research article protective effect of edaravone in primary...

Research ArticleProtective Effect of Edaravone in Primary Cerebellar GranuleNeurons against Iodoacetic Acid-Induced Cell Injury

Xinhua Zhou1 Longjun Zhu1 Liang Wang12 Baojian Guo1 Gaoxiao Zhang1 Yewei Sun1

Zaijun Zhang1 Simon Ming-Yuen Lee2 Pei Yu1 and Yuqiang Wang1

1 Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional ChineseMedicine Jinan University College of Pharmacy Guangzhou 510632 China2State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences University of MacauAvenue Padre Tomas Pereira SJ Macau

Correspondence should be addressed to Zaijun Zhang zaijunzhang163com

Received 15 August 2014 Revised 19 October 2014 Accepted 22 October 2014

Academic Editor Claudio Cabello-Verrugio

Copyright copy 2015 Xinhua Zhou et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect However it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronaldeath in vitro In the present study we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons(CGNs) and its possible underlyingmechanismsWe found that EDA attenuated IAA-induced cell injury in CGNsMoreover EDAsignificantly reduced intracellular reactive oxidative stress production loss of mitochondrial membrane potential and caspase 3activity induced by IAA Taken together EDA protected CGNs against IAA-induced neuronal damage which may be attributed toits antiapoptotic and antioxidative activities

1 Introduction

Stroke is the second leading cause of mortality among mostdeveloped countries [1] Cerebral ischemia is the most com-mon type of stroke and accounts for 87 of all stroke cases[2] Brain hypoxia and glucose deprivation are the primarypathophysiological features of cerebral ischemia which leadto cerebral infraction [3] After hypoxia the balance betweengeneration and clearance of reactive oxidative species (ROS)is compromised and overproduction of ROS as byproductsby mitochondria may result in activation of mitochondria-dependent apoptotic pathway in acute ischemia stroke [4]More recent lines of evidence have suggested that an exces-sive ROS generation may lead to mitochondrial membranedepolarization to induce the apoptosis cascade which resultsin functional and structural damage to neuronal cells [5]Mitochondrial dysfunction and excessive oxidative stress playa vital role in the pathogenesis of neurodegenerative diseasesincluding cerebral ischemia [3 6]Therefore neuroprotective

agents that scavenge free radicals and maintain mitochon-drial function should be an effective therapeutic strategy fortreatingROS-related disorders especially ischemic stroke [7]

Edaravone (EDA) has been used to treat acute cerebralinfraction since 2001 which is the first novel free radicalscavenger approved in Japan [8 9] EDA has been shownto quench hydroxyl peroxyl and superoxide radicals andinhibit free radical-mediated lipid peroxidative damage ina rat middle cerebral artery occlusion (MCAO) model [10]Furthermore recent mechanistic research found that EDAsuppressed delayed neuronal death induced by reperfusionreduced long-term inflammatory reaction and inhibited theexpression of vascular endothelial growth factor mediated bythe ischemic cascade [8 11] Thus the therapeutic benefit ofEDA is attributed not only to its antioxidative activity but alsoto its ability to regulate various signaling pathways

Several studies have shown that iodoacetic acid (IAA)treatment could closely mimic the hypoxicischemia condi-tion in nerve cells in vitro [12ndash14] IAA a chemical ischemia

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 606981 8 pageshttpdxdoiorg1011552015606981

2 Oxidative Medicine and Cellular Longevity

stimulus inhibits the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase irreversibly and induces cell deathaccompaniedwith an increase in ROS productionmitochon-drial dysfunction and loss of ATP [15ndash17] These responsesare very similar to changes observed in animal models ofischemic stroke [18] Cerebellar granular neurons (CGNs)are primary rat neurons obtained from cerebellum whichcontain the largest homogeneous neuronal population in thebrain which have been widely used as an in vitro modelfor investigating the cellular and molecular mechanismsunderlying neuronal apoptosis in neurodegenerative disor-ders [19ndash21] Previously researchers have demonstrated thatIAA reduced cell viability via increasing ROS production inCGNs [22 23] It has yet to be determined whether EDA canattenuate IAA-induced neuronal death in vitro Therefore weherein used the in vitroCGNsmodel exposed to IAA to studythe neuronal protective effects of EDA and to explore thepossibility of its underlying mechanisms of action

2 Materials and Methods

21 Chemicals and Reagents EDA was purchased fromAladdin Reagent Co (Shanghai China) MTT fluores-cein diacetate (FDA) propidium iodide (PI) 2101584071015840-dichloro-fluorescein diacetate (DCFH-DA) DNase poly-L-lysine andcytosine-D-arabinofuranoside were obtained from Sigma-Aldrich (St Louis MO USA) Basal medium Eagle (BME)fetal bovine serum penicillin and streptomycin were pur-chased from Invitrogen (Carlsbad CA USA) Mitochondrialmembrane potential assay kit and caspase 3 activity assaykit were obtained from Beyotime (Shanghai China) Thecytotoxicity detection kit (LDH) was purchased from RocheDiagnostics (Mannheim Germany)

22 Primary Cultures of Cerebellar Granule Neurons andDrug Treatment CGNs were isolated from postnatal 8-day-old Sprague-Dawley rat pups (15ndash20 g) from the AnimalCare Facility of Sun Yat-sen University according to theprotocol described by Bilimoria and Bonni [24] The densityof viable cells in suspension was determined by cell countusing trypan blue and adjusted to 2 times 106 cellsmL Neuronswere seeded into 96-well pates or 12-well plates which wereprecoated with 50120583gmL poly-L-lysine and maintained in ahumidified incubator with 5 CO

