Page - 76 - in Advances in Neuroimmunology

BrainSci. 2016,6, 16 response, sincean increase in thenumberofactivatedmicrogliawould likelyresult inapotentiated neuroimmuneresponseduringthesecondbinge. HippocampalBDNFconcentrationsweredeterminedinorder toassess the impactofmicroglia reactivityandchanges inmicroglianumberonthesurroundingenvironment. BDNFplaysapivotal role in neuronal integrity and its dysregulation is associatedwith neurodegeneration [73]. In the Con/EtOHgroup,BDNFwasdecreased, thenumberofmicrogliaweredecreasedandtherewasa significant correlationbetween thenumber ofmicroglia andBDNFprotein expression. However, in theEtOH/EtOHtreatedanimals,wheremicrogliaweremoreactivatedandtheirnumberswere increased,asignificantlyhigherBDNFconcentrationwasobserved, thoughthisvaluedidnotcorrelate significantly tomicroglianumber. It ispossible that the increase inBDNFconcentrations isdue tocells other thanmicroglia, suchasastrocytes,neurons,andotherCNScellssecretingBDNF[74]. Inaddition, theeffectofethanolonBDNFexpression isquitecomplex[75].Nevertheless, the interplaybetween the increased cytokine andneurotrophinproductionobserved in theEtOH/EtOHgroup requires furtherstudytounderstandits functional implications. The experimental design touse the sameanimals for both immunohistochemical andELISA experimentsallowedforaseriesofcorrelations tohelpdeterminewhataspectofethanolexposure, in thisAUDmodel,wasassociatedwithmicroglial reactivity.OX-42 immunoreactivitydidnotcorrelate to average dose per day or to the total dose of ethanol in either the Con/EtOHor EtOH/EtOH groups. This lackofcorrelation is importantas immunemodulatorssuchasLPShavedose-dependent responses inmicroglia reactivity [76]. The lackofcorrelationbetweenOX-42andtotaldoseofethanol suggests thatethanolpotentiates theOX-42responsebyactingasasecondarystimulusrather than anadditiveeffectof theaccumulativedose.Moreover,norelationshipbetweenthenumberof Iba-1+ cellsandOX-42 immunoreactivitywereobserved, supporting that increasedOX-42 immunoreactivity wasaresultofmicroglialactivationandnotanartifactof thechange incellnumber [77]. Correlations werealsoused toexamine the relationshipbetweenOX-42 immunoreactivityor Iba-1 cell number andfunctional indices (cytokine/neurotrophinproduction).However,neitherCR3receptor (OX-42) upregulationnor Iba-1+cellnumberwassignificantlycorrelatedwithTNF-αexpression. Interestingly, thebimodaldistributionofTNF-αproductionobservedintheEtOH/EtOHgroupdidmapontoBECs. AlthoughthemechanismbywhichBECsarerelatedtoTNF-αwerenotmeasured,atminimum, this correlationsuggests thatasBECsincreasewithrepeatedexposure,aprimedmicroglial statemaycause increasedpro-inflammatorycytokines. Finally, inrelationtoBDNF,onlymicrogliacellnumber in the Con/EtOHgroupshowedasignificantcorrelationwithBDNFconcentrationssupporting the idea that microglialdysfunctionandsubsequent lossof trophic factorsmaycontribute toneurodegeneration, especiallyalcoholicbraindamage[61]. Correlationsarenotbeing interpretedascausation,but theydo providedirection forwhataspectsofalcohol-exposure impactmicroglia reactivity leadingtoaprimed microglial state. Some evidence of classical activation has been observed in other AUD models, an effect thatmay be attributable to species differences and/or variations in the duration and pattern of exposure [30,69].Whilepreviousreports suggestedthat thedifference inmicroglia reactivitywasdue to theseaforementionedvariations inAUDmodels, thecurrentdata in this reportmoredefinitively indicates that it is the repeated insult thatmaydrive thegreatermicroglial response. Forexample, amodel of alcohol exposurewith lower total doses of alcohol dispersed over a longer period of timeproducedmoreOX-6positivecells thantheexposureusedherein,whereOX-6expressionmay havebeenananomaly inasingleanimal [30]. Theappearanceof theOX-6+cells,however, inboth models still appearedtobe thebushy, ramifiedmorphologyassociatedwitha low-levelorM2-like activation. Indeed, the only alcohol study, human or animal, where ED-1+microglia have been observed, is fromastudyinwhichratsunderwent fourcyclesofaMajchrowicz-likemodelwith three daysbetweenbinges.However, thehighmortalityrateandsevereweight lossof rats in that report make interpretationsdifficult.One interpretation is thatmicroglialactivationmayhaveoccurreddue to thestressof repetitivegavageand/orweight loss [57,78–80]. Thus, thecurrentstudyspecifically 76