Page - 104 - in Advances in Neuroimmunology

BrainSci. 2017,7, 14 2.MaterialsandMethods 2.1.Animals Fisher/NHsd344(F344) ratswerepurchasedfromHarlanLaboratories (Indianapolis, IN,USA). Theanimalswerehousedingroupsof3–5animals ina temperaturecontrolled(21 ◦C–22 ◦C)animal holdingroom,undera12-h light/12-hdark illuminationcycle (lightsonat7:00a.m.). Foodandtap waterwereprovidedadlibitum.TheInstitutionalAnimalCareandUseCommittee (IACUC)atSeton HallUniversity,SouthOrange,NJ,USAapprovedtheexperimentalprotocol. 2.2.MorphineandLPSAdministration A2+4regimenwasusedtoproducemorphinetolerance in7–8moold(250–350g)maleF344 rats [5,21–24]. Therats (n=16)wererandomlyassignedintotwogroups. Themorphine-tolerantgroup receivedtwo75mgmorphinesulfatepellets (NIDA,Rockville,MD,USA)onDay1viasubcutaneous (s.c.) implantationandfourpelletsonDay2,whereas thecontrolgroupreceivedplacebopelletson bothdays.OnDay5, the twogroupswererandomlyassignedtoreceiveeitherLPS(250μg/kg,Sigma, St. Louis,MO,USA)orsaline (vehicle) [5,21–24]. Thus, the fourexperimentalgroupswereplacebo-control+ saline,placebo-control+LPS,morphine-tolerant+saline,andmorphine-tolerant+LPS. Twohours after the treatmentwithLPSorsaline, theanimalswereeuthanizedandthebrainswereharvested. 2.3. RNAIsolationandPreparationof cDNA TotalRNAwasextractedfromthebrain tissueusingTRIZOL(Invitrogen,Carlsbad,CA,USA), followingthemanufacturer’sprotocol. ToremovecontaminatingDNA, the totalRNAsampleswere treatedwithRNase-freeDNase(Qiagen,Valencia,CA,USA), followedbyfurtherpurificationusingan RNeasyMiniKit (Qiagen,Valencia,CA,USA).TheRNAqualityandquantitywereassessedusing ananodropspectrophotometer (ThermoScientific,Waltham,MA,USA).AnequalamountofRNA (400ng) fromeachsamplewas thenconverted intofirst-strandcDNAusingaRT2 First StrandKit (SABiosciences,Frederick,MD,USA)foraPCRarray. 2.4. Real-TimePCRArray Theexpressionof84keygenesinvolvedinthefunctionof inflammasomes,generalNLRsignaling, andcytokineandchemokinegeneswasquantifiedusingacustomPCRarrayandRT2SYBRGreen FluoresceinqPCRMasterMix(SABiosciences,Frederick,MD,USA),accordingto themanufacturer’s protocol.UsinganABIPrism7900HTFastDetectionSystem(AppliedBiosystems,Foster,CA,USA), real-time PCRwas performed by first denaturing the PCRmix at 95 ◦C for 10min, followed by 40cyclesat95 ◦Cfor15sandat60 ◦Cfor1min. 2.5. PCRArrayDataAnalysis Theexpressionof eachgenewasnormalized tohousekeepinggenesandcalculatedusing the ΔΔCtmethod. The threshold andbaseline valueswere setmanually, and the resulting threshold cycle values (Ct) were analyzed using the PCR array data analysis template supplied on the manufacturer’s website [25]. Themean fold change inmRNA expression from 3 to 5 biological replicateswasconsideredsignificantatp<0.05. Thegeneprofilesignatureswerecreatedforevery twogroupscompared. 2.6. LINCSAnalysis Thedifferentiallyexpressedgeneswere input into theQueryApp(apps.lincscloud.org/query), as described previously [26,27]. Based on the LINCSdatabase, LincsCloud utilized gene profile signaturesgeneratedfromthePCRarray togenerateareport, includingprobabilityoutcomes in terms ofgeneknockdowneffectsanddrugmimics. Thescoresgiven in thereportevaluatedhowmucha 104