2in air at 37∘C Twenty-

four hours after plating cytosine-D-arabinofuranoside (finalconcentration 10120583M) was added to the culture mediumto arrest the proliferating of nonneuronal cells All animalstudies were conducted according to guidelines of the Exper-imental Animal Care andUse Committee of JinanUniversityThe experimental protocols were approved by the EthicsCommittee for Animal Experiments of Jinan University

Unless otherwise stated on day 7 of in vitro culture CGNswere pretreated with EDA (3 10 and 30 120583M) for 2 h IAA(50120583M) was then added for incubation for another 4 h toinduce cell injury

23 Immunofluorescence Immunofluorescence staining with120573III-tubulin antibody was used to observe the morphology

changes of the neurite Briefly CGNswere washedwithHBSSand were fixed with 4 paraformaldehyde for 15min at 4∘CThe cells were then permeabilized with 01 Triton X-100for 5min The fixed neurons were incubated in 10 horseserum for 1 h to block nonspecific protein interactions Theprimary antibody (rabbit anti-mouse 120573III-tubulin antibody1 400 Abcam) was added and the cells were incubated atroom temperature for 3 h After washing the neurons wereincubated with the secondary antibody (goat anti-rabbit IgG-FITC 1 100 Invitrogen) for 30min at room temperatureAfter washing twice with HBSS the coverslips were mountedwith antiquenching mounting medium and the neuritesmorphology changes of neurons were visualized using afluorescence microscope at times400 magnification

24 MTT Assay The cell viability was assessed using theMTTassay according to conditions described previously [25]The absorbance at 570 nm was measured using a WallacVictor3Vmicroplate reader (PerkinElmerNetherlands) Cellviability was expressed as a percentage of the MTT reductionof control

25 LDH Release Assay The cytotoxicity inflicted to cellsby IAA was also assessed by the LDH release assay Deter-mination of total and released LDH activity was performedaccording to the instructions accompanying the cytotoxicitydetection kit (Roche) LDH releasedwas normalized to a totalLDH release and the results were shown as a percentage oftotal LDH activity

26 FDAPI Double Staining and Hoechst Staining Viableneurons were stained with fluorescein formed from FDAwhich is deesterified only by living cells PI can penetratecell membranes of dead cells to intercalate into double-stranded nucleic acids Briefly neurons were washed twicewith ice-cold PBS After incubation with 10120583gmL of FDAand 5 120583gmL of PI for 15min the neurons were examinedand photographed using a fluorescence microscope (NikonInstruments Inc Melville NY)

Chromatin condensation was detected by staining thecell nucleus with Hoechst 33342 The cultures were fixedwith 4 paraformaldehyde and washed with PBS beforestaining with Hoechst 33342 for 30min Thereafter cellmorphology was observed under a fluorescent microscope(Zeiss Oberkochen Germany) The percentage of apoptoticnuclei from five random fields in each well of differenttreatment groups was quantified and averaged

27 Measurement of Mitochondrial Membrane Potential(Δ120595119898) The dye JC-1 was used as a molecular probe to

measureΔ120595119898The staining procedurewas performed accord-

ing to the manufacturerrsquos instructions with minor modifi-cation Briefly CGNs were washed with HBSS and stainedwith 2120583M JC-1 for 10min Fluorescence intensity was mea-sured on a microplate reader using 488 nm excitation and529 nm590 nm dual emissions The mitochondrial accumu-lation of JC-1 is dependent on Δ120595

119898and is reflected by a shift

in 529 nm and 590 nm emissions Mitochondrial membrane

Oxidative Medicine and Cellular Longevity 3

depolarization is indicated by a decrease in the ratio of590 nm to 529 nm emissions

28 ROS Measurement Intracellular ROS were measuredusing the redox-sensitive fluorescent probe DCFH-DAwhich is hydrolyzed to nonfluorescent DCFH by intracellularesterase DCFH is rapidly oxidized to the fluorescent DCFwhen reacting with intracellular ROS CGNs were washedwith PBS and were incubated with 20120583M DCF-DA for 1 hFluorescence was measured on a microplate reader at anexcitation wavelength of 495 nm and an emission wavelengthof 515 nm

29 Caspase 3 Activity Assay CGNs were scraped off inHBSS collected by centrifugation and lysed at 4∘C in cell lysisbuffer containing 20mMEDTA 20mMTris (pH 75) and 1Triton X-100 Lysates were centrifuged at 12000 g at 4∘C for5min Caspase 3 assay was performed on 96-well microplatesusing substrate peptides Ac-DEVD-pNA according to themanufacturerrsquos instructionThe release of p-NAwas qualifiedby determining the absorbance at 405 nm

210 Statistical Analysis All measurements were repeated 3timesDatawere expressed asmeanplusmn SEMandwere analyzedusing GraphPad Prism V50 (GraphPad Software Inc SanDiego CA USA) One-way analysis of variance (ANOVA)andDunnettrsquos test were used to evaluate statistical differencesThe value of statistical significance was set at 119875 lt 005

3 Results

31 Effect of EDA on IAA-Induced CGNsDeath We evaluatedthe protective effect of EDA on IAA-induced primary neurondeath by immunofluorescence-based morphological analy-sis MTT reduction and LDH leakage-based cell viabilityassay and FDAPI double staining As shown in Figure 1(a)IAA clearly destroyed the gross morphology of the CGNscell body and neuritic network Pretreatment with EDAnotably protected neurons from IAA-induced neurotoxi-city (Figure 1(a)) For 4 h treatment IAA concentration-dependently decreased the cell viability of CGNs The sur-vival rate reduced approximately by 50 when CGNs wereexposed to 50 120583M IAA (Figure 1(b)) The cell viability ofCGNs pretreated with 3ndash30 120583M EDA significantly increasedin a concentration-dependent manner (119875 lt 005 versus IAAtreatment alone Figure 1(c)) and was up to 85 of control at30 120583MEDA treatment Pretreatment of EDA also reduced thelevel of LDH release compared with IAA-treated alone group(119875 lt 005 Figure 1(d)) Moreover cotreatment of EDA withIAA also significantly inhibited the decrease of cell viabilityinduced by IAA in a concentration-dependent manner (119875 lt005 versus IAA treatment alone Figure 1(e))

