Glial cell linederived neurotrophic factor protect Parkinson model rat substantia nigra dopaminergi.docx
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GlialcelllinederivedneurotrophicfactorprotectParkinsonmodelratsubstantianigradopaminergi
Glialcellline-derivedneurotrophicfactorprotectParkinsonmodelratsubstantianigradopaminergicneuronsmechanismanalysis
Author:
DingYanxia,LIUHong-Mei,WangHongjun,WangYanjiang,highDianshuai
[Keywords]dopaminergicneuronsAbstract:
ObjectiveTostudytheglialcellline-derivedneurotrophicfactor(glialcellline-derivedneurotrophicfactor,GDNFprotectdopaminergic(dopamine,DAneuronsprocess,thecalcium-bindingproteinD28K(calbindin28K,CBandastrocytesthepossibleroleofinterstitialcells.Methodsrightstriatumofratsinjectionof6-hydroxydopamine(6-OHDA)PreparationofParkinson’sdisease(Parkinsondisease,PDmodelinjectionafterthe7thday,basedonbehavioranalysisscreeningmodelmouse,modelmicewererandomlydividedintothreegroups:
controlgroup,PBSgroup(therightsideofthesubstantianigrainjectionofPBSandGDNFgroup(rightsubstantianigrainjectionsofGDNFtheblankcontrolanimals,respectively,in6-OHDAinjectionafter7days,14dayssubstantianigra,21daysand28days,thePBSgroupandGDNFgroupanimalsinthefirst14daysafterthe6-OHDAinjection,respectively,21and28days,linesegmentscontinuouscoronalparaffin,tyrosinehydroxylase(tyrosinehydroxylase,TH,CBandglialfibrillaryacidicprotein(glialfibrousacidproteinofGFAPimmunohistochemistrystaining,respectivelyDAneurons,includingtheCBneuronsandastrocytes,lightmicroscopyandcellcount,statisticallyprocessingresults①THimmunohistochemicalstainingresults:
6-OHDAinjectionafter28days,theexpressionoftherightsideoftheGDNFgroupratsubstantianigraTHpositiveneuronswassignificantlymorethanthecontrolgroupandPBSgroup;②CBimmunohistochemicalstainingresults:
CBpositiveneuronssevendaysafterinjectionof6-OHDA,therightsideoftheblankcontrolgroupratssubstantianigracanstillbeobserved,afterthepointintimeofthecontrolgroupandthePBSgrouparenotobserved,therightbooksandGDNFratseachtimepointthesubstantianigrainthedetectionofCBpositiveneurons;3ofGFAPimmunohistochemistryresults:
blankcontrolgrouponthe7thdayaftertheinjectionof6-OHDAsmallamountofGFAPpositivecellscouldbeobservedafterinjection14daysexpressionofGFAPpositivecellsreachedapeak,andthengraduallyreduceto28daysaftertheinjectionhasbeenbasicallyundetectable;thePBSgroupGFAPexpressionconsistentperformanceofthenumberofpositivecellsintheblankcontrolgroup,andthetheGDNFgroupratsubstantianigraintheThedetectionofthepointintimecanbeobservedinalargenumberofreactivehyperplasiaofastrocytesconclusionCBand(orastrocytesmaybeinvolvedinGDNFtoprotectandpromotethesurvivalofPDmodelratsubstantianigraDAneurons.
Keywords:
dopaminergicneurons;glialcellline-derivedneurotrophicfactor;astrocytes;calcium-bindingproteinD28KAbstract:
ObjectiveToexplorethepossiblerolesofastrocytesandcalbindinD28K(CBintheprocessforglialcellline-derivedneurotrophicfactor(GDNFtoprotectdopaminergicneurons(DAneuronsinratsubstantianigra(SNagainst6-hydroxydopamine(6-OHDA)neurotoxicity.MethodsRatmodelofParkinsondisease(PD)wasestablishedbyster-eotaxicalinjectionof6-OHDAintotherightstriatum,withthequalifiedmodelsscreenedbypassingthebehavioraltests.Themodelratswererandomlydividedintothreegroups:
PDcontrol,PBStreatmentcontrolandGDNFtreatmentgroups,toundergovariedtreatments.Theanimalsweredecapitated1-4weeksafter6-OHDAinjectiontoproceedwiththehistologicstudyonthecontinuouscoronalsectionsoftheratmidbrainbymeansofimmunohistochemistryusingthaantibodiesagainsttyrosinehydroxy-lase(TH,CBandglialfibrillaryacidicprotein(GFAPtodetecttheDAneurons,CB-containingneuronsandastrocytesre-spectively.Results(1ThedensityofTHpositivecellswassignificantlyhigherintheGDNFtreatmentgroupthanthatinthecontrolgroups.(2TheCBpositiveneuronscouldonlybeseenonthe7thdayafter6-OHDAinjectioninthecontrolgroups,buttheypersistedintheGDNFtreatmentgroupthroughthewhole28-daycourseofexperiment.(3Inthetwocontrolgroups,theGFAPpositivecellsemergedonthe7thdayafter6-OHDAinjection,reachedtheclimaxstaininganddesityonthe14thday,begantodeclineonthe21thdayandalmostdisapearedonthe28thday.However,intheGDNFtreatmentgroup,thephe-nomenonofactivationofastrocytescouldbeevidencedthroughouttheexperimentalcourse.ConclusionCBand.orastrocytesmaybeinvolvedinthemechanismforGDNFtoprotectDAneuronsagainst6-OHDAneurotoxicity.
