DOC

Establishment of a mechanical injury model of rat hippocampal neurons in vitro

By Kristen Diaz,2014-02-18 20:06
7 views 0
Establishment of a mechanical injury model of rat hippocampal neurons in vitro

Establishment of a mechanical injury model

    of rat hippocampal neurons in vitro

    ChineseJournalofTraumatology2006:9(J):2933?29?

    Establishmentofamechanicalinjurymodelofrat hippocampalneuronsinvitro

    rANGXiaofeng杨小锋,CAOFei曹飞,PANDe-sheng潘德生,LIUWeiguo刘伟

    ,HUWeiwei胡未伟,

    ZHENGXiu-jue郑秀珏,ZHAOXuequn赵学群andLtJShiting吕世亭

    objective:ToestablishasireIpie,reproducible,and practicalmechanicalinjurymodelofhippocampalneurons ofSprague-Dawleyratsinvitro.

    Methods:Hippocampalneuronsisolatedfrom

    1-2.dayoldratswereculturedinvitro.Mild,moderateand severemechanicalinjuriesweredeliveredtotheneuronsby syringeneedletearing,respectively.Thecontrolneurons weretreatedidenticallywiththeexceptionoftrauma.CeIl damagewasassessedbymeasuringthePropidiumIodide (PI)uptakingatdifferenttimepoints(0.5,1,6,12and 24hours)afterinjury.Theconcentrationofneuronspecific enolasewasalsomeasuredatsometimepoints.

    Results:Pathologicalexaminationshowedthat degeneration.degradationandnecrosisoccurredinthe injuredculturedneurons.Comparedwiththecontrol raumaticbraininjuryisoneofthemajorcauses

    ofmortalityandmorbidity.Manyeffortshave

    beenmadetoevaluatetherecoverypotentialafter traumaticbraininjuryandtoidentifythepatientsunder

theriskofdevelopinglongtermneurologicaldisorders.

    Duringthepastdecade,modelsoftraumaticbrain injurywithfinereproducibilityandcontrollability gainedincreasinginterestsbothinexperimentaland clinicalneurotraumatology.Recently,moreandmore modelsofmechanicalinjurytobraincellsinvitrohave beenestablishedandstudied.Somedatademonstrated thatthehippocampusplaysacriticalroleinlearning, cognitionandmemory.'Studiesalsoindicatedthat thehippocampusisuniquelyvulnerabletohypoxia, ischemiaandtraumaticbraininjury...Theaimofthis DepartmentofNeurosurgery,SecondAffiliatedHospital, MedicalCollegeofZhejiangUniversity,Hangzhou310009, China(YangXF,CaoF,PanDS,LiuWG,HuWW,Zheng XJ,ZhaoXQandLaST)

    $Correspondingauthor:Tel:13606504517,Email:

    yangxf@zju.edu.cn

    group,theratioofPI-positivecellsintheinjuredgroups increasedsignificantlyaRer30minutesofinjury(P< 0.05).Moreseverethedamagewas,morePI-positive neuronsweredetected.Comparedwiththecontrolgroup, theconcentrationofneuronspecificenolaseintheinjured cultureincreasedsignificantlyafterlhourofinjury(P< 0.05).

    Conclusions:Theestablishedmodelofhippocampal neuroninjuryinvitrocanberepeatedeasilyandcan simulatethedamagemechanismoftraumaticbraininjury, whichcanbeusedinthefutureresearchoftraumaticbrain injury.'

    Kwords:Neurons;Culture;Rats

ChinJTraumatol2006:9():2933

    studyistoestablishamechanicalinjurymodelofrat hippocampalneuronsinvitro.