The FDAPI double staining was performed to furtherdetermine whether EDA attenuated cell injury after IAAtreatment in CGNs Simultaneous use of two fluorescentdyes allows a two-color discrimination of the populationof live cells from the necrotic-cell population As shownin Figure 2(a) IAA significantly increased cell necrosis as

stained by red fluorescence accompanying decrease in greenfluorescent cells Consistent with the result ofMTT-based cellviability assay pretreatment of EDA remarkably attenuatedCGNs necrosis induced by IAA (Figures 2(a) and 2(b))

32 EDA Prevents IAA-Induced CGNs Apoptosis Apoptosisis morphologically characterized by cell shrinkage chro-matin condensation To identify whether EDA reverses IAA-induced CGNs apoptosis we used Hoechst 33342 stainingto evaluate nuclear condensation As shown in Figure 3(a)normal untreated cells appeared circular or elliptical whereno condensation of the nucleus was observable In con-trast bright condensed dots known as apoptotic bodies (asindicated by arrows) were clearly identified when treatedwith IAA Pretreatment of EDA could mitigate IAA-inducedapoptosis (Figure 3(a)) In addition the count of apoptoticnuclei revealed that EDA significantly reduced IAA-inducedapoptosis concentration-dependently (Figure 3(b))

33 Effects of EDA on IAA-Induced Intracellular ROS Genera-tion IAA induces cell death accompanied with an increasein ROS production through irreversible inhibition of theglyceraldehyde 3-phosphate dehydrogenase [15ndash17] To inves-tigate whether EDA reduces ROS level after IAA treatment inCGNs intracellular ROS production was detected by DCFH-DAprobeAs shown in Figure 4 after exposure to IAA for 4 hthe level of ROSwas increased by 33-fold in CGNs comparedwith untreated control Pretreatment with EDA significantlysuppressed this increase in intracellular ROS level At 30120583MEDA almost completely inhibited ROS production to a levelequal to that of the normal control

34 The Effects of EDA on Δ120595119898in CGNs Induced by IAA

It has been reported that IAA induces mitochondrial dys-function [17] To investigate the protective effect of EDAagainst IAA-induced mitochondrial damage Δ120595

119898in CGNs

was assessed using JC-1 probe As shown in Figure 5 IAAtreatment resulted in a profound loss of Δ120595

119898in CGNs

When the cells were pretreated with EDA the Δ120595119898increased

significantly and concentration-dependently compared withthat of the IAA alone-treated group

35 EDA Attenuates IAA-Induced Caspase 3 Activation inCGNs Activation of caspase 3 plays a key role in cellularapoptosis Figure 6 showed that IAA treatment caused adramatic increase in caspase 3 activity When CGNs werepreincubated with EDA the elevated caspase 3 activityinduced by IAAwas significantly reduced in a concentration-dependent manner

4 Discussion

In the present study we demonstrated that EDA protectedagainst IAA-induced neurotoxicity in CGNs Subsequentexperiments to explore the mechanisms underlying the neu-roprotective effect revealed that EDA significantly decreasedneuronal apoptosis and intracellular ROS overproductionmaintained Δ120595

119898 and attenuated caspase 3 activity in CGNs


IAA IAA + EDA 3 IAA + EDA 10 IAA + EDA 30Ctrl

120573II

I-Tu

bulin

(a)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

Ctrl 25 50 100(120583M)

IAA for 4h

(b)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30

EDA

lowastlowastlowastlowast

lowast

50120583M IAA posttreatment for 4h

(c)

40

60

20

0

(120583M)Ctrl 3 10 30


EDA

LDH

rele

ase

lowastlowast

lowastlowast

lowast

(d)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30

EDA50120583M IAA cotreatment for 4h

lowastlowastlowast

lowastlowast

lowast

(e)

Figure 1 Protective effect of EDAagainst IAA-induced cells damage inCGNs (a)Grossmorphological change of CGNs cell body and neuriticnetworkwas determined by fluorescent immunostainingwith antibody against120573III-tubulin (400xmagnification) CGNswere pretreatedwithor without EDA for 2 h and then incubated with 50 120583M IAA for another 4 h (b) IAA-induced decrease in cell viability by MTT assay CGNswere exposed to 25 50 and 100 120583M of IAA for 4 h Cell viability was measured by MTT reduction assay (c) EDA pretreatment attenuatesIAA-induced neuronal loss in a concentration-dependent manner CGNs were pretreated with or without EDA for 2 h and then incubatedwith 50 120583M IAA for another 4 h Cell viability was measured by MTT reduction assay (d) Effect of EDA pretreatment on IAA-induced LDHrelease Cells were treated as in (c) and LDH release level was detected by cytotoxicity detection kit (e) Cotreatment of EDAwith IAA inhibitscell viability decrease induced by IAA CGNs were cotreated with EDA and IAA for 4 h Cell viability was measured by MTT reduction assay119875 lt 0001 versus control (Ctrl) lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 versus IAA alone group


PIM

erge

FDA

Ctrl IAA IAA + EDA 3 IAA + EDA 10 IAA + EDA 30

(a)

