Page - 88 - in Advances in Neuroimmunology

BrainSci. 2016,6, 25 2.7. StatisticalAnalysis Data (expressed as mean ˘ standard error of mean (SEM)) were evaluated for statistical significance with ANOVA with Fisher’s least significant difference (LSD) tests for individual comparisonsofgrouppairsasappropriate. Individualdatapoints thatwere threestandarddeviations fromtheir respectivegroupmeanswereremovedfromthegroupprior toanalysis. p-values<0.05 wereconsideredstatisticallysignificant. 3.Results 3.1. TimeCourseofExpressionofCytokineandTLR4mRNAs inCortexFollowing1-HourofRestraintStress inSprague–Dawley (S–D)Rats Previous studies have shown that acute stress can affect neuroimmune function in brain [13,14,18–22,24–26,41]. Therefore, our initial investigationwas to determine if the restraint stressutilized inpreviousbehavioral studies [5]would induceaneuroimmuneresponse. Figure1A showstheexperimentalprotocol fordeterminingcytokinemRNAchangesafter60minofrestraint stress in theabsenceofWCE.AsshowninFigure2, eachmRNAassayedwas increased2–4hafter the restraint stress. TheexpressionofCCL2mRNAafterstresswassignificantly increasedabovecontrol by121%at2handby111%at4h(p<0.05). TheexpressionofTNFαmRNAafterstresswas increased abovecontrolby2h(99%)(p<0.01). Likewise, IL-1βmRNAwaselevatedby92%abovecontrolby 2h(p<0.01). CytokinemRNAsgraduallyreturnedtocontrol levelsby8handremainedthere forup to48hafter theacute-stressexposure (Figure2). BecauseTLR4hasbeenimplicated in inductionof cytokines [11,15,41–45],wealsoexaminedwhethermRNAforTLR4wouldbealteredby theacute restraint-stress. Figure2DshowsthatTLR4mRNAexpressionwassignificantlyelevatedby68%above control4hfollowingthestresschallenge(p<0.05)withreturntocontrol levelsby8h. 3.2.DeterminationofCytokineProteinLevels inCortexafterRestraintStress Todeterminewhether increases incytokineproteinsaccompaniedthe increases inCCL2, IL-1β, andTNFαmRNAsinducedbyrestraint stress,proteinsweremeasured incortex4hafter therestraint stress challenge to theS–Drats (Table 1). Cytokineprotein levels, unlike cytokinemRNAs, in the controlswerenotstatisticallyaltered in theS–Drats (Table1). Subsequently, this sameassessmentwas performedforWistar rats todetermine if this rat strainmightexpressachange incytokineproteins followingthe60minofrestraintstress. In theWistarrats,as in theS–Drats, cytokineproteinswerenot increasedbystress (p>0.05). Because increases incytokineprotein levelswerenotobservedineither rat strain [38],onlyS–DratswereusedintheexperimentsassessingexpressionofcytokinemRNAs inducedbystressorWCE. Table1.Effectofacutestressoncytokineproteins inbrain. Group CCL2 IL-1β TNFα S-DNon-Stressed 12.9 (0.3) 0.37 (0.05) ND S-DStressed 13.7 (0.6) 0.28 (0.02) ND WistarNon-Stressed 42.2 (2.3) 1.48 (0.22) 0.11 (0.03) WistarStressed 58.1 (15.4) 1.05 (0.84) 0.12 (0.03) Dataaremean+/´standarderrorofmean(SEM)protein/mgtotalprotein fromcerebral cortexof rats that wererestrainedfor1handsacrificed4hlater.CCL2:Chemokine(C–Cmotif) ligand2; IL-1β: Interleukin-1-beta; TNFα: TumorNecrosisFactoralpha;ND:notdetectable;S–D:Sprague–Dawley. 3.3. Effect ofStress orWCEonSelectedCytokineandTLR4mRNAsinCortex In priorwork,WCE increased anxiety-like behavior [46] and elevated cortical cytokines [38]. This studydirectlycomparedthemagnitudeof theWCEeffectoncytokinemRNAwith thatproduced by stress. Figure 3 shows that anacute 60min restraint stress increased cortical cytokinemRNAs 88