Seite - 75 - in Advances in Neuroimmunology

BrainSci. 2016,6, 16 potentiatesOX-42immunoreactivity.ApotentiatedincreaseinOX-42immunoreactivity,orCR3density, byethanol isparticularly interestingbecauseCR3is intimately involved inmicroglialpriming[33]. The increasedupregulationofCR3in theEtOH/EtOH(doublebinge) ratscomparedwithCon/EtOH (single binge) rats suggests that binge ethanol exposure acts as a priming stimulus tomicroglia. Morphology, thoughnotspecificallyquantified,appearedconsistentwitha lowgrade/phenotypeof activationascellswereramifiedandnotamoeboid(e.g.,Figure1) [26].Abushy, ramifiedmicroglial morphology isalsoconsistentwith thatobservedinotherpathologies that reportaprimedmicroglia state [33,64,71]. Furthermore,despite thepotentiationofCR3receptordensity, nochanges inED-1 orOX-6expressionwereseenfollowingthesecondbinge. The lackofvisibleED-1+orOX-6+cells concurswith other reports in thismodel that donot showsigns of classicalmicroglial activation followingethanolexposure [22,31,60]. Becausemultipleendpointsshouldbemeasuredtounderstandthephenotypeofmicrogliaafter insult, functionaloutputssuchashallmarkpro-andanti-inflammatorycytokinesweremeasuredto betterunderstandthe typeofmicroglialactivationassociatedwithasecond“hit”ofethanolexposure. Nochange in theconcentrationof thehallmarkanti-inflammatorycytokine, IL-10,wasobservedin eitherethanolexposuregroupin thehippocampusorentorhinal cortex. The lackof IL-10response during intoxicationconfirmspreviousfindings in thismodel,althoughIL-10 isdecreased inamouse AUDmodel [22,23].However,upregulationofTNF-α in thehippocampus in theEtOH/EtOHgroup comparedwithallothergroupssuggests that thesecondbingepromotedapro-inflammatorystate. Thisfinding ishighlydistinct frommultiplepreviousreportsusingMajchrowicz-likemodelswhere no effect of ethanolwas observed on TNF-α concentrations [22,24,31] and highlights the impact of repeated ethanol exposure onpro-inflammatory cytokineproduction andmicroglial activation. The potentiation of TNF-α expression by the secondhit of ethanol,much like themorphological indices, isacommonresponse formicroglia thatareprimedandthenhitwithasecondaryperipheral immune insult [28,64,72]. In fact, alcohol andotherdrugsof abusehavebeen shown toprime the TNF-αresponsetoother immunestimulators [23,63],but thesefindingspecificallysuggest thatalcohol exposurecanactasboth theprimingandsecondarystimulusresulting inan increase inTNF-α. In theMajchrowiczmodel,microglia losswasobservedduringthe lastdaysof intoxication[61], whereasmicrogliaproliferationoccursafter thecessationofalcoholexposure,ontheseconddayof abstinence [31,60]. Therefore, Iba-1+cellnumberwasassessedtodeterminehowmultiplecyclesof ethanolaffectsmicroglianumber. Thesingleethanolbinge (Con/EtOHgroup)reducedthenumberof Iba-1+microglia, inboth thehippocampusandentorhinalcortex.Ourrecentworksupports that this reduction is likelyduetodegenerationofmicroglia following4-daybingeexposure [61]. Interestingly, the secondbinge (EtOH/EtOHgroup) resulted inan increasednumberof Iba-1+microglia in the hippocampuscomparedtoeither thecontrolgrouporsinglebinge (Con/EtOH)group. It isplausible that the increase in Iba-1+ cells in theEtOH/EtOHgroupobserved in thehippocampus isdue to microglial proliferation at twodays following the first binge [22,31,60]. This effect also suggests that ethanol does not significantly reduce these newly proliferatedmicroglial cells. It is of note that the effect of ethanol onmicroglianumbervariedby region: in the entorhinal cortex, both the singleanddoublebingeresulted inadecrease in thenumberofmicrogliaconsistentwithourrecent report [61]. The lackof increasedIba-1+cells in theentorhinalcortexofEtOH/EtOHgroupis likely related to thefinding thatmicroglianeitherproliferatedramatically at twodayspost-binge in the entorhinalcortexnor is thereasignificant increase inmicroglianumberaftersevendays;,however, bothproliferationand increased Iba-1+cellshavebeenobserved in thehippocampusat this same timepoint [22,60].Whymicrogliaproliferate in thehippocampusbutnotentorhinal cortexafterbinge ethanol exposure ispuzzling. Neurons in theentorhinal cortexdegeneratemore robustly,peaking at fourdaysofexposure [5,7,8],which is followedbyothersignsofreactivemicrogliosis [22].More studiesarenecessarytofullyunderstandthedynamiceffectsofalcoholonmicroglianumber,especially consideringtherecentdiscoveries thatmicrogliacontribute tosynapserefinementandplasticity [25]. Thesedatasupport thehypothesis thatasecondbingealcoholexposureexacerbates themicroglial 75