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Astrogliosis in the brain of obese Zucker rat: A model of metabolic syndrome

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Astrogliosis in the brain of obese Zucker rat: A model of metabolic syndrome
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  NeuroscienceLetters 543 (2013) 136–141 ContentslistsavailableatSciVerseScienceDirect Neuroscience   Letters  j   o   urnalhomepage:www.elsevier.com/locate/neulet Astrogliosis   in   the   brain   of    obese   Zucker   rat:   A   model   of    metabolic   syndrome Daniele   Tomassoni a , ∗ ,Innocent   Ejike   Nwankwo b ,Maria   Gabriella   Gabrielli a ,Siddhartha   Bhatt c ,Abdul   Bari   Muhammad c ,   Mustafa   F.Lokhandwala c ,Seyed   Khosrow   Tayebati b ,Francesco   Amenta b a SchoolofBioscienceand   Biotechnology,Universityof    Camerino,62032Camerino,Italy b SchoolofMedicinalandHealthProducts,UniversityofCamerino,62032Camerino,Italy c HeartandKidneyInstitute,CollegeofPharmacy,Universityof    Houston,Houston,TX77204,USA h   ig   h   l   ig   h   t   s •  Metabolic   syndrome   isassociated   with   impaired   cognitivefunction. •  The   obese   Zucker   rat(OZR)representsamodelof    type2diabetes. •  Study   has   investigated   brainastrogliosis   inOZRscompared   with   their   littermate   lean   Zucker   rats(LZRs). •  Results   indicating   the   occurrence   of    brain   injury   accompaniedby   astrogliosis   inOZRs. •  OZRsmay   also   representa   model   for   assessing   the   influence   of    metabolic   syndrome   onbrain. a   r   t   i   c   le   in   f   o  Articlehistory: Received24January2013Receivedinrevisedform11March2013Accepted14March2013 Keywords: MetabolicsyndromeObeseZuckerratsBrainAstrogliosis a   b   s   t   ra   ct Metabolic   syndrome   (MetS)   is   a   disorder   characterizedprimarily   by   the   development   of    insulin   resistance.Insulinresistanceand   subsequent   hyperinsulinemia,   srcinating   from   abdominal   obesity,   increasestheriskof    cerebrovascular   and   cardiovasculardisease   andall-cause   mortality.Obesityis   probably   a   riskfactor   for   Alzheimer’s   diseaseand   vascular   dementia   and   isassociated   with   impaired   cognitive   function.TheobeseZucker   rat(OZR)represents   amodel   of    type2diabetes   exhibiting   amoderate   degree   of    arterialhypertensionand   of    increasedoxidative   stress.   To   clarify   the   possiblerelationships   between   MetS   andbrain   damage,   the   presentstudy   has   investigated   brainmicroanatomy   inOZRs   compared   with   theirlittermatecontrolslean   Zucker   rats   (LZRs).   Male   OZRs   and   LZRsof    12weeksof    agewere   used.   Their   brainwasprocessed   forimmunochemical   and   immunohistochemical   analysis   of    glialfibrillary   acidic   protein(GFAP).In   frontalandparietal   cortex   of    OZRsasignificant   increase   inthe   number   of    GFAP   immunoreactiveastrocyteswas   observed.   