The comparative effects between tocotrieonol-rich fraction (TRF) and α- tocopherol on glutamate toxicity in neuron-astrocyte mono- and co- culture systems

Background: 
Vitamin E, which can be categorized into tocotrienols and tocopherols, is known to protect cells from glutamate neurotoxicity. Studies have shown that tocotrienol-rich fraction (TRF) protecting the brain against oxidative damage more efficient than α-tocopherol. The role of astrocyte in promoting neuronal survival and recovery after glutamate neurotoxicity is also increasingly appreciated. 
 
Aims: 
To elucidate the effects of TRF and α-tocopherol and the synergism between astrocyte and neuron against glutamate neurotoxicity. 
 
Methods: 
Astrocyte and neuron were subjected to glutamate injury followed by TRF and α-tocopherol treatments (100 – 300 ng/ml). Effects of TRF and α-tocopherol on nerve cell viability and glutathione contents against glutamate toxicity were examined. The synergism between astrocyte and neuron was elucidated through co-culture model. Statistical analysis was performed using one way ANOVA. 
 
Results: 
Both TRF and α-tocopherol improved approximately 10% of glutamate-injured astrocyte and neuronal cell viability. In co-culture model, TRF and α-tocopherol provided nearly complete protection from glutamate toxicity. Besides, TRF and α-tocopherol treatments significantly restored at least 20% of glutathione contents in glutamate-injured neurons. In the presence of astrocyte, 300 ng/ml TRF and α-tocopherol completely restored glutathione contents in glutamate-injured neuron. 
 
Conclusions: 
TRF and α-tocopherol had shown promising neuroprotective effects in astrocyte and neuron from glutamate toxicity. Great scavenging effect of both TRF and α-tocopherol against glutamate toxicity was observed in neuron. Similar protective effects between TRF and α-tocopherol were observed. Co-culture model demonstrated the synergistic properties between neuron and astrocyte. Supplementation of TRF and α-tocopherol in co-culture further improved the recovery process.

such as ischemic insults and traumatic brain damage which could be related to the etiology of most of the neurodegenerative diseases 1 . Glutamate toxicity in nerve cells exists in two forms which are receptor-initiated excitotoxicity 2 and non-receptor mediated oxidative glutamate toxicity 3 . Receptor-initiated excitotoxicity involves over-stimulation of glutamate receptor (GluRs) which leads to excessive Ca 2+ influx and activates a cell death cascade 4 . On the hand, non-receptor mediated oxidative glutamate toxicity involves the breakdown of the cystine/glutamate antiporter (x c -) mechanism, which leads to the depletion of glutathione (GSH) and subsequently causes oxidative stress and cell death 3 .
Nervous system, which is rich in both unsaturated fats and iron, makes it particularly susceptible to oxidative stress and damage 5 . Oxidative stress plays a significant role in the modulation of critical cellular functions, such as apoptosis program activation, ion transport and calcium mobilization 6 , which often leading to cell death 7 . However, cell death caused by oxidative stress can be prevented by administration of free radical scavenging antioxidants, such as vitamin E. Vitamin E, which is a fat-soluble antioxidant, prevents lipid peroxidation in biological membranes. It exists in eight forms, namely α-, β-, γ-and δ-tocopherols and α-, β-, γ-and δ-tocopherols 8 . Alpha-tocotrienol has been found to be 40 to 60 times more potent in preventing lipid peroxidation as compared to α-tocopherol 9 . On the other hand, many studies demonstrated that tocotrienol-rich fraction (TRF) possesses powerful antioxidant 10 , anti-inflammation 11 and cholesterol-lowering properties 12 .Tocotrienols differ from tocopherols by possessing an unsaturated isoprenoid side chain instead of a saturated phytyl tail. The unsaturated side chain of tocotrienol is claimed to allow better penetration and distribution into saturated lipid layers such as brain and liver 13 . In previous studies, α-tocopherol was shown to protect cells from glutamate-induced cell death in micromolar concentration. However, in recent studies, α-tocotrienol had shown better protection from glutamate-induced cell death in nanomolar concentration by inhibiting glutamate-induced early activation of c-Src kinase 14 . This finding showed that α-tocotrienol has potent signal transduction regulatory properties which was independent of its antioxidant properties. Thus, the interest to study the neuroprotection of tocotrienols from glutamate toxicity, either by antioxidant activity or by antioxidant-independent activity has increased with time.
Nerve system consists of glial and neuron. Astrocyte, subtype of glial cell, is known to protect neuron from oxidative stress through transcriptional up-regulation of glutathione synthesis and removal of extracellular glutamate 15 . Astrocytes death after ischemia or reperfusion may strongly affect neuronal survival due to the absence of trophic and metabolic support to neurons and astrocytic glutamate uptake 16 . Thus, the role of astrocytes in promoting neuronal survival and recovery after a cerebral insult is becoming increasingly appreciated.

