The Combinational Treatment of Antioxidants Protects Brain Endothelial Cells Against Oxygen-Glucose Deprivation Followed by Reperfusion Induced Injury

Introduction

Ischemic stroke is caused by a reduction of blood flow and re- sults in the deficiency of glucose and oxygen supply to the brain.

Increased levels of reactive oxygen species (ROS) are the major reason of tissue injury after cerebral ischemia. ROS are consid- ered regulatory molecules and known to act as second messen- gers during ischemia-reperfusion (I/R). ROS are generated dur- ing ischemia-reperfusion injury and promote the apoptosis pathway.

Antioxidant attenuates oxidative damage induced by I/R through decreasing mechanism of ROS production.

Specifi- cally, dehydroascorbic acid (DHA) and glutathione (GSH) play an important physiological role in cells as a antioxidant, free rad- ical scavenger.

A high intracellular GSH concentration protects cells against ROS by reacting directly with free radicals

and GSH is involved in a variety of mechanism related with human diseases.

DHA inhibited cell death by increasing the GSH lev-

els.

Melatonin is synthesized in the pineal gland and has been known to exert direct and indirect antioxidant actions.

Mela- tonin reduces the harmful cellular actions of ROS in I/R barin injury.

In in vivo cerebral ischemia model, previous research- es demonstrates that melatonin treatment attenuates brain dam- age against ischemia-hypoxia induced injury.

Previous stud- ies confirmed that melatonin treatment led to the maintenance of higher contents of GSH through its regulation of the AA-GSH cycle. Melatonin strongly is involved in AA-GSH cycle.

In the present study, we examined the synergetic beneficial effect of melatonin on the combinational treatment of DHA and GSH.

The blood brain barrier (BBB) includes biochemical mecha- nisms that control the exchange of materials between blood and the brain. BBB is crucial in central nervous system considering regulation of the brain microenvironment for neuron’s optimal functioning.

Tight junctions are important BBB structural components that seal the gaps between adjacent endothelial cells

Vascular Neurology 2013;5:35-41 ISSN 2092-6855

The Combinational Treatment of Antioxidants Protects

Brain Endothelial Cells Against Oxygen-Glucose Deprivation Followed by Reperfusion Induced Injury

So Mang Kang,

Juhyun Song,

Won Taek Lee,

Kyung Ah Park,

Kyoung Min Lee,

Jong Eun Lee

1Department of Anatomy, ²BK21 Plus Project for Medical Sciences, Yonsei University College of Medicine, Seoul, Korea

3Seoul National University College of Medicine, Seoul, Korea

Received November 15, 2013 Revised November 21, 2013 Accepted November 27, 2013 Correspondence Jong Eun Lee, PhD Department of Anatomy, Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea

Tel +82-2-2228-1646, 1659 Fax +82-2-365-0700 E-mail [email protected]

Antioxidants including dehydroascorbic acid (DHA), glutathione (GSH) and melatonin have cel- lular protective effects on murine brain endothelial (bEnd.3) cells after oxidative stress and isch- emic injury. Up to date, there are no studies that the combinational treatment of these antioxi- dants boosts the cellular protective effect on bEnd.3 cells. In this study, we investigated the protective effect of antioxidant combinational treatment on bEnd.3 cells after oxygen-glucose de- privation followed by reperfusion (OGD/R) induced injury. The effect of antioxidants combina- tional treatment on the bEnd.3 cells was examined by cell viability test, western blot analysis, im- munocytochemistry. The combinational treatment of DHA (1 mM), GSH (1 mM) protects bEnd.3 cells after OGD/R induced injury and also melatonin (10 μM) treatment boosts the pro- tective effect in combinational treatment of DHA and GSH on bEnd.3 cells after OGD/R induced injury. Western blot analysis suggests that the combinational treatment of DHA, GSH and melato- nin attenuated the expression of vascular endothelial growth factor (VEGF), phosphorylation of ERK compared with each single treatment after OGD/R induced injury. Claudin 5, hypoxia in- ducible factor-1α (HIF-1α) and VEGF immunostaining data suggests the preventive effect of anti- oxidants combinational treatment. In conclusion, the combinational treatment of antioxidants el- evates the cellular protective effect on bEnd.3cells against OGD/R induced injury.