Similar   findings   were   foundinthehippocampus,   where   an   increased   numberof    GFAP   immunoreactive   astrocytesweredetected   inthe   CA1   and   CA3subfields   anddentate   gyrusof OZRs   compared   tothe   LZRs.   These   findings   indicatingthe   occurrence   of    braininjuryaccompanied   byastrogliosisinOZRs   suggest   that   these   rats,   developed   as   an   animal   model   of    type2diabetes,   may   alsorepresent   amodel   for   assessing   the   influence   of    MetS   onbrain.   Theidentification   of    neurodegenerativechangesinOZRsmay   represent   the   first   step   for   better   characterizing   neuronalinvolvement   inthis   modelof    MetS   and   possibletreatment   for   countering   it. © 2013 Elsevier Ireland Ltd. All rights reserved. 1.Introduction Metabolicsyndrome(MetS),alsoknownasdysmetabolicsyndrome,syndromeXor,in   somecases,asinsulinresis-tancesyndromeischaracterizedbyobesity,insulinresistance, ∗ Correspondingauthorat:SchoolofBioscienceandBiotechnology,Universityof Camerino,ViaGentileIIIda   Varano,62032Camerino(MC),Italy.Tel.:   +390737403320;fax:+390737403325. E-mailaddresses: daniele.tomassoni@unicam.it,daniele.tomassoni@gmail.com(D.Tomassoni). dyslipidemiaandhypertension.InMetStheoverallriskof    car-diovascularmorbidityandmortalityis   increased[25].MetS ischaracterizedbyacombinationof    abdominalobesity,ele-vatedbloodpressure,insulinresistanceorglucoseintoleranceandatherogenicdyslipidemia.Thedyslipidemiaresultsfromraisedplasmatriglycerides(TGs),lowplasmaconcentrationsof high-densitylipoprotein(HDL)-cholesterol,increasedremnantlipoproteins,elevatedapolipoproteinB,smalllow-densitylipopro-tein(LDL)andsmallHDLparticles[11,21].   LargeclinicalstudieshaveconsideredMetSanditsindividualcomponentsa   causeof increasedcerebrovasculardisease,coronaryheartdisease,andall-causemortality[18].Inductionof    neuroinflammation,increased 0304-3940/$–seefrontmatter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.03.025  D.Tomassonietal./    NeuroscienceLetters 543 (2013) 136–141 137 productionoffreeradicals,alterationsinneurotrophicfactorsandreductionofinsulintransportintothebrainhavebeenreportedinpatientswithMetS[10,15].ObesityhasbeensuggestedasariskfactorforAlzheimer’sdis-easeandvasculardementiaandhasbeenassociatedwithpoorercognitiveperformanceinpopulation-basedinvestigations.More-over,combinationofobesityandarterialhypertensioncouldimpairperformanceacrossvariouscognitivedomains.Hypertensionitself hasalsobeenassociatedwithcognitivedeclineaswellaswithstrokeanddementia[30]andthetreatmentofhypertensionpro- tectsagainstdementia,especiallyin   theelderly.Hyperlipidemiahasbeensuggestedtoincreasetheriskofdementia,althoughthereisnoagreementof    literatureonit[9].ElevatedHDL-cholesterol levelshavebeenassociatedwitha   significantlydecreasedriskof dementia[17].Thereis   alsoevidenceof    anassociationbetweenTGslevelsandanincreaseintheriskof    dementia.Type2diabetesmellitus(T2DM)comprisesa   strongriskfactorforcognitivedecline,dementiaandAlzheimer’sdisease[18].TheobeseZuckerrats(OZRs),witha   mutationinleptinrecep-torsrepresenta   modelof    T2DMexhibitinga   moderatedegreeofarterialhypertensionandincreasedoxidativestress.OZRswerecharacterizedbythesimultaneousoccurrenceof    obesity,hyperglycemia,hyperinsulinemia,hyperlipidemiaandmoderatehypertension,similartoMetS[1].   