Ctrl 3 10 300

20

40

60

80

EDA

IAA

Nec

rotic

cells

()

(120583M)


lowast

(b)

Figure 2 EDA attenuates CGNs neurosis induced by IAA CGNs were preincubated with or without EDA for 2 h followed by exposure to50 120583M IAA for another 4 h (a) CGNs were stained with FDA and PI (200x magnification) (b) Quantitative analysis of necrotic cells fromthree representative photomicrographs was represented as a percentage of the total number of cells counted 119875 lt 0001 versus Ctrl lowast119875 lt 005and lowastlowast119875 lt 001 versus IAA alone group

Cerebral ischemia impairs the normal neurological func-tions which are triggered by a complex series of biochemicaland molecular mechanisms such as excitotoxicity oxidativestress and apoptosis along with histological changes [26]Cerebral ischemia triggers two general pathways of apoptosisthe intrinsic pathway originating frommitochondrial releaseof cytochrome c and associated stimulation of caspase 3and the extrinsic pathway originating from the activationof cell surface death receptors resulting in the stimulationof caspase 8 In addition cerebral ischemia and reperfusiongenerate ROS which causes DNA damage Many studieshave shown that EDA attenuated apoptosis in neuronalcells Song et al demonstrated that EDA suppressed theBad protein overexpression and increased Bcl-2 protein

expression repaired the mitochondrial dysfunction andmaintained ATP level in PC12 cells [27] Xiong et al showedthat EDA inhibited protein Bax expression and attenuateddownregulation of Bcl-XL induced by rotenone [28] Chenet al also found that EDA inhibited cobalt chloride-inducedapoptosis in PC12 cells via the regulation of Bcl-2 familyand reduced caspase 3 activity [4] Consistent with previousfindings our present study showed that EDA also signifi-cantly reduced IAA-induced CGNs apoptosis via suppressionof caspase 3 activation and maintaining Δ120595

119898 However

different from other studies which applied oxygen-glucosedeprivation rotenone and cobalt chloride as causes of cellinjury our present study used IAA to induce cell damage Ithas been reported that oxygen-glucose deprivation induced


Ctrl IAA

IAA + EDA 3 IAA + EDA 10 IAA + EDA 30

(a)

10

20

30

40

50

60

0

Cell

apop

tosis

()

(120583M)Ctrl 3 10 30

EDA

IAA

IAA

lowastlowast

lowastlowast

lowast

(b)

Figure 3 EDAattenuates IAA-inducedCGNs apoptosis CGNswere preincubatedwith orwithout EDA for 2 h followed by exposure to 50 120583MIAA for another 4 h (a) Cell apoptosis was assessed by Hoechst 33342 staining and observed by fluorescent microscopy (400xmagnification)Apoptotic nuclei with condensed chromatin were indicated by red arrows (b) The amount of apoptosis nuclei was counted from threerepresentative photomicrographs andwas represented as a percentage of the total number of nuclei counted 119875 lt 0001 versus Ctrl lowast119875 lt 005and lowastlowast119875 lt 001 versus IAA alone group

0

100

200

300

400

EDAIAA

(120583M)


lowast

Ctrl 3 10 30IAA

( o

f con

trol)

DCF

fluo

resc

ence

inte

nsity

Figure 4 Effect of EDA on IAA-induced ROS production in CGNsCGNs were preincubated with or without EDA for 2 h followed byexposure to 50 120583MIAA for another 4 h Intracellular ROS generationwas evaluated by DCFH-DA probe 119875 lt 0001 versus Ctrl lowast119875 lt005 and lowastlowast119875 lt 001 versus IAA alone group

mitochondrial dysfunction and oxidative stress in neurons[29] and stimulated Ca2+-dependent glutamate release incortical slice cultures [30] Both rotenone and cobalt chlo-ride as well as IAA are mitochondrial inhibitors howeverthey inhibit mitochondrial function at different sites ofthe mitochondrial respiratory chain and are inhibitors ofmitochondrial respiratory complex I prolyl hydroxylase andglyceraldehyde 3-phosphate dehydrogenase respectively [1517 31 32]

Free radical production is enhanced in both the ischemiccore and penumbra following stroke injury and this isbelieved to cause much of the damage seen in these regions

0

20

40

60

80

100

120

EDA

IAA

(120583M)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Ctrl 3 10 30

JC-1

fluor

esce

nce i

nten

sity

ratio

( o

f con

trol)

Figure 5 Effect of EDA on IAA-induced Δ120595119898loss in CGNs CGNs

were preincubatedwith orwithout EDA for 2 h followed by exposureto 50 120583M IAA for another 4 h The Δ120595

119898was determined by JC-1

119875 lt 00001 versus control lowastlowastlowast119875 lt 001 versus IAA alone group

[1] Normally oxidative stress is being caused by the imbal-ance between free radical production and degradation Brainis most susceptible to oxidative stress due to high consump-tion of oxygen [33] Natural formation of oxidants duringmitochondrial electron transport and autooxidation of someneurotransmitters and in ischemic attacks of events duringischemia can result in oxidant formation and subsequenttissue damage [34] Normally free radicals are removed byantioxidant enzymes in living cells The antioxidant defensesinclude superoxide dismutase (SOD) glutathione peroxidase(GPX) catalase (CAT) and glutathione (GSH) SOD cataly-ses the dismutation of the highly reactive superoxide anion(O2

∙minus) to O2and H

2O2 CAT reacts with peroxide to form


Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)