Vascular Neurology 2013;5:35-41 Key Wordsaa Dehydroascorbic acid, Glutathione, Melatonin,

Oxygen-glucose deprivation followed by reperfusion, Murine brain endothelial cells.

and regulate cellular permeability.

We investigated whether the antioxidants combinational treatment preserves the tight junction protein. Hypoxia inducible factor-1α (HIF-1α) is a key regulator of hypoxia-induced gene expression and induces a va- riety of mechanisms including angiogenesis, cell survival, cell death after hypoxia.

In addition, vascular endothelial growth factor (VEGF) has a detrimental effect in endothelium permea- bility leading to vasogenic edema.

Therefore, activation of ERK contributes to cell death

and regulates the expression of VEGF.

We examined whether antioxidant combinational treat- ment contributes to the expression of VEGF and phosphoryla- tion of ERK1/2. In the present study, we investigated the syner- getic protective effect of antioxidants combinational treatment on bEND3 cells against OGD injury.

Materials and Methods

Cell culture

Murine brain endothelial cells (bEnd.3 cells, Manassas, VA, USA) were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone Laboratories, Lo- gan, UT, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA) and 100 units/mL of penicillin/streptomycin (Hyclone Laboratories, Logan, UT, USA), at 37°C in a humidified atmosphere in the presence of 5%

CO

. Culture medium was changed every 2 days.

Drug treatment

DHA was dissolved in 0.1 M bicarbonate buffer and GSH and Melatonin were melted in phosphate-buffered saline (PBS). Cul- tured bEnd.3 cells were divided into four groups as follows: 1) Control group, incubated with normal media; 2) Experimental group, cultured in non-treated medium for 6 hours after 6 hours of OGD injury; 3) combinational treatment of DHA and GSH group, corresponding to cells incubated in the presence of DHA and GSH for 6 hours (1 mM DHA+1 mM GSH) after OGD in- jury; and 4) combinational treatment of DHA, GSH and MEL group, treated DHA, GSH and MEL for reperfusion 6 hours (1 mM DHA+1 mM GSH + 10 μM Melatonin).

Oxygen-glucose deprivation and reperfusion Confluent cells were transferred to an anaerobic chamber (Forma Scientific, Marietta, OH, USA) (O

tension, 0.1%), washed three times with PBS. Then culture medium was re- placed with deoxygenated glucose-free balanced salt solution and cells were incubated for 6 hours. Following OGD injury, cells were incubated for 6 hours under normal growth condi- tions with or without drug treatment.

Cell viability assays

Cells (5×10

cells/well) were seeded into 48-well plates, and went through all experiment procedures, including pretreat-

ment, OGD injury and reperfusion. After that, the cells were rinsed twice with PBS, then 3-[4,5-dimethylthiazol-2-yl]-2,5-di- phenyl tetrasodium bromide (MTT, 2 mg/mL, Sigma, St. Louis, MO, USA) was added to each well. After 6 hours of incubation, medium were removed and dimethyl sulfoxide (DMSO) was added for solubilizing purple formazan product formed by MTT treatment. Then, supernatant from each well (200 μL/well) was replaced into 96-well plates for reading the absorbance with an ELISA reader at 570 nm. All experiments were repeated at least three times with similar results. The mean absorbance for the triplicate cultures of each drug was calculated, and the mean blank value was subtracted from these. Cell viability in the con- trol medium without any treatment was represented as 100%.

Cell viability displayed the relative value compared with the con- trol group.

Hoechst 33258 and propidium iodide staining Cell viability was evaluated by staining bEnd.3 cells with Hoechst 33258 dye (Sigma, St. Louis, MO, USA) and propidium iodide (PI; Sigma, St. Louis, MO, USA). Hoechst dye was added to the culture medium (2-3 μg/mL) and then the samples were kept at 37.8°C for 30 min. PI solution was then added (2-5 μg/mL) just before observing with an fluorescent microscope (BX51;

Olympus) equipped with epifluorescence and a UV filter block.

Hoechst-positive, PI-positive and Hoechst-positive, PI-negative cells were counted as dead and live cells, respectively.