Itis   thoughtthatOZRscorre-spondtoearly-stagecardiovascularcomplicationsassociatedwithmetabolicsyndrome.Thepurposeof    thepresentstudywastoinvestigatebrainmicroanatomyinOZRscomparedwiththeirlit-termateleancontrolsZuckerrats(LZRs)to   clarifythepossiblerelationshipsbetweenmetabolicsyndromeandbraininjury. 2.Materialsandmethods  2.1.Animalsandtissuetreatment  MaleobeseZuckerratsandthelittermateLZRsaged12weekswerepurchasedfromHarlan(Indianapolis,IN).TheanimalswerekeptintheUniversity’sAnimalCareFacilityandanimaluseprotocolswereapprovedbyInstitution’sAnimalCareandUseCom-mittee.Ratswerefastedovernightpriortothedayof    experiment.Onthedayofexperiments,animalswereanesthetizedwithInactin(100mg/kgbodywt.,i.p.)andbloodsampleswerecollectedfromthecarotidartery.Inthebloodsampleglucoseandlipidlevelsweremeasuredwithautomatedglucoseanalyzer(AccuCheck)andtriglyceridereader(BioScanner2000,PolymerTechnologySystemInc.,Indianapolis,IN),respectively.Insulinlevelwasdeterminedbyusinga   radioim-munoassaykitandmanufacturer’sprotocol(LincoResearch,St.Charles,MO).Thiobarbituricacidreactivesubstances(TBARS)were Fig.1. PanelA:   Valuesof    glucose,insulin,totalcholesterolandtriglyceridesinplasmaof    obeseZuckerrats(OZRs)andleanZuckerrats(LZRs).PanelB:TBARSlevelsintheplasmaofOZRscomparedtoLZRs.Eachvalueisthemean ± SEMofthesingle   animal.*  p   <   0.05vs.LZRs. measuredusingcommercialkits(Cayman,ChemicalCompany,AnnArbor,MI,   USACat.No.10009055).Ratswerethendecapitatedandthebrainwas   dissectedoutanddividedintotwohalvesbycuttingtheinterhemisphericscissure.Theareaof    frontalcortexandhippocampusoftherighthemi-spherewereisolatedandimmediatelyfrozenindryiceandkeptat − 80 ◦ Cuntiluseforperformingimmunochemicalanalysis.Lefthemispherewas   fixedin4%   bufferedformalinsolution,frozenorembeddedinparaffin.Groupsof    fiveserialconsecutive12  mthicksectionswereusedformorphometricanalysisandimmunohisto-chemistryasdetailedbelow.  2.2.WesternBlotanalysis Samples(0.1 ±   0.02g)   offrontalcortexandhippocampuswerehomogenizedin   a   MixerMillMM300(Qiagen,Hilden,Germany)with800  l   of    lysisbuffer[28].   Aftertwocentrifugationsat Fig.2. Westernblotof    glialfibrillaryacidicprotein(laneA)and  -actinusedasinternalstandard(laneB)inpreparationsof    frontalcortex(1and2)   andhippocampus(3and4).   Theresultsofdensitometricanalysis(panelC)areexpressedasopticaldensityvalues.Eachvalueis   the   mean   ± SEMoffive   differentindependentexperiments.1and3:LZRs;   2and4:OZRs*  p <0.05vs.LZRs.  138  D.Tomassonietal./NeuroscienceLetters 543 (2013) 136–141 Fig.3. Sectionsofthefrontalcortexprocessedfortheimmunohistochemicaldemonstrationof    glialfibrillaryacidicprotein(GFAP)incontrolleanZuckerrats(AandB)andobese   Zuckerrats(C   andD).IV:zonefourthof    frontalcortexcorrespondingto   VIlayer;Calibrationbar:AandC:50  m;   BandD:25    m. 13,000rpm(10min   at4 ◦ C)aliquotsof    thesupernatantwereusedforproteinassayusinga   