    METHoDS

    In.iurydevice

    Theinjurydevicemadeofstainlesssteelwas designedbyourselvesonthebasisoftheprocedures describedbyFadeneta1.Briefly.itconsistedofa baseboard.asleeveconduitandtworodswith graduatedscale.Thefirstrod(RodA)wasfixedoff thebaseboardhorizontally,andthesecondrod(Rod B)wasplacedvertically.RodBcouldslide perpendicularlytoRodA.Thesleeveconduitcould slidealongRodBandcouldbefastenedtoRodBat anyexpectedpositionbyasleevenut.Asyringe needlewasfastenedtothesleeveconduit.Therefore. bychangingthedistanceanddepthinwhichtheneedle moved,theextentoftheinjurycouldberegulated precisely(Fig.1).

    ChineseJournalofTraumatology2006;9(J):29-33 Fig.1.Thedeviceisusedtodeliverinjury Hippocampaineuronsculturedinvitro

    Weused1-2-dayoldSprague-Dawley(SD)rats (providedbythe.ExperimentalAnimalCenterof MedicalCollegeofZhejiangUniversity,Hangzhou, ZhejiangProvince,China)forthisexperiment.In brief,thehippocampaltissuesoftheratswere dissociatedandminced,and5ml0.25%trypsinin Hanksbalancedsaltsolution(HBSS)wasadded.

    Thenthetissueswereincubatedat37oCfor20 minutes,followedbycentrifugationat(1000r/min) 4~Cfor2minutes.Thesupernatantwasremovedand thesedimentwaswashedrepeatedlyinordertoremove trypsin.Thecellswereresuspendedwithpipettetip andplatedonto110plasticcultureplates(35mmin diameter1containingpoly-l-lysine-coatedcoverslipsat adensityofl000cells/cm.and10mlEagles's minimumessentialmedium(MEM)culturemedium containing10%horseserumwasaddedtoeachplate. 10no1/Lcytosinearabinosidewasalsoaddedfor inhibitingthedivisionofnon.neuronalcells.Thenthe culturewasmaintainediflanincubator(5%CO,, 37~C).Theculturemediumwaspartiallyreplaced onceaweek.

    Mechanicalinjurytohippocampaineurons Thehippocampalneuronswereinjured

    mechanicallyat7daysaftercultureinvitro.The cultureplatesweredividedrandomlyintoan experimentalgroup(n=75)andacontrolgroup (n=35).Theexperimentalgroupwasthendivided equallyintothreesubgroupsaccordingtotheextentof injury:mild,moderateandseveregroup.Inthemild group,asterile20-gaugeneedlewasdrawnevenly acrossthecelllayertoproduceatearof6mminlength byusingtheinjurydevicedescribedabove.Inthe moderategroup,twoparalleledtearsweremade.In theseveregroup,twotransversalandtwolongitudinal tearsweremade.Thecontrolgroupunderwentallthe samemanipulationexcepttrauma.

Neuronviabilityassay

    Cellinjurywasassessedinalltheexperimentsat differenttimepoints(0.5,1,6,12and24hours) afterinjurybystainingwithPropidiumIodide(PI).PI stainingsolutionwasaddedtotheplatesataworking concentrationof5g/m1.3minuteslater,themedium solutionwasreplacedbyMEM,andthentheculture wasplacedatroomtemperaturefor30minutes.Then thePI.1abeledcellswerecountedunderaninverted fluorescencemicroscope.Ifthecellwasdeador injuredandthecellmembranewasdamaged,P1would enterthecellandstainthenucleus.resultingina brightredfluorescence.Foreachplate, thedegreeof

    injurytoneuronswasevaluatedbytheratioofPI.

ChineseJournalofTraumatology2006;9(J):2933?3l?

    positivecellstothetotalcellscounted.'Thatisto say,thehighertheratiowas,themoreseverethe injurywas.

