-inflammatory activity, as well as effects of reducing blood lipids, all of which were helpful for TBI treatment (Li et al. 2008). Furthermore, compared with other sulfated polysaccharides, fucoidans are widely available from various kinds of cheap sources. All these data strongly indicate that LMWF might be an ideal candidate for clinical drug investigation for TBI patients.

Oxidative stress-induced neuronal damage has long been implicated in both aging process and neurological disorders (Balaban et al. 2005; Cheng et al. 2012). Neuronal cells are critically dependent on mitochondrial integrity based on high rates of metabolic activity and need to response promptly to activity-dependent neurotransmission and hot spots of energy consumption, such as presynaptic and postsynaptic sites (Hiebert et al. 2015; Robertson et al. 2007). Previous studies have revealed that oxygen lipid and protein peroxidation induced by free radicals is one of the most important factors in the pathology of oxidative stress after TBI (Tyurin et al. 2000; Awasthi et al. 1997). Our results showed that the increased protein carbonyl, MDA, and 4-HNE levels after TBI were all attenuated by LMWF treatment, indicating the involvement of anti-oxidative mechanisms. It is well known that many endogenous antioxidant enzymes, such as CAT, SOD, and GPx, work together to provide a line of defense against oxidative damage, but this important defense mechanism is incapable of scavenging the over-generated free radicals under secondary brain injury conditions (McConeghy et al. 2012; Chen et al. 2014). As expected, the preserved activities of these antioxidant enzymes were observed after LMWF treatment in both cortical and hippocampal areas. Together with the suppressed cytochrome c release, ROS generation and preserved MMP level, these data suggested that LMWF significantly enhanced mitochondrial function, which in turn decreased oxidative stress and increased endogenous antioxidant system activity, leading to reduced neuronal apoptosis and brain edema after TBI.

Sirt3 is a member of the sirtuin family of protein deacetylases that is preferentially localized to mitochondria (Onyango et al. 2002). It targets various mitochondrial proteins for lysine deacetylation and regulates important cellular functions such as energy metabolism, aging, and stress response (McDonnell et al. 2015; Bause and Haigis 2013). Many previous studies have shown that the mitochondrial Sirt3 has an important role in warding off the vicissitudes of aging, and that mice lacking Sirt3 develop severe age-related pathologies in the heart and brain (Pillai et al. 2010). In the present study, we found that both Sirt3 mRNA and protein were down-regulated in aged mice. In consistent with our results, a recent study showed that Sirt3 is suppressed with aging, and up-regulation of Sirt3 in aged hematopoietic stem cells significantly improved their regenerative capacity (Brown et al. 2013). With a conserved catalytic domain, Sirt3 binds and deacetylates several metabolic and respiratory enzymes that regulate important mitochondrial functions (Onyango et al. 2002). Sirt3-mediated deacetylation activates enzymes responsible for quenching ROS, including MnSOD, CAT, and IDH2, and thereby exerts a profound protective action against oxidative stress-dependent pathologies. More recently, overexpression of Sirt3 was shown to preserve mitochondrial function through inhibiting mitochondrial Ca2+ overloading and promoting mitochondrial biogenesis (Dai et al. 2014a, b). As the observed protective effects of LMWF here were shown to be associated with inhibited oxidative stress and preserved mitochondrial function, we speculated that Sirt3 might be involved in LMWF-induced protection. The results of RT-PCR and Western blot showed that expression of Sirt3 was significantly increased by TBI and LMWF, and additional increases in both mRNA and protein levels of Sirt3 were observed in LMWF-treated and TBI-injured mice. The activated Sirt3 in our experimental models might be an endogenous protective mechanism, which was enhanced by LMWF treatment. This hypothesis was demonstrated by previous data using in vitro neuronal injury model (Hu et al. 2014), and also confirmed by our results showing that knockdown of Sirt3 partially ablated the neuroprotection induced by LMWF. All these data provide strong evidence that activation of Sirt3 might contribute to the neuroprotection induced by LMWF against TBI in aged mice.

In summary, our present study demonstrates that pretreatment with LMWF, a fucose-containing sulfated polysaccharide derived from brown algae species, displays neuroprotective effects against TBI in aged mice. It inhibited oxidative stress and mitochondrial dysfunction through up-regulating the expression of Sirt3 after TBI. These results demonstrated the feasibility for future treatment of TBI with LMWF, which needs to be further determined in clinical trials.

Acknowledgments

The authors would like to thank Dr. Terry Chen for his technical support for the experiments and the preparation of the manuscript.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Tao Wang and Mang Zhu contributed equally to this work.

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