Figure 6 EDA inhibits caspase 3 activation in CGNs CGNs werepreincubated with or without EDA for 2 h followed by exposure to50 120583M IAA for another 4 h Cells were lysed and caspase 3 activitywas measured as described in Section 29 119875 lt 0001 versus controland lowastlowast119875 lt 001 versus IAA alone group

water and molecular oxygen GPX catalyses the reduction oforganic peroxides using GSH and thereby protects cells fromoxidative damage Many studies have shown that protectiveeffect of EDA was associated with the increased SOD activityand CAT GSH levels in PC12 cells [35 36] Our present studyindicated that EDA significantly decreased ROS productioninduced by IAA in CGNs However that EDA defensesROS production induced by IAA in CGNs through directscavenging ROS or through indirect elevating antioxidativeenzymes needs to be further clarified

5 Conclusion

In conclusion the neuroprotective effect of EDA on IAA-induced neurotoxicity in CGNs was due to its antiapoptoticand antioxidative effects However the precise molecularevents involved in its antioxidative and antiapoptotic actionsneed to be further elucidated

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work is partially supported by grants from theNational Natural Science Foundation of China (NSFC81303251 to Zaijun Zhang) Chinarsquos ldquo125rdquo InnovativeDrug Project (2012ZX09103101-055 to Yuqiang Wang) theMunicipal Science and Technology Project of Guangdong(2012A080201009 to Yuqiang Wang) and the InternationalScience and Technology Collaborative Project of GuangdongDepartment of Education (gjhz1102 to Yuqiang Wang)

References

[1] T M Woodruff J Thundyil S-C Tang C G Sobey S MTaylor and T V Arumugam ldquoPathophysiology treatmentand animal and cellular models of human ischemic strokerdquoMolecular Neurodegeneration vol 6 article 11 2011

[2] Q Tang R Han H Xiao J Shen Q Luo and J Li ldquoNeuropro-tective effects of tanshinone IIA andor tetramethylpyrazine incerebral ischemic injury in vivo and in vitrordquo Brain Researchvol 1488 pp 81ndash91 2012

[3] S D Chen D I Yang T K Lin F Z Shaw C W Liou and YC Chuang ldquoRoles of oxidative stress apoptosis PGC-1120572 andmitochondrial biogenesis in cerebral ischemiardquo InternationalJournal of Molecular Sciences vol 12 no 10 pp 7199ndash7215 2011

[4] J-X Chen T Zhao and D-X Huang ldquoProtective effects ofedaravone against cobalt chloride-induced apoptosis in PC12cellsrdquo Neuroscience Bulletin vol 25 no 2 pp 67ndash74 2009

[5] T Kalogeris Y Bao and R J Korthuis ldquoMitochondrial reactiveoxygen species a double edged sword in ischemiareperfusionvs preconditioningrdquo Redox Biology vol 2 pp 702ndash214 2014

[6] K Niizuma H Endo and P H Chan ldquoOxidative stressand mitochondrial dysfunction as determinants of ischemicneuronal death and survivalrdquo Journal of Neurochemistry vol109 supplement 1 pp 133ndash138 2009

[7] J Fang H Qin T Seki et al ldquoTherapeutic potential ofpegylated hemin for reactive oxygen species-related diseasesvia induction of heme oxygenase-1 results from a rat hepaticischemiareperfusion injury modelrdquo Journal of Pharmacologyand ExperimentalTherapeutics vol 339 no 3 pp 779ndash789 2011

[8] P A Lapchak ldquoA critical assessment of edaravone acuteischemic stroke efficacy trials is edaravone an effective neuro-protective therapyrdquo Expert Opinion on Pharmacotherapy vol11 no 10 pp 1753ndash1763 2010

[9] M Mittal D Goel K K Bansal and P Puri ldquoEdaravonemdashciticoline comparative study in acute ischemic stroke (ECCS-AIS)rdquo Journal of Association of Physicians of India vol 60 no11 pp 36ndash38 2012

[10] K Kikuchi K-I Kawahara S Tancharoen et al ldquoThe freeradical scavenger edaravone rescues rats from cerebral infarc-tion by attenuating the release of high-mobility group box-1 inneuronal cellsrdquoThe Journal of Pharmacology and ExperimentalTherapeutics vol 329 no 3 pp 865ndash874 2009

[11] S Ikeda K Harada A Ohwatashi and Y Kamikawa ldquoEffectsof edaravone a free radical scavenger on photochemicallyinduced cerebral infarction in a rat hemiplegic modelrdquo TheScientific World Journal vol 2013 Article ID 175280 5 pages2013

[12] P Sandner K Wolf U Bergmaier B Gess and A KurtzldquoHypoxia and cobalt stimulate vascular endothelial growth fac-tor receptor gene expression in ratsrdquo Pflugers Archiv EuropeanJournal of Physiology vol 433 no 6 pp 803ndash808 1997

[13] W ZouM YanWXu et al ldquoCobalt chloride induces PC12 cellsapoptosis through reactive oxygen species and accompanied byAP-1 activationrdquo Journal of Neuroscience Research vol 64 no 6pp 646ndash653 2001

[14] J M Noel J P Fernandez de Castro P J DeMarco Jr et alldquoIodoacetic acid but not sodium iodate creates an inducibleswine model of photoreceptor damagerdquo Experimental EyeResearch vol 97 no 1 pp 137ndash147 2012

[15] P Maher K F Salgado J A Zivin and P A Lapchak ldquoA novelapproach to screening for new neuroprotective compounds for


the treatment of strokerdquo Brain Research vol 1173 no 1 pp 117ndash125 2007

[16] O Sperling Y Bromberg H Oelsner and E Zoref-ShanildquoReactive oxygen species play an important role in iodoacetate-induced neurotoxicity in primary rat neuronal cultures and indifferentiated PC12 cellsrdquo Neuroscience Letters vol 351 no 3pp 137ndash140 2003

