Page - 66 - in Advances in Neuroimmunology

BrainSci. 2016,6, 16 accreditedvivariumwith a 12h light:dark cycle. Ratswere allowed to acclimate to thevivarium for twodays followedby three days of handling before any experimentation. Except during the bingeperiods,animalshadad libitum foodandwateraccess. Followingacclimation, ratsunderwent amodifiedversionof theMajchrowiczAUDmodelsimilar topreviouslypublishedreports [40–42]; however, animalsused in this studyunderwent theMajchrowicz4-dayparadigmtwice separated bysevendays.Ratsweredividedinto fourgroupsofcomparableweightsassummarizedinTable1. Briefly, ratsweregavaged intragastricallywith either ethanol (25%w/v) or control diet (isocaloric dextrose) inVanillaEnsurePlus® (AbbottLaboratories;Chicago, IL,USA)every8h. Initially, each rat in an ethanol group received 5g/kgof ethanol, but subsequent doseswere titratedusing the individual rat’sbehavioral intoxicationscoreonasix-point scale identical toprevious reports [40]. Control rats receivedanaverageof thevolumegivento theethanolgroup.All ratswere thengiven sevendaysof recoverywith ad libitumaccess to foodandwater. Aseven-dayrecoveryperiodwas chosenbecausemicroglial activation is elevated for aweekafter ethanol exposure [22], andseven daysallowedanimals torecover fromwithdrawalandregainbodymass lostduringthepriorbinge. Thus,on the11thday, theMajchrowiczbingemodelwasrepeatedwithrats receivingeitherethanolor controldiet (Table1).Aseparategrouphadad libitumaccess to foodandwater throughoutallperiods. Forallgroups,bodyweightswereassesseddailyduring thebingeprocedures. Thepercentdifference inweightat thestartandendof the15-daytreatmentperiodwascalculated. Table1.ExperimentalDesign. Group Binge1(4Days) Recovery(7Days) Binge2(4Days) Con/Con(n=10) ControlDiet ControlDiet Con/EtOH(n=11) ControlDiet Adlibitumchow EthanolDiet EtOH/EtOH(n=8) EthanolDiet EthanolDiet Adlibitum (n=4) N/A N/A 2.2. BloodEthanolConcentrationDetermination Todeterminebloodethanolconcentrations (BECs), tailbloodwascollectedninetyminutesafter the seventhsessionof ethanoldosingduringBinge1and/orat euthanasia (Binge2). Bloodswere centrifuged for 5minat 1800ˆg to separateplasma fromredbloodcells and immediately stored at´20 ˝C.BECsweredetermined fromsupernatant serumonanAM1AlcoholAnalyser (Analox; London,UK)calibratedagainsta300mg/dLexternal standard. Eachsamplewasrun in triplicateand theaverageof theserunswascalculatedandexpressed inmg/dL˘SEM. 2.3. TissueProcessing Ratswere euthanizedwithin 2–4 h of their final gavage by rapid decapitation. Brainswere extractedanddissected into twohemisphereson ice. The lefthemispherewasfixedbyimmersion in 4%paraformaldehyde inphosphatebuffer (pH=7.4) for2h, rinsedandstored inphosphatebuffered saline at 4 ˝Cuntil use in immunohistochemical experiments. The right hemispherewas further dissectedtoremovethehippocampusandentorhinalcortex. Extractedregionsweresnapfrozenon dry iceandstoredat´80˝Cuntiluse inenzymelinkedimmunosorbentassays (ELISAs). 2.4. Immunohistochemistry Immunohistochemicalproceduresweresimilar topreviousreports [22,31]. The lefthemisphere wassectionedina1:12seriesat40μmthicknesswithavibratingmicrotome(LeicaVT1000S;Wetzlar, Germany)andsectionswerestored incryoprotectantat´20˝C.Adjacentseriesofevery12thsection wereprocessedfor immunohistochemistry. Briefly,afteraseriesofwashes (TBS,pH=7.5),quenching ofendogenousperoxidases (0.6%H2O2 inTBS)andblockingofnonspecificantibodybinding(TBS, 0.1%tritonX-100,and3%horseorgoatserumasappropriate), tissueserieswas incubatedovernight in 66