Objectives
To elucidate the role of TRF and α-tocopherol in recovery processes by supplementation to glutamate-injured astrocyte, neuron as well as neuron in co-culture system.
To examine the synergism between astrocyte and neuron against glutamate toxicity with the supplementation of TRF and α-tocopherol.

Cell culture
Human neurons (SK-N-SH) and human astrocytes (DBTRF-05MG) were obtained from American Type Culture Collection (ATCC, USA). Neurons and astrocytes were cultured in MEM and RPMI 1640, respectively. Both cell lines were supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) throughout the experiments. Cultures were maintained at 37ºC in 5% CO 2 and 95% air in a humidified incubator. In order to mimic the IJBAR (2013) 04 (06) www.ssjournals.com in situ spatial interaction between neurons and astrocytes, neurons were co-cultured with astrocytes in co-culture membrane insert model prior to treatment.

TRF, α-tocopherol and glutamate preparation
TRF and α-tocopherol were freshly prepared in absolute ethanol with concentration of 100, 200 and 300 µg/ml (10 3 × working concentration) prior to treatment. Glutamate solutions with various concentrations were prepared in phosphate buffer saline (PBS).

Mono-culture
Neurons and astrocytes were individually seeded in 6 well plates at cell density of 1 × 10 6 cells per well (total medium per well = 2 ml) followed by 24 hours incubation in humidified incubator at 37°C, 5% CO 2 . After 24 hours incubation, neurons and astrocytes were exposed to 60 mM and 180 mM glutamate, respectively for 30 minutes prior to TRF and α-tocopherol treatments. After 30 minutes of glutamate treatment, cells were treated with 100, 200 and 300 ng/ml of TRF and α-tocopherol in the respective wells. Positive control (glutamate-injured cells) was treated with equal volume of absolute ethanol (0.1%, v/v) to replace TRF or α-tocopherol. Meanwhile, negative control (without any treatment) was added with equal volume of absolute ethanol (0.1%, v/v) while glutamate was replaced with PBS. Plate was then incubated in humidified incubator at 37°C, 5% CO 2 for another 24 hours and ready for cell viability assay and glutathione assay.

Co-culture
Astrocytes were seeded in 6 well culture inserts which were placed in an empty 6 well plate. Meanwhile, neurons were seeded in 6 well plate. Both cells were seeded with cell density of 1 × 10 6 cells per well. Both cell lines were then incubated in humidified incubator at 37°C, 5% CO 2 for 2 hours to allow cell attachment. After that, inserts containing astrocytes were transferred to the 6 well plate containing neurons. The cultures were left to adapt for 1 day prior to treatment. On the second day, TRF and α-tocopherol treatments were carried out as described earlier in section 2.3.1.

Determination of cell viability
MTT cell viability assay was carried out after 24 hours of TRF and α-tocopherol treatments. Culture medium in 6 well plate was removed followed by addition of 2 ml fresh medium. In co-culture model, inserts containing astrocytes were removed before removal of culture medium and addition of fresh medium. Then, 500 µl MTT (2 mg/ml) was added to each well followed by 4 hours incubation at 37°C with 5% CO 2 . A total volume of 1 ml DMSO was then added to each well to dissolve the formazan formed. Plate was gently agitated for 5 minutes before being transferred to a sterile 96 well plate for optical density measurement. Negative controls were measured as 100% cell viability.