[17] H SunaM Arai Y Tsubotani A Hayashi A Setiawan andMKobayashi ldquoDysideamine a new sesquiterpene aminoquinoneprotects hippocampal neuronal cells against iodoacetic acid-induced cell deathrdquo Bioorganic andMedicinal Chemistry vol 17no 11 pp 3968ndash3972 2009

[18] P Lipton ldquoIschemic cell death in brain neuronsrdquo PhysiologicalReviews vol 79 no 4 pp 1431ndash1568 1999

[19] W Cui W Li Y Zhao et al ldquoPreventing H2O2-induced apop-

tosis in cerebellar granule neurons by regulating the VEGFR-2Akt signaling pathway using a novel dimeric antiacetyl-cholinesterase bis(12)-hupyridonerdquo Brain Research vol 1394pp 14ndash23 2011

[20] W CuiW Li R Han et al ldquoPI3-KAkt and ERK pathways acti-vated by VEGF play opposite roles in MPP+-induced neuronalapoptosisrdquoNeurochemistry International vol 59 no 6 pp 945ndash953 2011

[21] Y Du K R Bales R C Dodel et al ldquoActivation of a caspase3-related cysteine protease is required for glutamate-mediatedapoptosis of cultured cerebellar granule neuronsrdquoProceedings ofthe National Academy of Sciences of the United States of Americavol 94 no 21 pp 11657ndash11662 1997

[22] S Gonzalez-Reyes M Orozco-Ibarra S Guzman-Beltran EMolina-Jijon L Massieu and J Pedraza-Chaverri ldquoNeuropro-tective role of heme-oxygenase 1 againts iodoacetate-inducedtoxicity in rat cerebellar granule neurons role of bilirubinrdquo FreeRadical Research vol 43 no 3 pp 214ndash223 2009

[23] L M Reyes-Fermın S Gonzalez-Reyes N G Tarco-AlvarezM Hernandez-Nava M Orozco-Ibarra and J Pedraza-Cha-verri ldquoNeuroprotective effect of 120572-mangostin and curcuminagainst iodoacetate-induced cell deathrdquo Nutritional Neuro-science vol 15 no 5 pp 34ndash41 2012

[24] P M Bilimoria and A Bonni ldquoCultures of cerebellar granuleneuronsrdquo Cold Spring Harbor Protocols vol 3 no 12 2008

[25] Z J Zhang L C V Cheang M W Wang and S M-Y LeeldquoQuercetin exerts a neuroprotective effect through inhibition ofthe iNOSNO system and pro-inflammation gene expression inPC12 cells and in zebrafishrdquo International Journal of MolecularMedicine vol 27 no 2 pp 195ndash203 2011

[26] E Auriel and N M Bornstein ldquoNeuroprotection in acuteischemic strokemdashcurrent statusrdquo Journal of Cellular and Molec-ular Medicine vol 14 no 9 pp 2200ndash2202 2010

[27] Y SongM Li J-C Li and E-QWei ldquoEdaravone protects PC12cells from ischemic-like injury via attenuating the damage tomitochondriardquo Journal of Zhejiang University Science B vol 7no 9 pp 749ndash756 2006

[28] NXiong J XiongGKhare et al ldquoEdaravone guards dopamineneurons in a rotenone model for Parkinsonrsquos diseaserdquo PLoSONE vol 6 no 6 Article ID e20677 2011

[29] A Almeida M Delgado-Esteban J P Bolanos and J MMedina ldquoOxygen and glucose deprivation induces mitochon-drial dysfunction and oxidative stress in neurones but not inastrocytes in primary culturerdquo Journal of Neurochemistry vol81 no 2 pp 207ndash217 2002

[30] S Fujimoto H Katsuki T Kume S Kaneko and A AkaikeldquoMechanisms of oxygen glucose deprivation-induced gluta-mate release from cerebrocortical slice culturesrdquo NeuroscienceResearch vol 50 no 2 pp 179ndash187 2004

[31] Q Ai Y Jing R Jiang et al ldquoRotenone a mitochondrial res-piratory complex i inhibitor ameliorates lipopolysaccharideD-galactosamine-induced fulminant hepatitis in micerdquo Interna-tional Immunopharmacology vol 21 no 1 pp 200ndash207 2014

[32] S S Karuppagounder and R R Ratan ldquoHypoxia-induciblefactor prolyl hydroxylase inhibition robust new target oranother big bust for stroke therapeuticsrdquo Journal of CerebralBlood Flow ampMetabolism vol 32 no 7 pp 1347ndash1361 2012

[33] C Ikonomidou and A M Kaindl ldquoNeuronal death and oxida-tive stress in the developing brainrdquo Antioxidants and RedoxSignaling vol 14 no 8 pp 1535ndash1550 2011

[34] M Di Carlo D Giacomazza P Picone D Nuzzo and P LSan Biagio ldquoAre oxidative stress andmitochondrial dysfunctionthe key players in the neurodegenerative diseasesrdquo Free RadicalResearch vol 46 no 11 pp 1327ndash1338 2012

[35] Y-H Pan Y-C Wang L-M Zhang and S-R Duan ldquoProtec-tive effect of edaravone against PrP106-126mdashinduced PC12 celldeathrdquo Journal of Biochemical andMolecular Toxicology vol 24no 4 pp 235ndash241 2010