    Contentofneuron-specificenolase(NSE) NSEintheculturemediumwasmeasuredwith enzymelinkedimmunosorbentassay(ELISA)at differenttimepoints(0.5,1,6,12and24hours) afterinjury,justasdescribedpreviously. Statisticalanalysis

    UsingSPSS10.0softwarepackage,analysisof variance(ANOVA)followedbyStudent.Newman. KeulstestwasmadetocomparethePI.positiveratio andNSEconcentrationindifferentgroups.APvalue

    lessthan0.05wasconsideredstatisticallysignificant. RESULTS

    Morphologicalchangesofhippocampalneurons afterinjury

    Mechanicalinjurycouldinducedegenerationand deathoftheneurons.Within2.3minutesafter mechanicalinjury,manyneuronalcellbodiesneara teardevelopedpronouncedswellingandgranularity underaphasecontrastmicroscope.Withtimepassing by,theswellingoccultednotonlyintheneurons adjacenttothetear,butalsointhepart12mmaway

    fromthetear.Overthenext2024hours,many

    swollenneuronsfurtherdegenerated,andtheaxonal processesappearedlooseinsteadoftheirnormaltight andcabledappearance.Especiallyinthemoderateand severegroups,moreandmoreneuronsdisplayed membraneblebbingandlysis,cellularcontent leakage,cellshrinkage,chromatincondensation, nueleosomeformation,evennecrosis.Sometimesinthe mildinjurygroup,noobviouspathologicalchanges wereobservedexceptthe"beaded"lookofthe dendrites.

    PIstaining

    Inthecontrolgroup,theratioofPIpositivecells

    remainedconstantlylowatanygiventimepoint.At30 minutesafterinjury,thePIpositiveratiooftheinjury

    groupsincreasedsignificantly,comparedwiththatof thecontrolgroup.Withtheinjurydegreeincreasing, theratioofPIstainedcellsalsoincreased.From30 minutesto24hoursafterinjury,theratioofPI.positive

    cellsinanyinjurygroupwashigherthanthatofthe controlgroupatanytime(P<0.05).From1to24 hoursafterinjury,themoderateandsevereinjury groupsexhibitedasignificantincreaseinPIpositive

    ratiocomparedwiththatofthemildinjurygroup(P< 0.05).From1to12hours,thesevereinjurygroup showedasignificantlyhigherPIpositiveratiothanthat

    ofthemoderateinjurygroup(P<0.05,Table1). SEmeasurement

    NSElevelsweremeasuredintheculturemediaat differenttimepointsfrom30minutesto24hourswith ELlSAmethod.At30minutesafterinjury,compared withthatofthecontrolgroup,theNSElevelinany injurygroupdemonstratednosignificantdifference (P>0.05).After1hourofinjury,theNSElevel showedsignificantdifferencebetweentheinjurygroups andthecontrolgroup(P<0.05).After6hoursof injury,theNSEconcentrationinthemoderateand severeinjurygroupsincreasedsignificantlycompared withthatofthemildinjurygroup(P<0.05).The NSElevelinthesevereinjurygroupwassignificantly higherthanthatofthemoderateinjurygroupat6hours and12hoursafterinjury(P<0.05.Table2). DISCUSSION

    Instudiesoftraumaticbraininjury,amodelwith finereproducibilityandcontrollabilityisoften indispensable.Theapplicableinjurymodelsinclude experimentalanimalmodelsandcelliniurymodels culturedinvitro.Recently.researchershaveattached greatimportancetotheinvitromodelsbecausethey

    allowfortheprecisecontroloftheextracellular environment,easyandperhapsrepeatedaccesstothe cells,andlowerassociatedcosts.Themostoften employedinvitromodelsincludechemicalinjury models(e.g.,excitatoryaminoacid,NOandfree radicalinducedinjurymodels)andmechanicalinjury models(e.g.,stretchinduced,fluidpercussionand

    transectionmodels1.Chemicalinjurymodelshave oftenbeenappliedtosimulateneuronaldamage inducedbyhypoxiaandischemia.whilemechanical injurymodelsarecommonlyusedtoinvestigatethe mechanismoftraumaticbraininjuryinvitro,suchas structuralandpath0physiologicalchangesoftheinjured cells.Inastretch.inducedinjurymode1.cellsare culturedintheflexplatewithaelasticmembrane bottom,andthenareinjuredbyforoeofcompressed air,whichmakesthesilasticmembranedeformed

?