Western blot analysis

After pretreatment, OGD injury and restoration, the cells were rapidly washed with ice-cold PBS, scraped and collected.

Cell pellets were lysed with ice-cold RIPA buffer (Sigma, St.

Louis, MO, USA). The lysates were centrifuged at 13200 rpm for 1 h at 4°C to yield whole-cell extracts. The protein content of the cell lysates was quantified using BCA method (Pierce, Rockford, IL, USA). Protein (20 μg) was separated on a 10% SDS-poly- acrylamide (PAGE) gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% bovine serum albumin prepared in Tris-buffered saline/Tween [TBS-T;

20 nM Tris (pH 7.2); 150 mM NaCl; 0.1% Tween 20] for 1 h at room temperature (RT), immunoblottings (overnight at 4°C) were reacted with primary antibodies that specifically detect VEGF (1 : 1000), ERK (1 : 1000), p-ERK (1 : 2000), Beta actin (1 : 2000) (all purchased from Millipore, Billerica, MA, USA). Then, the immunoblots were reacted with HRP-linked anti-mouse and -rabbit IgG antibodies purchased from Abcam (Cambridge, MA, USA) for 1 h at RT, and enhanced chemiluminescence was per- formed by ECL (Pierce, Rockford, IL, USA).

Immunocytochemistry (ICC)

The expression of VEGF, claudin-5 and HIF-1α in bEnd.3

cells was confirmed by immunocytochemistry (ICC). The ex-

pression of VEGF, claudin-5 and HIF-1α in bEnd.3 cells was

confirmed by immunocytochemistry. All the experimental groups was washed 3 times with phosphate-buffered saline, fixed with 4% paraformaldehyde for 3 hours, and then washed with PBS. bEnd.3 cells were permeabilized with 0.025% Triton X-100 and were blocked for 1 hour at room temperature with dilution buffer (Invitrogen life science, Carlsbad, CA, USA). Primary an- tibody rabbit anti VEGF (1 : 500, Millipore, Billerica, MA, USA), rabbit anti HIF-1 α (1 : 500, Santa Cruz Biotechnology, Santa Cruz, CA, USA) prepared in the dilution buffer was added to the samples and incubated for 3 hours at room temperature. Pri- mary antibody was removed and cells were washed 3 times for 3 minutes each with PBS. Later samples were incubated with FITC-conjugated goat anti rabbit second antibodies (1 : 200, Jackson Immunoresearch, West Grove, PA, USA) or Rhoda- mine-conjugated donkey anti rabbit second antibodies (1 : 500, Millipore, Billerica, MA, USA) for 2 hours at room temperature.

Cells were washed again 3 times for 3 minutes each with PBS and stained with 4’,6-diamino-2-phenylindole (1 : 100, Invitro- gen life science, Carlsbad, CA, USA) for 10 minutes at room temperature. The fixed samples were imaged using Zeiss LSM 700 confocal microscope (Carl Zeiss Microimaging, Thorn- wood, NY, USA).

Statistical analysis

Statistical comparisons were performed by using an indepen- dent t-test for 2 groups or analysis of variance with the SPSS software. Values are expressed as mean±S.E.M of 3-6 experi- ments. Differences were considered significant at p<0.1, p<0.05,

Results

Combination treatment of antioxidants enhances cell viability of bEND3 cells against OGD/R Induced Injury

We examined MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyl tetrasodium bromide] assay commonly used as cell viability test for confirming the difference of cell viability between groups after OGD injury. Fig. 1A shows that DHA group and GSH group and MEL group elevate the cell viability than it of EC group. All single treatment groups have a cellular protective ef- fect in bEND3 cells against OGD/R injury. All combinational treatment groups (D+G, G+M, M+D, D+G+M groups) increase the cell viability of bEND3 cells compared to single treatment groups (DHA, GSH, MEL group). Especially, D+G+M group significantly elevates the cell viability of bEND3 cells compared with D+G group. Melatonin boosts the cell viability of bEND3 cells in the combination treatment group of DHA and GSH after OGD injury (Fig. 1A). Fig. 1B indicates that EC group shows that PI positive cells (red color) as a marker of death cells in- creases markedly in comparison with NC group. In comparison EC group, D+G group and D+G+M group decreases consider-

ably PI positive cells (Fig. 1B). Fig. 1B suggests that the combina- tional treatment contributes to reduction of death cells on bEND3 cells after OGD injury (p<0.1, p<0.05, p<0.001).