[36] G-L Zhang W-G Zhang Y Du et al ldquoEdaravone amelioratesoxidative damage associated with A12057325-35 treatment in PC12cellsrdquo Journal of Molecular Neuroscience vol 50 no 3 pp 494ndash503 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


stimulus inhibits the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase irreversibly and induces cell deathaccompaniedwith an increase in ROS productionmitochon-drial dysfunction and loss of ATP [15ndash17] These responsesare very similar to changes observed in animal models ofischemic stroke [18] Cerebellar granular neurons (CGNs)are primary rat neurons obtained from cerebellum whichcontain the largest homogeneous neuronal population in thebrain which have been widely used as an in vitro modelfor investigating the cellular and molecular mechanismsunderlying neuronal apoptosis in neurodegenerative disor-ders [19ndash21] Previously researchers have demonstrated thatIAA reduced cell viability via increasing ROS production inCGNs [22 23] It has yet to be determined whether EDA canattenuate IAA-induced neuronal death in vitro Therefore weherein used the in vitroCGNsmodel exposed to IAA to studythe neuronal protective effects of EDA and to explore thepossibility of its underlying mechanisms of action

2 Materials and Methods

21 Chemicals and Reagents EDA was purchased fromAladdin Reagent Co (Shanghai China) MTT fluores-cein diacetate (FDA) propidium iodide (PI) 2101584071015840-dichloro-fluorescein diacetate (DCFH-DA) DNase poly-L-lysine andcytosine-D-arabinofuranoside were obtained from Sigma-Aldrich (St Louis MO USA) Basal medium Eagle (BME)fetal bovine serum penicillin and streptomycin were pur-chased from Invitrogen (Carlsbad CA USA) Mitochondrialmembrane potential assay kit and caspase 3 activity assaykit were obtained from Beyotime (Shanghai China) Thecytotoxicity detection kit (LDH) was purchased from RocheDiagnostics (Mannheim Germany)

22 Primary Cultures of Cerebellar Granule Neurons andDrug Treatment CGNs were isolated from postnatal 8-day-old Sprague-Dawley rat pups (15ndash20 g) from the AnimalCare Facility of Sun Yat-sen University according to theprotocol described by Bilimoria and Bonni [24] The densityof viable cells in suspension was determined by cell countusing trypan blue and adjusted to 2 times 106 cellsmL Neuronswere seeded into 96-well pates or 12-well plates which wereprecoated with 50120583gmL poly-L-lysine and maintained in ahumidified incubator with 5 CO

2in air at 37∘C Twenty-

four hours after plating cytosine-D-arabinofuranoside (finalconcentration 10120583M) was added to the culture mediumto arrest the proliferating of nonneuronal cells All animalstudies were conducted according to guidelines of the Exper-imental Animal Care andUse Committee of JinanUniversityThe experimental protocols were approved by the EthicsCommittee for Animal Experiments of Jinan University

Unless otherwise stated on day 7 of in vitro culture CGNswere pretreated with EDA (3 10 and 30 120583M) for 2 h IAA(50120583M) was then added for incubation for another 4 h toinduce cell injury

23 Immunofluorescence Immunofluorescence staining with120573III-tubulin antibody was used to observe the morphology

changes of the neurite Briefly CGNswere washedwithHBSSand were fixed with 4 paraformaldehyde for 15min at 4∘CThe cells were then permeabilized with 01 Triton X-100for 5min The fixed neurons were incubated in 10 horseserum for 1 h to block nonspecific protein interactions Theprimary antibody (rabbit anti-mouse 120573III-tubulin antibody1 400 Abcam) was added and the cells were incubated atroom temperature for 3 h After washing the neurons wereincubated with the secondary antibody (goat anti-rabbit IgG-FITC 1 100 Invitrogen) for 30min at room temperatureAfter washing twice with HBSS the coverslips were mountedwith antiquenching mounting medium and the neuritesmorphology changes of neurons were visualized using afluorescence microscope at times400 magnification

24 MTT Assay The cell viability was assessed using theMTTassay according to conditions described previously [25]The absorbance at 570 nm was measured using a WallacVictor3Vmicroplate reader (PerkinElmerNetherlands) Cellviability was expressed as a percentage of the MTT reductionof control

25 LDH Release Assay The cytotoxicity inflicted to cellsby IAA was also assessed by the LDH release assay Deter-mination of total and released LDH activity was performedaccording to the instructions accompanying the cytotoxicitydetection kit (Roche) LDH releasedwas normalized to a totalLDH release and the results were shown as a percentage oftotal LDH activity

26 FDAPI Double Staining and Hoechst Staining Viableneurons were stained with fluorescein formed from FDAwhich is deesterified only by living cells PI can penetratecell membranes of dead cells to intercalate into double-stranded nucleic acids Briefly neurons were washed twicewith ice-cold PBS After incubation with 10120583gmL of FDAand 5 120583gmL of PI for 15min the neurons were examinedand photographed using a fluorescence microscope (NikonInstruments Inc Melville NY)

Chromatin condensation was detected by staining thecell nucleus with Hoechst 33342 The cultures were fixedwith 4 paraformaldehyde and washed with PBS beforestaining with Hoechst 33342 for 30min Thereafter cellmorphology was observed under a fluorescent microscope(Zeiss Oberkochen Germany) The percentage of apoptoticnuclei from five random fields in each well of differenttreatment groups was quantified and averaged

27 Measurement of Mitochondrial Membrane Potential(Δ120595119898) The dye JC-1 was used as a molecular probe to

measureΔ120595119898The staining procedurewas performed accord-

ing to the manufacturerrsquos instructions with minor modifi-cation Briefly CGNs were washed with HBSS and stainedwith 2120583M JC-1 for 10min Fluorescence intensity was mea-sured on a microplate reader using 488 nm excitation and529 nm590 nm dual emissions The mitochondrial accumu-lation of JC-1 is dependent on Δ120595

