Page - 86 - in Advances in Neuroimmunology

BrainSci. 2016,6, 25 2.4. BrainTissueCollectionandReal-TimePCRAnalysis forTissuemRNA Following experimental procedures, ratswere rapidlydecapitated andbrain tissue stored at ´80˝Cforsubsequentextractions forPCR.To initiatePCRprocedures, totalRNAwasextractedwith Trizol (Invitrogen, Carlsbad, CA, USA) from homogenized dissected brain regions from control and ethanol-treated experimental brain sections followed by use of the SV total RNA isolation system(Promega,Madison,WI,USA).This tissuewas thenusedforreverse transcriptionPCRusing theSuperscript First StrandorSuperscript III First StrandSynthesis Supermix (LifeTechnologies, GrandIsland,NY,USA)[38]. Theprimersequencesusedfor thecortexwerechemokine (C–Cmotif) ligand2(CCL2)=51-TCACGCTTCTGGGCCTGTTG-31 (forward)and51-CAGCCGACTCATTGGGATC ATC-31 (reverse); Interleukin-1β (IL-1β)=51-GAAACAGCAATGGTCGGGAC-31 (forward)and51-AA GACACGGGTTCCATGGTG-31 (reverse); tumornecrosisfactor-α(TNFα)=51-ATGTGGAACTGGCAG AGGAG-31 (forward) and 51-ACGAGCAGGAATGAGAAGAGG-31(reverse); Toll-like-receptor-4 (TLR4)=51-GCCGGAAAGTTATTGTGGTGGT-31 (forward)and51-ATGGGTTTTAGGCGCAGAGTT T-31 (reverse);β-actin, 51-ATGGTGGGTATGGGTCAGAAGG-31 (forward)and51-GCTCATTGTAG AAAGTGTGGTGCC-31 (reverse). SYBRgreenPCRmastermix (AppliedBiosystems, FosterCity, CA, USA) was used for real-time PCR analysis of the cortex on the Bio-Rad MyiQ (Bio-Rad, Hercules, CA, USA). For other brain regions (and the CP154,526 study), mRNA analyses were optimizedwithTaqMan® (ThermoFisherScientific,Waltham,MA,USA)expressionassays—CCL2 (Rn00580555_m1), TNFα (Rn01525859_g1), IL-1β (Rn00580432_m1), TLR4 (Rn00569848_m1), andβ-actin (Rn00667869_m1)—and sampleswere run on a StepOnePlus real time PCRmachine (LifeTechnologies,GrandIsland,NY,USA).Foralldata, thecycle time(Ct)valueswerenormalized withβ-actin toassess therelativedifferences inexpressionbetweengroups.Ctvaluesofβ-actinnever differedacrossgroups thereforeβ-actinwasanappropriatechoiceasahousekeepinggene.Calculated valueswereexpressedasrelativechangetoadesignatedcontrol setas100%. 2.5. Enzyme-linked ImmunosorbentAssay (ELISA) forCytokines Becausechanges in levelsof cytokineproteins in theS–Dratshavebeenfoundtocorrelatepoorly withmRNAchanges (see [38,39]) initially only expression ofmRNAs for cytokineswas assessed. Nonetheless,becauseof interest in therelationshipbetweenmRNAandproteins inducedbystress, ELISAassays forcytokineproteins incortexwerefirstperformed4hafterstress in the timecourse determination in theS–Drats (Figure2). Subsequently,assaysofcytokineproteinswereperformed4h afteranacuterestraintstress toWistarrats (CharlesRiver,Raleigh,NC,USA).Eachcortical sample was homogenized in Iscove’sModifiedDulbeccoMedium (Invitrogen, #12440046, Carlsbad, CA, USA)containing1 tabletper50mLof the completeprotease inhibitor cocktail (RocheDiagnostics #11697498001, Indianapolis, IN,USA).Homogenizedspecimenswere thencentrifugedat12,000ˆg for10minat4˝Candthesupernatantscollectedandstoredat´80˝Cuntil theELISAdetermination wasmade. ELISAkitswerepurchasedfor IL-1βandTNFα fromR&DSystems, (Minneapolis,MN, USA),andfor theCCL2fromBDBio-Sciences (SanJose,CA,USA).ELISAprocedureswereperformed according to themanufacturer’s instructions. Standards for IL-1βandTNFαwere seriallydiluted 4times toconcentrationsof1.95pg/mLand0.78pg/mL,respectively,andthestandardforCCL2was usedassupplied.All tissuecytokine levelswerecorrectedforproteinusingPierce®BCAProteinassay (ThermoScientific,Rockford, IL,USA). 86