60 50 40 30 20 10

0 EC DHA GSH MEL D+G G+M M+D D+G+M

Cell viability (%)

†

* *

†

A

Figure 1. The cell viability measurement of bEND3 cells after OGD/R induced injury. A: The cell viability is lower in EC group due to OGD/R injury compared to NC group (100%). Also, the cell viability is higher combination treatment groups than single treatment groups. Especially, D+G group and D+G+M group sig- nificantly increases the cell viability of bEND3 cells after OGD/R injury. B: Hoechst and PI immunostaining shows that PI positive cells (red color) as a maker of death cells decreases by treating combinational antioxidants compared to EC group. PI positive cells increases in EC group compared with NC group due to OGD/R injury. PI positive cells decreases in D+G group and D+G+M group compared with EC group. Hoeschst: blue, PI: red.

*p<0.1, ^†p<0.05, ^‡p<0.001. bEND3: brain endothelial, OGD/R: ox- ygen-glucose deprivation followed by reperfusion, NC: normal control group, EC: experimental group, DHA: DHA treatment group, GSH: GSH treatment group, MEL: melatonin treatment group, D+G: DHA and GSH treatment group, G+M: GSH and Melatonin treatment group, M+D: Melatonin and DHA treatment group, D+G+M: DHA and GSH and Melatonin treatment group.

B

Hoeschst

NC

EC

D+G

D+G+M

PI Merge

Combination treatment of antioxidants reduces the expression of HIF-1

and VEGF on bEND3 cells against OGD/R induced injury

To measure the expression of HIF-1α and VEGF on bEND3 cells against OGD injury, we conducted ICC using HIF-1α and VEGF antibodies. The expression of HIF-1α as a inducer of hy- poxia related genes increases conspicuously in EC group and D+G group reduces HIF-1α positive cells (red color) compared to EC group (Fig. 2A). Specifically, D+G+M group reduces HIF- 1α positive cells among all groups (Fig. 2A). The expression of HIF-1α is attenuated by antioxidant combination treatment un- der hypoxia condition. Fig. 2B shows that the expression of VEGF (red color) increases evidently in EC group and decreases in combination treatment groups (D+G group and D+G+M group) (Fig. 2B). The expression of HIF-1α and VEGF increases markedly in EC group and decreases in combinational treat- ment groups including D+G group and D+G+M group. The ex- pression pattern of HIF-1α and VEGF is similar in all groups.

Combination treatment of antioxidants preserves tight junction protein on bEND3 cells against OGD/R induced injury

To measure the expression of claudin 5 known as tight junc- tion protein on bEND3 cells after OGD injury, we conducted using ICC claudin 5 antibody. Fig. 3 indicates that the expression of claudin 5 (green color) decreases substantially in EC group compared to NC group. Claudin 5 as tight junction protein re- duces against OGD injury. However, in combinational treatment groups, claudin 5 protein is preserved in comparison with EC group. The combinational treatment of DHA, GSH and Melato- nin has a preventive effect of tight junction protein on bEND3 cells against OGD injury.

Combination treatment of antioxidants reduces vascular permeability of bEND3 cells through VEGF and ERK1/2 pathway against OGD/R induced injury

To determine the protein expression of VEGF and phosphor- ERK1/2 on bEND3 cells after OGD injury, we conducted west- ern blot analysis using VEGF and phosphor-ERK1/2 antibodies.

Fig. 4A shows that the expression of VEGF considerably increas-

Figure 2. Immunocytochemistry (ICC) to investigate the expression of HIF-1α and VEGF on bEND3 cells after OGD/R induced injury. A:

HIF-1α positive cells increases in EC group due to OGD/R injury compared to NC group. In addition, HIF-1α positive cells decreases in D+G group and D+G+M group compared to EC group. HIF-1α decreases more in D+G+M group than D+G group. HIF-1α: red, DAPI: blue.

