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BrainSci. 2016,6, 15 Somewhatsurprisingly,consideringtheimportanceofastrocyticIL-6inneuroinflammation[2,4,25,26], wedidnotobservedramaticeffectsofastrocytic IL-6deficiencyoneitherastrocytesormicrogliaat the timesstudied. This resultmakes itunlikely thatchanges inglial cell reactivityareunderlyingthe differences inclinical signsbetweenAst-IL-6KOmiceandfloxedmice.Nevertheless,overall staining andcellmorphologiesare limitedapproaches,andmoredetailedstudiesareneededtoassessother rolesofglial cells (other thanIL-6productionbyastrocytes) inclinical signsofEAE.Finally, regarding thenumberofvessels,weobservedadecrease inAst-IL-6KOmice,which is inagreementwithresults inmiceoverexpressingIL-6,whichshowextensiverevascularization,both inbasal conditions [25,26] andafter an injury [34], indicativeof a role of IL-6 invasogenesis. Other studies also support the idea that IL-6promotesvasogenesis [35]. Probably, this is secondary to thenumberand/or typeof inflammatorycellspresent in thespinalcord, sincenodifferenceswereobservedinmalemice. The extent of the reductionof IL-6 in astrocytes of theAst-IL-6KOmice invivohasyet to be determined.However, studiesofculturedastrocytes fromtheAst-IL-6KOmicedemonstrated that the astrocytesaredeficient in IL-6production. In thesestudies,analysisofculturesupernatantafter24h ofstimulation(10ng/mLofLPSand10ng/mLof INF-γ) showedthatastrocytes fromfloxedmice producedapproximately13ng/mLof IL-6,whereasastrocytes fromAst-IL-6KOmiceonlyproduced 2ng/mLIL-6 [17,18]. Regardlessof theextentof theastrocytic IL-6deficiency in theAst-IL-6KOmice, adelayinonset toclinicalsymptomswasevident infemales, includinglessdemyelinationat20–22dpi inaccordancewith the lowerclinical scores.However, thiswasa transienteffect, andsometimeafter 20–22dpi, theEAEwassimilar inbothgenotypes (as indicatedbythe lackofsignificantdifferences in demyelinationat46dpi), indicatingtheastrocytic IL-6no longerplayedarole. 5.Conclusions Inconclusion,wehaveshownthat lackofastrocytic IL-6 isnotsufficient topreventEAEdisease, but it is able todelay thediseaseandtoameliorateclinical scoringandthe inflammatorymilieu in femalemice. These results support the idea that the localCNSproductionof IL-6 is important in thisdisease. Several interestingquestionsremaintobeaddressed. Forexample,dueto thedelayed onsetofclinical signsofdisease inAst-IL-6KOfemales, itwillbe important toanalyze indetail the primingandinflammatory infiltrates in theCNSof femaleandmaleAst-IL-6KOmiceandtheirfloxed controls. Furthermore, studiesofmalesat laterdpimayrevealgenotypicdifferencesat laterstages. Moreover,acomparison of the immunizedAst-IL-6 KO andfloxedmicewith aCFA immunized control groupcould reveal specificEAEeffects inAst-ILK-6KOmice. Future studieswill address theseandotherrelevant issuesrelative to theroleofastrocytic IL-6 in theEAE. Acknowledgments: This work was supported by SAF2011-23272 and SFA2014-56546-R to Juan Hidalgo. MariaErtagratefullyacknowledgesaPhDfellowshipfromUniversitatAutònomadeBarcelona. AuthorContributions:M.E.,M.G.,S.J.,A.M.andG.C.were involvedinseveralaspectsof theexperimentscarried out.M.E. ledmostof theexperimentalwork. J.H.conceivedof theexperiments.M.E.andJ.H.wrote thepaper. Conflictsof Interest:Theauthorsdeclarenoconflictof interest. The foundingsponsorshadnorole in thedesign of the study; in the collection, analyses or interpretationofdata; in thewritingof themanuscript; nor in the decisiontopublish theresults. References 1. Hirano,T.;Taga,T.;Nakano,N.;Yasukawa,K.;Kashiwamura,S.; Shimizu,K.;Nakajima,K.;Pyun,K.H.; Kishimoto,T.Purification tohomogeneityandcharacterizationofhumanB-celldifferentiation factor (BCDF orBSFp-2).Proc.Natl. Acad. Sci.USA1985,82, 5490–5494. [CrossRef] [PubMed] 2. Gruol,D.L.;Nelson,T.E.Physiologicalandpathological rolesof interleukin-6 in thecentralnervoussystem. Mol.Neurobiol. 1997,15, 307–339. [CrossRef] [PubMed] 3. Schöbitz, B.; deKloet, E.R.; Sutanto,W.; Holsboer, F. Cellular localization of interleukin 6mRNAand interleukin6receptormrna inratbrain.Eur. J.Neurosci. 1993,5, 1426–1435. [CrossRef] [PubMed] 26