BIO-RADproteinassay(BIO-RAD,Munich,Germany).Equalamountsofprotein(40  g)wereseparatedby8%sodiumdodecylsulphatepolyacrylamidegel   electrophoresisandtransferred.Transblottedmembraneswereincubatedwithapolyclonalanti-GFAP(Mousemonoclonal,Millipore ® ,   USA,Cat.No.MAB3402)diluted1:1000,in   PBS0.1M,   BSA(1%)andTween-20(0.05%).ImmunochemistryproductwasvisualizedusingasHRP-conjugatedantibody(goatanti-mouseIgGCat.No.A0168,SigmaChemicalCo.,St.Louis,MO)   followedbyachemiluminescencedetectionsystem(LiteAblot ® plus,Cat.EMP011005,Euroclone,Italy)withcomputer-drivendensitometry.To   normalizeproteinloadings,membraneswerestrippedandincubatedwithamono-clonalanti-  -actinantibody(cloneAC-74;SigmaChemicalCo.,St.Louis,MO).BandintensitiesweremeasuredbydensitometrywithanIAS2000imageanalyzer(Biosystem,Rome,Italy).DensitometryvaluesforGFAPwerenormalizedtotherespectiveactinintensityforeachsample.  2.3.Immunohistochemistry Consecutivesections(12  m)wereprocessedforimmunohis-tochemicaldetectionofdifferentproteinlocalization.Thefirstsectionofeachgroupoffiveconsecutivesectionsof    thelefthemi-spherewasstainedwithNissl’smethod(cresylviolet1.5%)toverifymicroanatomicaldetails;thesecondandfourthsectionswereusedfortheimmunohistochemicaldetectionof    GFAPimmunoreactivitydiluted1:500with0.3%PBS-TritonX-100,whereasthefifthonewasusedforcontrol.Aftertheincubationwithspecificbiotinilatedsecondaryantibodytheproductofimmunoreactionwas   revealedbyindirectavidin–biotinimmunohistochemistryusingthe3,3-diaminobenzidineasa   chromogen.Sectionswereviewedunderalightmicroscopeata   finalmagnificationof    200 × .Viaa   TVconnec-tion,imagesweretransferredfromthemicroscopetothescreenofanIAS2000imageanalyzerandusedforassessingtheintensityofimmunereaction.Thenumberof    astrocyteswasreferredtothevolumeof    correspondingbrainareas.For   furtherdetailsonimageanalysisof    GFAPimmunohistochemistry,seeTomassonietal.[28].Analysisof    parenchymalastrocytes,whicharemorediffusedthantheperivascularones[28]   was,centeredonfrontalcortex,parietalcortexandhippocampuscellelements.Perivascularastro-cyteanalysiswasextendedtothewholebrain.  2.4.Dataanalysis Valuesforindividualanimalswithinthegroupsinvestigatedaremeansofmeasurementsof    theparametersconsideredandthenor-mal   distributionof    datawasassessedbytheKolgomorov–Smirnovtest.Groupmeanswerethenderivedfromindividualmeans.Statis-ticalanalysiswasperformedbyanalysisof    variance(ANOVA).Thesignificanceof    differencesbetweenmeanswasassessedbyDun-can’smultiplerangetest,taking  p <   0.05astheminimumlevelof significance. 3.Results Thevaluesof    glucose,insulin,totalcholesterolandtriglycerideswerehigherinOZRscomparedtotheleanLZRs(Fig.1,   panelA)indicatinga   conditionof    dismetabolismsimilarlyto   MetS.TBARSlevelsincreasedin   theplasmaof    OZRscomparedtoLZRs(Fig.1,panelB)indicatinganincreaseoftheoxidativestressinthisanimalmodel.Immunoblotsof    frontalcortexandhippocampusforGFAPwereboundtoa   singlebandof    approximately50kDa(Fig.2).Expo- sureof    membranestonon-immunemouseIgGsdidnotcausethedevelopmentofbandsof    immunoreactivity(datanotshown).Themigrationpatternofimmunoreactionswassimilarinthetwo   ani-mal   groupsinvestigated(Fig.2).Theintensityof    thebandof    GFAP,normalizedforthecorresponding  -actinexpression,increasedinOZRsratscomparedto   LZRsratsbothin   thefrontalcortexandinthehippocampus(Fig.2AandC).  