Glutathione assay
Cells were treated with TRF or α-tocopherol against glutamate toxicity in 6 well plate as described earlier in section 2.3.1 and section 2.3.2. After 24 hours of TRF and α-tocopherol treatments, plate was then ready for glutathione assay. The cell lines were washed twice in PBS followed by cell detachment with trypsin. Cells were then collected through centrifugation at 700 × g for 5 minutes at 4°C. The remaining procedure was performed according to the manufacturer's instructions. Lastly, total glutathione was measured at wavelength of 450 nm with microplate reader (BioTex-ELx800, USA). Glutathione content in negative control samples was calculated as 100%.

Statistical analysis
Data were reported as mean ± SEM of 3 independent experiments. Comparisons between groups were made by using one way analysis of variance (ANOVA) post hoc analysis (SPSS 17.0). A p-value less than 0.05 was considered as statistically significant.

Effects of TRF and α-tocopherol on glutamate treatment
The half maximal inhibitory concentration (IC 50 ) of glutamate for neurons and astrocytes were 80 mM and 230 IJBAR (2013) 04 (06) www.ssjournals.com mM, respectively based on the finding from dose response study (data not shown). In order to cause cell injury, IC 20 of glutamate was used throughout this study. 60 mM glutamate was used to cause injury on neuron while 180 mM glutamate was used to cause injury on astrocyte. Figure 1. Effects of TRF and α-tocopherol on neurons, (A) astrocytes, (B) neurons in co-culture model, (C) against glutamate toxicity in term of cell viability. Data are presented as mean ± SEM of 3 independent experiments (n = 3 in each experiment). *p<0.05, compared with control group; ∆ p<0.05, TRF and α-tocopherol-treated group compared with glutamate-treated group; # p<0.05, TRF-treated group compared with α-tocopherol-treated group.

Cell viability
At concentrations of 100 to 300 ng/ml, both TRF and α-tocopherol increased neuronal cell viability against glutamate toxicity in a dose-response manner ( Figure 1A). Both TRF and α-tocopherol treatments at concentration of 300 ng/ml recovered approximately 10% of the neuronal cell viability upon glutamate treatment. On the other hand, both TRF and α-tocopherol treatments at concentration of 100 and 200 ng/ml recovered more than 10% of astrocyte cell viability from glutamate toxicity ( Figure 1B). In contrast with neuron, only minor increment of astrocyte cell viability was observed in TRF treatment with concentration of 300 ng/ml. In the presence of astrocyte (co-culture), TRF and α-tocopherol further improved neuronal cell viability to nearly 100% cell viability from glutamate toxicity ( Figure 1C). In terms of cell viability, TRF and α-tocopherol showed similar protecting effect in both cell lines against glutamate challenge.
GSH concentration of both cell lines decreased at least 30% after 24 hours of glutamate challenge (Figure 2A and  2B). At concentrations of 100 to 300 ng/ml, TRF and α-tocopherol significantly restored glutathione content of neuron after glutamate challenge (Figure 2A). At concentration of 300 ng/ml, TRF completely restored glutathione content of glutamateinjured neuron. Similar finding was observed with α-tocopherol treatment at concentration of 200 ng/ml. However, αtocopherol treatment at concentration of 300 ng/ml did not exert better effect in restoring glutathione content as compared to 200 ng/ml. In astrocyte, both TRF and α-tocopherol did not restore glutathione content upon glutamate challenge ( Figure  2B). In co-culture model, TRF and α-tocopherol at concentration of 200 and 300 ng/ml further improved the glutathione content in neuron ( Figure 2C). At concentration of 300 ng/ml, both TRF and α-tocopherol completely restored glutathione content in neuron in the presence of astrocyte.