Keywords:
:
dopaminergicneuron;glialcellline-derivedneurotrophicfactor;astrocyte;calbindinD28k
Parkinson’sdisease(Parkinson,disease,PDcharacteristicpathologicalchangesindopaminergic(ofdopamineinthebrain,progressivedegenerationofDAneuronsdeath.Neuroprotectivetherapyiscommittedtoblockordelaytheprocessofneuronaldegenerationpromotetheremnantsofneuronsrestorationandrepair.retrospectiveanalysisofthebrainofPDpatientsshowconsiderablenumberofDAneuronsisstillinthesurvivalstatus[1],whichprovidesatheoreticalbasisfortheneuroprotectivetreatmentatthesubstantianigra.glialcellline-derivedneurotrophicfactor(glialcellline-derivedneurotrophicfactor,GD-NFsofaridentifiedinthemidbrainDAcanneuronsneurotrophicrolestrongestfactoroneofalargenumberofstudieshaveshownthat,GDNFcanpreventorreversePDprogressivedegenerationofthenigrostriatalDAsysteminanimalmodels[2],neuroprotectivetherapyforPDpreferredneurotrophicfactor.,however,thebodyGDNFishowtoprotectandpromotetheDAneuronssurvivedsofartostudyverylesscalcium-bindingproteinD28K(calbindinD28K,CBisacalcium-bindingproteinfamilymembers,canbecombinedwithcalciumionswithinthecell,thecellsfromthetoxiceffectsofhighcalciumproduced,thuscytoprotective3]Inthisstudy,usingimmunohistochemicalstaining,detectionofGDNFintheprotectionofDAneuronsprocess,CBandastrocytemarkerglialfibrillaryacidicprotein(glialfibrillaryadietwhichprotein,GFAPexpression,substantianigraDAexploreGDNFprotectionofneuronsprocess,theCBandthepossibleroleofastrocytes,soastofurtherexploretheGDNFprotectionmechanismofDAneuronsprovideatheoreticalbasis.
1Materialsandmethods
1.1AnimalsandreagentshealthyadultmaleSprague-Dawley(SDrats,weighing200to250g,providedbytheExperimentalAnimalCenterofXuzhouMedicalCollege.RecombinanthumanofGDNF,6-hydroxydopamine(6-hydroxydopamine,6-OHDA,miceKangDatheratTHmonoclonalantibody,mouseanti-ratCBmonoclonalantibody,rabbitanti-ratGFAPmonoclonalantibodyandABCkit(SigmaChemicalCo.;stereotaxicinstrument(Kopf,Japan;closedwithgoatserumstocksolution(BeijingZhongshan.
1.2stereotaxicinjectionintraperitonealinjectionofsodiumpentobarbital(50mg.kganesthesia,anesthesiaago,5~10minintraperitonealinjectionofatropine(breathinganimal0.1mg.kgtofacilitatesurgery.AccordingtoPaxinosandWatson(1986)Spectrumselectcoordinaterightstriatuminratsinjectionof6-OHDA:
6-OHDAsolutionwasformulatedinsterilesaline(containing0.02%ofascorbicacid,thefinalconcentrationof3g.Lof4μl.coordinates(mm:
P(anteriorfontanelle+1.6;L(nexttoopen1.5;V(subdural4.8and5.6.7daysafterinjection,basedonbehavioralsciencedetectionscreening50PDmodelratswererandomlydividedintothreegroups:
blankcontrolgroup(n=20,theexperimentalcontrolgroup(ipsilateralsubstantianigraattherightsideofthesubstantianigrainstereotacticinjection:
ofGDNFsolutiontosterileinjectionofPBS,n=15,andtheexperimentalgroup(ipsilateralsubstantianigrainjectionsofGDNF,n=15.the0.01mol.LPBSpreparation,itsfinalconcentration5mg.L,of2μl,experimentalcontrolgroupinjectedwiththesamevolumeofsterile0.01mol.LPBS.coordinates(mm:
P,-4.9;L,and1.8;V8.1with5μltheHamitonmicroinjectorextractliquidinjectionspeed0.5μl.min,Notecompletionoftheneedlefor5minand1mm.minspeedslowexit.
1.3Behaviordetection
1.3.1postureasymmetryChiangdescribedin[4],filedtherat-tailedratswerevacantintheexperimentalstagesideabout10cmtoobserveratheadorbodyrotationdirection,doit20times,whichisrotatedtotheleftTheshareoffrequencyastheevaluationindex.
1.3.2forelimbinitiationcannotbebasedonOlssonetal[5],andLind-neretal[6]describedthesecondhalfoftheratbodyholdup,andbodyweightinratsonlysupportedbythesideoftheforelimbs,record10swithinratforelimbwalkingtimes,andthenusethesamemethodstomeasuretheothersideoftheforelimbrightlimbasymmetryscoreastheevaluationindex,calculatedasfollows:
(numberofstepsontheright-thenumberofstepsontheleftbothsidesofthetotalnumberofsteps.
1.4drawnslicedblankcontrolanimalsafterthe6-OHDAinjection,respectivelyfirst7days,14days,21daysand28days,PBSgroupandGD-NFgroupsofanimals,respectively,14daysafterthe6-OHDAinjection,21daysand28day(eachtimepointn=5,4%anesthetizedbyintraperitonealinjectionofsodiumpentobarbital,ascendingaorticcannulationthroughtheleftventricle,4%paraformaldehydefixedtothebackoftheheadfixedinthesamefixativefor24hours.conventionalThedehydration,transparent,waxdippedandembedding.substantianigrasegmentcontinuouscoronalslices,slicethickness6μm,slicingpacketcollection.
1.5Immunohistochemicalstainin