B: VEGF positive cells increases in EC group due to OGD/R injury compared to NC group. VEGF positive cells decreases in combination group such as D+G group and D+G+M group compare with EC group. Especially, VEGF known as vascular permeability maker decreases in D+G+M group compared to D+G group. VEGF: red, DAPI: blue.HIF-1α: Hypoxia inducible factor-1α (HIF-1α), VEGF: vascular endothe- lial growth factor, bEND3: brain endothelial, OGD/R: oxygen-glucose deprivation followed by reperfusion, EC: experimental group, NC: nor- mal control group, DAPI: 4’,6-diamidino-2-phenylindole.

A

DAPI

NC

EC

D+G

D+G+M

HIF-1α MERGE

B

DAPI

NC

EC

D+G

D+G+M

VEGF MERGE

es in EC group compared to NC group. Also D+G+M group shows significantly decreases compared with MEL group and D+G group (Fig. 4A). Fig. 4A suggests that melatonin treatment boosts the decrease of VEGF expression in combinational treat- ment of DHA and GSH. Fig. 4B shows that the phosphorylation of ERK1/2 increases in EC group compared with NC group and decreases considerably in D+G group. Especially, the phosphor- ylation of ERK1/2 decreases conspicuously in D+G+M group in comparison with all groups. Fig. 4B indicates that melatonin is associated with the decreased phosphorylation of ERK in com- binational treatment of DHA and GSH group.

Discussion

Reactive oxygen species (ROS) are important neurotoxic fac- tors in cerebral ischemia-reperfusion injury. ROS increase the susceptibility of brain tissues to ischemic damage and induce a variety of molecular cascades and finally results in BBB hyper- permeability, brain edema, hemorrhage, inflammation and cell death.

BBB disruption are commonly observed in the patients

with various neurological disorders including stroke and de- mentia.

Previous researches suggested that antioxidants pre- serve BBB disruption and decreases ROS after cerebral ischemia reperfusion after stroke in vivo

and in vitro.

DHA as an ox- idized form of ascorbic acid (AA) prevents cellular apoptosis

and also ameliorates endothelial dysfunction related to nitric ox- ide (NO) activity.

In in vivo stroke model, DHA significantly reduce the infarct volume and neurologic deficits.

DHA is taken up through the glucose transport mechanism

and is regenerat- ed to AA at the expense of the GSH within the cells.

DHA in-

Figure 3. Immunocytochemistry (ICC) for confirming expression of claudin 5 on bEND3 cells in OGD/R induced injury. Claudin 5 positive cells (green) decreases in EC group compared to NC group due to OGD/R injury. Also, claudin 5 increases in combina- tional groups such as D+G group and D+G+M group compare with EC group. Specifically, claudin 5 known as tight junction pro- tein preserves in D+G+M group compared to D+G group. Claudin 5: green, DAPI: blue. bEND3: brain endothelial, OGD/R: oxygen- glucose deprivation followed by reperfusion, EC: experimental group, NC: normal control group, D+G: DHA and GSH treatment group, D+G+M: DHA and GSH and Melatonin treatment group, DAPI: 4’,6-diamidino-2-phenylindole.

DAPI

NC

EC

D+G

D+G+M

Claudin 5 MERGE

Figure 4. Western blot analysis for confirming vascular permeabili- ty of bEND3 cells in OGD/R induced injury. A: The protein expres- sion of VEGF, vascular permeability maker, increases in EC group owing to OGD/R injury compared to NC group. In antioxidant com- binational treatment group, the protein expression of VEGF de- creases compared with EC group. In detail, the expression of VEGF decreases more in D+G+M group compared to D+G group.

(B) The protein expression of ERK and phosphor-ERK increases in EC group. The protein expression of phosphor ERK decreases in D+G group and D+G+M group. *p<0.001, ^†p<0.05, ^‡p<0.1, bEND3:

brain endothelial, OGD/R: oxygen-glucose deprivation followed by reperfusion, VEGF: vascular endothelial growth factor, p-ERK:

phospho-extracelluar single-regulated kinases, NC: normal control group, EC: experimental group, MEL: only melatonin treated group, ERK: extracelluar single-regulated kinases, D+G: DHA and GSH treatment group, D+G+M: DHA and GSH and Melatonin treatment group.

