Seite - 19 - in Advances in Neuroimmunology

BrainSci. 2016,6, 15 Athoroughunderstandingof theroleof thecellularcontext in thisandotherdiseases isnecessary toclarify theputativerolesof IL-6 in theCNS[15,16].Werecentlycharacterizedtheroleofastrocytic IL-6 in normal (basal) conditions byusing transgenicmicewith astrocyte-specific IL-6 deficiency, showingeffectsonbehaviorandbodyweightinasex-dependentmanner[17,18].TheroleofastrocyticIL-6 duringpathologicalsituationsremainedtobeassessed,andhere,wereport theinitialstudieswithEAE. 2.MaterialsandMethods 2.1.Animals Thegenerationofastrocyte-IL-6KO(Ast-IL6KO)andfloxed littermatemice,whichservedas controls,wasasdescribedpreviously [17].AnumberofstudieshaveshownthatGFAPisprimarily expressed in astrocytes of theCNS,withminimal expression inperipheral regions [19]. Allmice werekeptunderconstant temperaturewith freeaccess to food(Harlanglobaldiet2918)andwater. Ethicalapproval for the use and experimentation of allmice in this studywas obtained from the AnimalandHumanExperimentationEthicsCommitteeof theUniversitatAutònomadeBarcelona (no 4017,approved3September2015). 2.2. EAEInductionandClinicalEvaluation For the inductionofEAE, two-month-oldmaleandfemalemicewereused. EAEwas inducedby active immunizationwithMOG35–55 peptide.OnDay0,allmice,under isofluraneanesthesia,were injectedsubcutaneously into thehindflankswithanemulsionof100μLMOG35–55 (3mg/mL)and 100μLCompleteFreund’sAdjuvant (CFA) (Sigma-Aldrich,St. Louis,MO,USA)supplementedwith 4mg/mLMycobacteriumtuberculosisH37RA(Difco,Detroit,MI,USA). Inaddition,animals received anintraperitoneal injectionof500ngpertussis toxin (Sigma-Aldrich,St. Louis,MO,USA),whichwas repeatedtwodays later. After immunization,micewereexamineddaily,weighedandscoredforEAE.TheEAEclinical score was assessed for each animal according to the following criteria: 0 = no signs of disease, 0.5=partial lossof tail tonus, 1= lossof tail tonus, 2=moderatehind limbparaparesis, 2.5=severe hindlimbparaparesis, 3=partialhindlimbparalysis, 3.5=hindlimbparalysis, 4=hindlimbparalysis pluspartial front legparalysis,4.5=moribund/totalparalysisand5=death. Finally, foreachanimal, wedeterminedthetimetodiseaseonset (clinicalscoreě1), timetopeakdisease,peak-score,cumulative score (sumofall scores fromdiseaseonset toDay20)andgradeofremission(differencebetweenpeak scoreandoutcome). Three independentEAEexperimentswerecarriedout (Table1). Experiment1wascarriedout for 0–22dayspost-immunization(dpi);Experiment2wascarriedout for0–20dpi; andExperiment3was carriedout for0–46dpi. Foreachexperiment, littermateswereused. Femalemice from20–22dpiwere groupedbeforecomparison to46dpimice.MaleAst-IL-6KOmicedidnotdiffer frommale floxed miceat20–22dpiand, thus,werenotexaminedat46dpi. Inallcases,all survivingmicewereeuthanized attheindicateddayspost-immunizationbydecapitation. Spinalcordswereimmediatelyremovedand fixedfor48hin4%paraformaldehydeandembeddedinparaffinfor immunohistochemistry(IHC)and histochemistry(HC)analyses. Spinalcordsfromadditionalhealthyfemalemicewereprocessedinparallel. Table1.Numberofmicepergenotype, sexandexperiment. Genotype Experiment1 (0–22dpi) Experiment2 (0–20dpi) Experiment3 (0–46dpi) Males Females Males Females Females Floxed 7 3 11 17 8 Ast-IL-6KO 3 7 8 8 8 dpi,dayspost-immunization;Ast, astrocyte. 19