Page - 72 - in Advances in Neuroimmunology

BrainSci. 2016,6, 16 3.4.DifferentialEffects ofTreatmentonNumberofMicroglia Stereologyandprofilecountswereusedtodeterminewhetherrepeatedethanolexposureaffected thenumberofmicrogliaduringethanolexposure(Figure5).One-wayANOVAsindicatedasignificant effectof treatment in theCA1(F(3,29)=161.6,p<0.0001),CA2/3 (F(3,29)=17.99,p<0.0001), and DG (F(3,29) = 69.98, p < 0.0001) fields, aswell as in entorhinal cortex (F(3,28) = 6.78, p = 0.001). Post-hocTukey’s tests indicateda significant increase in thenumberof Iba-1+cells throughout the hippocampus in theEtOH/EtOHgroupcomparedwithall othergroups (Figure5A–C).However, in theentorhinal cortexmicroglia cells in theEtOH/EtOHgroupweredecreasedcompared to the ad libitum and control groups butwere similar to the number seen inCon/EtOH treated animals (Figure 5D).A post-hocTukey’s test showed thatCon/EtOHrats haddecreased Iba-1+ cells in all regionsmeasuredascomparedtoCon/Conandad libitumgroups(Figure5) [61]. Importantly,because thenumberofmicrogliacanaffect immunoreactivity,acorrelationbetweenthenumberofmicroglia versusOX-42 immunoreactivitywasrun,butnosignificant relationshipwasobserved. Figure5.MicroglialCellCountsDifferentiallyAlteredbyEthanolExperience. Stereologicalestimates indicatean increase in thenumberofmicroglia in theEtOH/EtOHgroupinthe(A) cornuamonis1 (CA1), (B) cornuamonis2/3 (CA2/3), and(C)dentategyrus (DG)comparedwithallothergroups. However, thenumberofmicrogliaintheCon/EtOHgroupwasdecreasedthroughoutthehippocampus. In the (D) entorhinal cortex,microgliaweredecreased inboth theCon/EtOHandEtOH/EtOHgroups comparedtoboth thead libitumandCon/Congroups. *p<0.05comparedtoad libitumandCon/Con group;#p<0.05versusCon/EtOH. 3.5. IncreasedPro-InflammatoryCytokineExpression inEtOH/EtOHGroup ELISAswereusedtoassess the functional stateofmicroglia, specifically theanti-inflammatory cytokine, IL-10,andthepro-inflammatorycytokine,TNF-α. Nochangeswereseen in IL-10during intoxicationamonganygroups ineither thehippocampus(F(3,28)=0.57,p=0.64)or theentorhinal cortex (F(3,24) = 0.50, p = 0.69; Figure 6A,B). However, one-way ANOVAs of TNF-α protein concentrations indicatedasignificanteffectof treatment in thehippocampus(F(3,28)=4.658,p=0.009) butnot theentorhinal cortex (F(3,24)=0.99,p=0.41). Post-hocTukey’s tests indicatedasignificant increase in TNF-α in the hippocampus in the EtOH/EtOHgroup compared to all other groups (Figure6C).Correlationsofbingeparametersversus immunohistochemical resultswererunwithin theEtOH/EtOHgroup to furtherprobe thedistributionofTNF-α concentrations (Table 5). BECs correlatedwithTNF-αconcentration(P(8)=0.807,p=0.016;Figure7). 72