0.7 0.6 0.5 0.4 0.3 0.2 0.1

0 NC EC MEL D+G D+G+M

Relative optical density

†

A

*

VEGF B actin

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

0 NC EC MEL D+G D+G+M

Relative optical density

†

†

‡

B

p-ERK ERK B actin

hibited H

O

-induced cell death by increasing the GSH levels.

GSH as a antioxidant plays also an important free radical scav- enger in cells.

GSH is involved in a variety of mechanism in- cluding cell survival pathway and protects cells against ROS.

Melatonin is synthesized in the pineal gland and has been known to involved in antioxidant actions.

Therefore, melato- nin reduces the cellular damage by ROS in I/R barin injury.

In in vivo cerebral ischemia model, previous researches demon- strates that melatonin treatment attenuates brain damage against ischemia-hypoxia induced injury.

Wang et al.

demonstrated that melatonin treatment induces the maintenance of higher contents of GSH through its regulation of the AA-GSH cycle.

Melatonin strongly contributes to AA-GSH cycle.

In the pres- ent study, we demonstrated that the cell viability was enhanced considerably by the combinational treatment of DHA, GSH and melatonin compared to antioxidant single treatment. In addi- tion, the combinational treatment of DHA, GSH and melatonin reduces markedly the death cells and prevents the cells after OGD/R injury. BBB damage aggravates by reperfusion after ischemia

and the tight junctions as BBB structural compo- nents

disrupts against ischemic injury. Argaw et al. demon- strated that disruption of claudin-5 contribute to BBB damage in various CNS pathological conditions and specifically VEGF me- diates these mechanism.

Tao et al. demonstrates that melatonin prevents tight junction integrity against ischemic injury by con- firming ZO-1 expression in endothelial cells.

Our results also indicates that claudin 5 as a tight junction protein was preserved by DHA and GSH and melatonin combination treatment. In this study, we could not determine that melatonin can boost the preventive effect of tight junction protein in combinational treat- ment of DHA and GSH group. However, the preventive effect of tight junction protein is almost the same between the combina- tional treatment of DHA and GSH and the combinational treat- ment of DHA and GSH and melatonin. Moreover, the expres- sion of HIF-1α and VEGF attenuated conspicuously in the combinational treatment of DHA and GSH and melatonin. The antioxidant combinational treatment prevents bEND3. cells against hypoxia injury suggesting that HIF-1α and VEGF induc- es hypoxia gene’s transcription

and triggers apoptosis pathway and exacerbate damaged region under hypoxia injury.

There- fore, VEGF induces activation of ERK1/2.

Slevin et al. demon- strated that increased ERK1/2 phosphorylation was noted in the vulnerable penumbra after acute ischemic stroke in humans and in animal models.

Also, previous study demonstrated that VEGF activates signaling pathways via the phosphorylation of ERK and inhibition of it also resulted in reduced cell death.

Al- though VEGF is a potential inducer in angiogenesis, it can have detrimental effects such as an increase in endothelium permea- bility leading to vasogenic edema.

Zhang and Chopp found that early postis chemic administration of VEGF significantly increased BBB permeability.

In the present study, findings of western blot shows that the combinational treatment of antioxi-

dants promotes reduction of VEGF expression in spite of the same exposure of hypoxia injury. These findings suggests that the antioxidant combinational treatment reduces the BBB per- meability and the possibility of making vasogenic edema. In ad- dition, by confirming the phosphorylation of ERK1/2, we con- cludes that the combinational treatment of DHA and GSH and melatonin inhibits cell death mechanism through increase of ERK1/2 expression like previous studies.

Like our findings, Narasimhan et al. also demonstrated that pERK1/2 expression decreased after OGD in endothelial cells that had decreased VEGF levels and cell death decreased by blocking VEGF.

In the present study, we demonstrated that the combinational treat- ment of DHA and GSH and melatonin increases the cell viability and preserves BBB permeability and also inhibits the apoptosis pathway associated with VEGF and ERK1/2. Hence, the combi- national treatment of DHA and GSH and melatonin boosts the preventive effect of bEND3 cells against OGD/R induced injury.

Acknowledgments

This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2009-0080364). This work was sup- ported by the Brain Korea 21 Plus Project for Medical Science, Yonsei Uni- versity.

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