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BrainSci. 2016,6, 15 Table2.EAEdisease. EAE Females Males Ast-IL-6KO Floxed Ast-IL-6KO Floxed Incidence 21/23 28/28 11/11 17/18 Mortality 3/23 0/28 0/11 0/18 Dayofonset 13.38˘0.59* 11.57˘0.29 12.27˘0.52* 11.65˘0.49 Resultsshownarepooleddata fromExperiments1–3separatedbysexandgenotype. Foreachanimal, time todiseaseonsetwasdefinedbyaclinical scoreě1. Forstatisticalanalysis,a two-wayANOVAforgenotype (floxedvs.Ast-IL-6KOmice)andsexas themainfactorswasperformed. *p<0.05vs.floxedmice. When the day of disease onset was analyzed using sex and genotype as the main factors, asignificantgenotypicdifferencewasobservedforbothsexes (Table2). Incontrast, thepeakscore, cumulativescoreandgradeof remissionat20dpiwerenotdifferentbetweengenotypes foreithersex (Table3). Theseresults suggest thatwhile thedisease isdelayedandtheseverity is somewhat lowerat theearlystages in femalemice, it isnotaffectedbyastrocytic IL-6deficiency lateron. Table3.EAEclinical course. ClinicalCourse Females (0–20dpi) Females (46dpi) Males (0–20dpi) Ast-IL-6KO (n=15) Floxed (n=20) Ast-IL-6KO (n=8) Floxed (n=8) Ast-IL-6KO (n=11) Floxed (n=18) Timeofpeakscore 14.30˘0.53 14.80˘0.49 22.5˘2.46* 15.5˘1.92 15.18˘0.80 13.76˘0.6 Peakscore 2.80˘0.23 3.45˘0.16 3.68˘0.50 3.18˘0.23 3.18˘0.18 3.14˘0.12 Cumulativescore 19.40˘2.55 24.16˘1.32 88.87˘21.84 72.18˘10.35 20.32˘1.64 22.61˘1.34 GradeofRemission 0.64˘0.13 0.91˘0.11 1.00˘0.34 1.43˘0.27 0.82˘0.26 20.32˘1.64 Results shownarepooleddata fromExperiments1–3separatedbysexandgenotype. Forsimplicity,data from 20–22dpiweregroupedandarereferredtoas20dpi. Foreachanimal,wedeterminedtimetopeakdisease, peak-score, cumulativescore (sumofall scores fromdiseaseonset toDays20and22combinedor46),andthe gradeof remission(differencebetweenpeakscoreandoutcome). Resultsare themean˘SEM.Forstatistical analysisat0–20dpi, a two-wayANOVAforgenotype (floxedvs.Ast-IL-6KOmice)andsexas themain factors wasperformed. For the femalesat46dpi,aone-wayANOVAforgenotype(floxedvs.Ast-IL-6KOmice)was performed. *p<0.05vs.floxedmice. 3.2. ReducedCellular Infiltrates andDemyelination in theSpinalCordofAst-IL-6KOFemaleMice Thenumberof inflammatory infiltrates in the longitudinal lumbar-cervical spinal cordofcontrol and EAE-induced animals was assessed in females and males at 20–22 dpi (for simplicity, data from20–22dpiweregroupedandare referred toas 20dpi). Femaleswerealsoassessedat 46dpi (Figure2A,C).The totalnumberof infiltrates inwhitematterwascounted inaCresylviolet/Luxol FastBluestainingandinaCD3IHCcounterstainedwithhematoxylin;ameanvalueofbothstainings was thencalculated foreachanimalandusedfor statistical analysis. AsshowninFigure2A, there isasignificantdecrease in thenumberof infiltrates inAst-IL6KOfemalesatboth20–22and46dpi comparedtofloxedfemalemice.Ast-IL-6KOmalesdidnotshowasignificantdifferencecomparedto floxedmales (Figure2A). Demyelination in spinal cordwhitematterwas assessedmeasuring the percentage of Luxol FastBlue-stainedarea (Figure2B,C).Healthyanimals (0dpi)had100%areacoveredbyLFBstaining. Following EAE,Ast-IL-6 KO females showed a significantly lower Luxol Fast Blue-stained area, indicatinggreaterdemyelination,comparedtofloxedfemalemiceat20dpi,butnotat46dpi(Figure2B). Ast-IL-6KOmalesdidnotshowasignificantdifference inLuxolFastBlue-stainedareacomparedto floxedmales (Figure2B). 22