Seite - 68 - in Advances in Neuroimmunology

BrainSci. 2016,6, 16 buffer/50mgof tissue; pH= 7.4), then tumor necrosis factor-α (TNF-α; Invitrogen, #KRC3011C; Camarillo, CA, USA) and interleukin-10 (IL-10; Invitrogen, #KRC0101) cytokine protein was determinedviaELISAaccordingto themanufacturer’s instructions. These twocytokineswereusedto assessprooranti-inflammatorymicroglia, respectively [43]. Brainderivedneurotrophic factor (BDNF) wasmeasuredin thehippocampus(Millipore,#CYT306;Billerica,MA,USA)as thehippocampus is moresusceptible toalcohol-inducedBDNFdysregulation [55,56]. All samplesandstandardswere runinduplicate.Absorbancewasmeasuredat450nmonaDXT880MultimodeDetectorplate reader (BeckmanCoulter; Brea, CA,USA).Cytokine concentrationswerenormalized to the total protein contentasdeterminedbyaPierceBCAProteinAssayKit (ThermoScientific;Rockford, IL,USA)and reportedaspg/mgof totalprotein˘SEM. 2.6. StatisticalAnalyses DatawereanalyzedandgraphedusingPrism(version5.04,GraphPadSoftware, Inc. La Jolla, CA,USA).Effectswereconsideredsignificantlydifferent ifp<0.05. Behavioral scoreswereanalyzed with aKruskal-Wallis test. All other analyses used aone-wayANOVAwith post-hocTukey’s test to compare groups if an effect of treatmentwas observed. Where appropriate, each regionof the hippocampusorentorhinal cortexwasconsidered independentand thereforeanalyzedseparately. Correlationswereconductedtoexaminetherelationshipofmicroglialmarkersofactivationandthe animalmodeldataaswellasmicroglialactivationandcytokineconcentration.Correlationswereonly runwithin theCon/EtOHorEtOH/EtOHgroupifpost-hocanalysesshowedasignificantdifference to controlgroups. Spearmananalyseswereusedfor intoxicationbehaviorscores (nonparametric),while Pearson’sanalyseswereusedforallother factors (parametric). 3.Results 3.1.AnimalTreatmentData Foranimalmodeldata, eachbingeperiodwasanalyzedindependently. Forexample,BECsfrom Binge1andBinge2fortheEtOH/EtOHgroupwereanalyzedseparately.Nodifferencesweredetected betweenanygroups ineither intoxicationscore (H(3)=5.60,p=0.07;grandmean=1.6˘0.1)or in BECs (F(2,24) = 0.78, p=0.32; grandmean=399.8˘ 12.4mg/dL) as shown inTable 2. However, one-wayANOVArevealeddifferences in theaveragedoseperday(F(2,24)=4.235,p=0.03).Apost-hoc Tukey’s test indicatedthatethanoldosesofBinge2 in theEtOH/EtOHratsweresignificantlyhigher thanethanoldosesof thesinglebinge(Con/EtOH)rats (Table2). Bodyweightswerealsoassessedto determinewhetherrestrictedcaloric intakeaffectedmicrogliaactivation[57,58].One-wayANOVA indicated that treatment affected weight change (F(2,24) = 4.235, p = 0.03) (Table 2). A post-hoc Tukey’s test showedthat theweightchangedifferedbetweenallof the liquiddietgroups(Con/Con, Con/EtOH,andEtOH/EtOH)comparedwith thead libitumgroup.Therewasasignificanteffectof receivingethanolonweight losscomparedwith theCon/Congroup,butnodifferencebetweenthe Con/EtOHandEtOH/EtOHgroupswasobserved(Table3). Table2.AlcoholModelData. Group IntoxicationBehavior (0–5Scale) Dose(g/kg/day) BEC(mg/dL) Con/EtOH(15thDay) 1.8˘0.1 9.6˘0.2 422.2˘21.1 EtOH/EtOHBinge1(4thDay) 1.7˘0.1 9.9˘0.4 378.7˘17.7 EtOH/EtOHBinge2(15thDay) 1.3˘0.2 11.0˘0.5 # 390.3˘24.0 # p<0.05comparedtoCon/EtOH. 68