D.Tomassonietal./    NeuroscienceLetters 543 (2013) 136–141 139 Fig.4. SectionsofCA1subfield(A,B,E,andF)anddentategyrus(C,D,G,andH)ofthehippocampusprocessedfortheimmunohistochemicaldemonstrationof    glialfibrillaryacidic   protein(GFAP)of    controlleanZuckerrats(A–D)andobeseZuckerrats(E–H).P:Pyramidalneurons;O:stratumoriens;R:stratumradiatum;M:   molecularlayer;G:granule   neurons.Calibrationbar:   A,C,E,andG:50  m;   B,   D,F,andH:   25    m. Inthefrontalcortexandintheparietalcortexof    OZRsa   signifi-cantincreasein   thenumberofGFAPimmunoreactiveastrocyteswasobserved(Table1andFig.3).Similarfindingswerefound inthehippocampus.Inthisarea,anincreasednumberof    GFAPimmunoreactiveastrocytesindifferentsubfieldsanddentategyrusofOZRswasfound(Table1andFig.4A–D)comparedtoLZRs (Fig.4D–H). Forouranalysis,immunoreactiveastrocytesweredividedinhyper-reactiveastrocytes(H/R)andhypertrophic/hyper-immunoreactiveastrocytes(H/H).H/Rastrocytesweredefinedthosedisplayinga   higherdegreeof    immunoreactionincellbodyandmaincellularprocesses.H/Hastrocytesweredefinedthoselargerinsize,displayinga   higherintensityof    immunoreactionandwithahighernumberandlengthofcellularprocesses.InLZRsastrocytes,generallynotpresentchangesin   size,shapeandnum-berof    arborizationsandonlyfewhypertrophicelementswereobserved(Figs.3and4).InOZRsthepresenceof    H/RandH/Hele-mentswas   a   commonfeatureinallsections.Inthefrontalcortexclustersof    H/RandH/Helementswereobservedin   thezoneIVnearthecorpuscallosumwherethenumberofastrocytesandthelengthofcellularprocessedwerehighercomparedtoLZRs(Fig.3DandE).   Intheparietalcortex(zoneIV)of    OZRs,anincreaseofH/R andH/Helementswas   noticeablecomparedtoLZRs(Table1).In thewhitematterof    thecorpuscallosumH/RandH/Hcellswereobserved(Fig.5).Inthecorpuscallosum,asshowninFig.5,   den-sitometricanalysisof    meanimmunoreactionareaofastrocytesdidnot   revealsignificantdifferencesbetweenLZRsandOZRs(datanotshown).  140  D.Tomassonietal./NeuroscienceLetters 543 (2013) 136–141 Fig.5. Sectionsofwhitematterofcorpuscallosum(A   andC)   andfimbriafornix(BandD)processedfortheimmunohistochemicaldemonstrationof    glialfibrillaryacidicprotein(GFAP)incontrolleanZuckerrats(AandB)andobeseZuckerrats(CandD).LV:   lateralventricle.Calibrationbar:25    m. Inthehippocampusof    OZRs,a   highernumberof    H/HelementswasfoundcomparedtoLZRs(Fig.4).Astrocytesof    OZRsdisplayedamarkedGFAPimmunoreactionin   thecellbodyandanelevatednumberofarborizations.Similarlytothecorpuscallosum,inthefimbriafornixofLZRshypertrophicelementscharacterizedbyH/R andH/Hcellswereobserved(Fig.5C).Nosignificantdifference wasfoundinthefimbriafornixastrocytesof    OZRscomparedtoLZRs(Fig.5).Themeanof    immunoreactionareaof    astrocyteswasdeterminedas112.3 ±   5.8and119.7 ±   5.3  m 2 