    32?ChineseJournalofTraumatology2006;9():2933

    rapidlyandtransiently.Theseverityofcellinjury dependsonthedegreeofdeformationorstretchofthe silasticmembrane.Fluidpercussioninjurymodelsare

    producedbyusingthestreamoffluidtoimpactcell cultureatapredeterminedvelocity.''''.

    Table1.RatioofPI?positivecellsatdifferenttimepointsafterinjury(%)

    $P<0.05comparedwiththecontrolgroup.$$P<0.05comparedwiththemildgroup.and

    $$$P<0.05comparedwiththemoderategroup Table2.ConcentrationofNSEatdifferenttimepointsafterinjury(ng/m1)

    $Jp<0.05comparedwiththecontrolgroup(t=2.84),$$P<0.05comparedwiththemildg

roup(t=2.58),and$$$P<0.05compared

    withthemoderategroup(t=4.15).

    Inthisstudy,weestablishedasimple.

    reproducibleandpracticalmodelofneuroninjuryin vitrothathascertainadvantages.Firstly.ithas uncomplicatedworkingprinciple,superior reproducibilityandcontrollability.Mechanicalcell injurycanbeachieved.At30minutesafterinjury,the PIpositiveratioofinjurygroupincreasedsignificantly. Withtheinjurydegreeincreased,theratioofPI. stainedcellsalsoincreased.Datafromourexperiments showedthattheresultsofthismodelwereconsistent withthepreviouslypublishedstudies.7,1',12Such

    experimentalresultsvalidatedthevalidityofthis mode1.Secondly.theactualmechanismoftraumatic braininiurycanbesimulatedatthecelllevel accurately.Andthirdly,differentinjurydegreesofthe neuronscanbeobtainedbyregulatingthedistanceand depthofneedlemoving.

    Inthisresearch,asyringeneedlefastenedtoa selfdevisedgadgetwasusedtotearthroughtheculture ofneurons.Theinjuryseverityofthecellswas assessedbystainingwithPI,whichwouldbindto nucleicacids(ithasaccesstothemthroughadamaged cellmembrane).Since30minutesafterinjury. neuronsappeareddegenerationandnecrosis.The numberofPIpositivecellswascloselyrelatedtothe injuryleve1.ThecytoplasmicenzymeNSE,whichis normallyf0undinthecytoplasmofneuronsandincells withneuroendocrinedifferentiation.willleaktothe

    extracellularspacethroughthedamagedmembrane whenthecellisseverelyiniured.Increased concentrationofNSEcanbedetectedinthe

    cerebrospinalfluidandtheperipheralbloodafter traumaticbraininjury.andisoftenrelatedtosevere neuropathologicaldysfunction.Earlierstudieshave revealedthatthechangeofNSEconcentrationinthe cerebrospinalfluidofbrain..injuredpatientswastime.. dependentandwasareliable,quantitative,and specificmarkerofneuronalinjury.,,'.Inour experiment,theconcentrationofNSEincreased immediatelyafterinjuryandreacheditspeakvalueat about6hoursaftermechanicalinjury.Furthermore.a significantcorrelationbetweenNSEreleaseandthe degreeofinjurywasobserved.

    Inthisstudy,we~cusedonestablishinga

    simple,reproducible,andpracticalmechanicalinjury modelofhippocampalneuronsinvitrothatcanbeused toinvestigatethepath0physi0l0gicalmechanismof traumaticiniuryatthecellularleve1.0urmodel appeamtoreproducesomecertainfeaturesoftraumatic braininjury,includingneuromorphologicaland neurochemicalabnormalities.Theinjurymodel describedherewillfacilitatefurtherresearcht0 investigateneuropathologicalchangesintraumaticbrain injuries,andtodeveloppharmacologicalprotective

ChineseJournalofTraumatology2006:9():2933?33?

    agentsagainsttheinjury

    REFERENCES

Report this document

For any questions or suggestions please email
cust-service@docsford.com