119898and is reflected by a shift

in 529 nm and 590 nm emissions Mitochondrial membrane






3 Results








119898in CGNs


119898in CGNs




4 Discussion





120573II

I-Tu

bulin

(a)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

Ctrl 25 50 100(120583M)

IAA for 4h

(b)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30

EDA


lowast


(c)

40

60

20

0

(120583M)Ctrl 3 10 30


EDA

LDH

rele

ase

lowastlowast

lowastlowast

lowast

(d)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30


lowastlowastlowast

lowastlowast

lowast

(e)



PIM

erge

FDA


(a)

Ctrl 3 10 300

20

40

60

80

EDA

IAA

Nec

rotic

cells

()

(120583M)


lowast

(b)




119898 However



Ctrl IAA


(a)

10

20

30

40

50

60

0

Cell

apop

tosis

()

(120583M)Ctrl 3 10 30

EDA

IAA

IAA

lowastlowast

lowastlowast

lowast

(b)


0

100

200

300

400

EDAIAA

(120583M)


lowast

Ctrl 3 10 30IAA

( o

f con

trol)

DCF

fluo

resc

ence

inte

nsity




0

20

40

60

80

100

120

EDA

IAA

(120583M)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Ctrl 3 10 30

JC-1

fluor

esce

nce i

nten

sity

ratio

( o

f con

trol)









Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)



5 Conclusion




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3 Results








119898in CGNs


119898in CGNs




4 Discussion





120573II

I-Tu

bulin

(a)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

Ctrl 25 50 100(120583M)

IAA for 4h

(b)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30

EDA


lowast


(c)

40

60

20

0

(120583M)Ctrl 3 10 30


EDA

LDH

rele

ase

lowastlowast

lowastlowast

lowast

(d)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30


lowastlowastlowast

lowastlowast

lowast

(e)



PIM

erge

FDA


(a)

Ctrl 3 10 300

20

40

60

80

EDA

IAA

Nec

rotic

cells

()

(120583M)


lowast

(b)




119898 However



Ctrl IAA


(a)

10

20

30

40

50

60

0

Cell

apop

tosis

()

(120583M)Ctrl 3 10 30

EDA

IAA

IAA

lowastlowast

lowastlowast

lowast

(b)


0

100

200

300

400

EDAIAA

(120583M)


lowast

Ctrl 3 10 30IAA

( o

f con

trol)

DCF

fluo

resc

ence

inte

nsity




0

20

40

60

80

100

120

EDA

IAA

(120583M)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Ctrl 3 10 30

JC-1

fluor

esce

nce i

nten

sity

ratio

( o

f con

trol)









Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)



5 Conclusion




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120573II

I-Tu

bulin

(a)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

Ctrl 25 50 100(120583M)

IAA for 4h

(b)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30

EDA


lowast


(c)

40

60

20

0

(120583M)Ctrl 3 10 30


EDA

LDH

rele

ase

lowastlowast

lowastlowast

lowast

(d)

120

80

40

0

Cel

l via

bilit

y (

of c

ontro

l)

(120583M)Ctrl 3 10 30


lowastlowastlowast

lowastlowast

lowast

(e)



PIM

erge

FDA


(a)

Ctrl 3 10 300

20

40

60

80

EDA

IAA

Nec

rotic

cells

()

(120583M)


lowast

(b)




119898 However



Ctrl IAA


(a)

10

20

30

40

50

60

0

Cell

apop

tosis

()

(120583M)Ctrl 3 10 30

EDA

IAA

IAA

lowastlowast

lowastlowast

lowast

(b)


0

100

200

300

400

EDAIAA

(120583M)


lowast

Ctrl 3 10 30IAA

( o

f con

trol)

DCF

fluo

resc

ence

inte

nsity




0

20

40

60

80

100

120

EDA

IAA

(120583M)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Ctrl 3 10 30

JC-1

fluor

esce

nce i

nten

sity

ratio

( o

f con

trol)









Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)



5 Conclusion




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PIM

erge

FDA


(a)

Ctrl 3 10 300

20

40

60

80

EDA

IAA

Nec

rotic

cells

()

(120583M)


lowast

(b)




119898 However



Ctrl IAA


(a)

10

20

30

40

50

60

0

Cell

apop

tosis

()

(120583M)Ctrl 3 10 30

EDA

IAA

IAA

lowastlowast

lowastlowast

lowast

(b)


0

100

200

300

400

EDAIAA

(120583M)


lowast

Ctrl 3 10 30IAA

( o

f con

trol)

DCF

fluo

resc

ence

inte

nsity




0

20

40

60

80

100

120

EDA

IAA

(120583M)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Ctrl 3 10 30

JC-1

fluor

esce

nce i

nten

sity

ratio

( o

f con

trol)









Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)



5 Conclusion




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctrl IAA


(a)

10

20

30

40

50

60

0

Cell

apop

tosis

()

(120583M)Ctrl 3 10 30

EDA

IAA

IAA

lowastlowast

lowastlowast

lowast

(b)


0

100

200

300

400

EDAIAA

(120583M)


lowast

Ctrl 3 10 30IAA

( o

f con

trol)

DCF

fluo

resc

ence

inte

nsity




0

20

40

60

80

100

120

EDA

IAA

(120583M)

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

Ctrl 3 10 30

JC-1

fluor

esce

nce i

nten

sity

ratio

( o

f con

trol)









Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)



5 Conclusion




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ctrl 3 10 300

20

40

60

80

100

120

EDA

IAA

IAA

(120583M)

lowastlowast

lowastlowast

lowastlowast

Casp

ase 3

activ

ity (

of I

AA

)



5 Conclusion




Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article protective effect of edaravone in primary...

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