forLZRsandOZRsrespectively.Ingeneral,thenumberof    perivascularastrocyteprofileswaslowerthanthenumberof    parenchymalastrocytes.Perivascularastrocytesanalysisinthewholebraindid   notrevealsignificantdifferencesbetweentwoanimalgroupsstudied.Themeanof   Table1 Numberandsizeof    GFAP-immunoreactiveastrocytesinthefrontalcortexandsub-fieldsofhippocampusofleanandobeseZuckerrats.LZRs( N  =6)   OZRs( N    =   6) Frontalcortex Astrocytesnumber(10 2 /mm 3 )19.1 ± 2.225.7 ± 1.9 a Meanimmuno-reactionarea(  m 2 )72.5 ± 3.898.2 ± 5.4 a Parietalcortex Astrocytesnumber(10 2 /mm 3 )21.2 ± 3.728.2 ± 2.5 a Meanimmuno-reactionarea(  m 2 )81.9 ± 3.1109.7 ± 6.4 a HippocampusCA1subfiled Astrocytesnumber(10 3 /mm 3 )35.7 ± 1.865.1 ± 2.7 a Meanimmuno-reactionarea(  m 2 )85.8 ± 4.0123.1 ± 8.2 a HippocampusCA3subfiled Astrocytesnumber(10 3 /mm 3 )42.9 ± 4.471.5 ± 2.7 a Meanimmuno-reactionarea(  m 2 )83.4 ± 3.5101.2 ± 3.6 a Dentategyrus Astrocytesnumber(10 3 /mm 3 )32.1 ± 3.449.1 ± 2.7 a Meanimmuno-reactionarea(  m 2 )87.6 ± 5.5105.2 ± 8.6 a LZRs,leanZuckerrats;OZRs,obeseZuckerrats.Dataarethemean ± S.E.M. a  p   <0.05vs.LZRs. immunoreactionareavalueswere89.7 ± 3.8and91.3 ± 5.2    m 2 forLZRsandOZRsrespectively. 4.Discussion OZRsweredevelopedasanimalmodelofT2DMandwerelargelyusedforinvestigatingmechanismsandpathophysiologyof    thedis-easeincludingpharmacologicaltreatmentof    it.Moreover,OZRswereconsideredamodelforassessingtheinfluenceofMetSinthebrainandthecorrelationwithdegenerativephenomenain   obesity[1,20,23].Astrocytesreacttotissueinjurybyincreasingtheexpressionof    GFAP,anintermediatefilamentproteinexpressedbynumerousastrocytes[8,24].ThispropertymakesGFAPimmunohistochem- istryausefulmarkerof    astrogliosis,a   phenomenonoccurringinbrainsundergoingneuronalloss,vascularandmicrovascularchanges[3,5].Anincreasedexpressionof    brainGFAPandof    GFAPmRNAwasreportedinassociationwithaging[6,14,19,24]ordis- ease[6,7,22].Astrogliosisin   brainofspontaneouslyhypertensiverats(SHR)andinstreptozotocin-treatedratswerethetwopatholo-giesexacerbatingthisphenomenon,indicatingtheoccurrenceof braininjuryin   hypertensionanddiabetes.ThefindingsofincreasedGFAPexpressionin   OZRssupportandextendourpreviousdataof    theoccurrenceof    astrogliosisinthemodelof    diabetesassociatedto   hypertensionrepresentedbySHR withdiabetesinducedbyinjectionofstreptozotocin[27].   Glialcellsrapidlychangetheirstructureandfunctionin   responsetoseveralfactors,suchaslossorimpairmentof    nervecells,changesof    neu-ronalmetabolicactivityandmodificationsof    synapticplasticity[29].   Astrocytescontributeto   thestabilityof    neuronalmicroenvi-ronment[12,13].   Increasedexpressionof    immunoreactivityof    theastrocytecytoskeletalproteinGFAPis   consideredasanindicatorof braininjury[13,26].   OurevaluationofGFAPchangesin   thefrontalcortex,parietalcortexandhippocampusof    OZRsincludedquanti-tativeWesternblotanalysisandimmunohistochemistryassociatedto   densitometricevaluation.Thisanalysishasshownin   12-week-oldOZRsanumericalincreaseofGFAP-immunoreactiveastrocytes,
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