Discussion
The model of glutamate-induced cell death had been widely used for the identification of agents that provide neuroprotective effect. In the present study, glutamate induced toxicity on neuron and astrocyte in a dose-dependent and time-dependent manner (data not shown). This study showed that 80 mM of glutamate was required to cause 50% of neuronal death which was similar to the study on SH-SY5Y neuronal cell, a subclone of SK-N-SH neuronal cells, against glutamate challenge for 24 hours 17 . On the other hand, a much higher concentration of glutamate was required to cause toxicity in astrocyte as compared to neuronal cell. This demonstrated that astrocytes were more resistant to glutamateinduced toxicity when compared with neurons, which can be explained by the distribution of glutamate transporters in neurons and astrocytes. Excessive extracellular glutamate re-uptake is needed from synaptic cleft into neurons or astrocytes through glutamate transporter to prevent neurotoxicity. Previous study showed that astrocytic glutamate transporters (EAAT1 and EAAT2) are responsible for more than 80% of glutamate uptake in the brain 18 . Current finding further support the idea of glutamate transporters in astrocyte play a bigger role to keep extracellular glutamate concentration low compared to neurons. Glutamate taken up by astrocytes will be converted into glutamine, a non-neuroactive species, via the glutamine synthetase pathway which occurs exclusively in astrocytes 19 . In addition, astrocytes were found to contain higher glutathione levels as compared to neurons both in culture and in vivo 20 which further explains higher glutamate concentration needed in causing toxicity in astrocytes.
In terms of cell viability, both TRF and α-tocopherol at low concentration (100 to 300 ng/ml) gave significant recovery effects on glutamate-injured neurons in a dose-dependent manner. TRF showed similar cytoprotective effects against glutamate-induced cell death with α-tocopherol in this study, which was consistent with the finding reported by Saito and colleagues 21 . On the other hand, cytoprotective effects of TRF or α-tocopherol were also observed in glutamateinjured astrocytes. Neurons co-cultured with astrocyte showed significant increment in terms of cell viability as compared to mono-culture model. This further supports the role of astrocytes in protecting neuron from glutamate toxicity as described earlier.
Glutathione is a key defense system in cells of the nervous system against oxidative damage. Low intracellular concentration of glutathione is one of the early markers of neurotoxicity. Activation of 12-LOX can be triggered by lowering glutathione level. It will lead to the production of peroxides, Ca 2+ influx and subsequently cell death 22 . In this study, the loss of glutathione content in glutamate-injured astrocyte was not restored by TRF or α-tocopherol treatment. However, increment of glutathione production was observed in glutamate-injured neuron upon TRF or α-tocopherol treatment. Both cell lines responded differently toward TRF and α-tocopherol treatments in terms of glutathione production. Increment of glutathione content in neuron suggested that scavenging effect of TRF and α-tocopherol at low concentration (100 to 300 ng/ml) may occur prior to that of glutathione in neuron. In addition, there were studies showing that supplementation of vitamin E increased glutathione levels and also activities of glutathione reductase (GR) and glutathione-S-transferase (GST) decreased activity of glutathione peroxidase (GP) of rat brain. These observed changes in GR, GST and GP activity can lead to an increase of glutathione levels 23 . This showed that vitamin E did not only exert neuroprotective effects by scavenging ROS, but also exhibited antioxidant-independent protective effects. Furthermore, glutathione content of glutamate-injured neurons increased significantly in the presence of astrocytes in this study. Astrocyte is known to provide cysteine, which is the rate-limiting substrate for glutathione synthesis, to neurons 24 . In the presence of astrocyte, TRF and α-tocopherol treatment further increased glutathione content in glutamate-injured neuron. There was a study that showed cultured glial cells released cysteine into the medium which suggested that neurons maintain glutathione level by taking up cysteine provided by glial cell 25 . Moreover, glutamine synthesized in astrocyte from glutamate will be released and transferred back to neurons for reconversion to glutamate; namely glutamate-glutamine cycle 26 .

Conclusion
Neurons were more vulnerable to glutamate toxicity as compared to astrocyte. Both TRF and α-tocopherol at concentration of 100 to 300 ng/ml inhibited astrocyte and neuronal cell death against glutamate toxicity. Great scavenging effect of both TRF and α-tocopherol against glutamate toxicity was observed in neuron. Similar effect between TRF and αtocopherol in recovering neurons and astrocytes from glutamate toxicity was observed. Co-culturing models have demonstrated that neuronal survival is dependent on astrocytes survival. In the presence of astrocytes, neurons were more resistant to glutamate toxicity.