Page - 5 - in Advances in Neuroimmunology

BrainSci. 2016,6, 19 fromastrocyteculturespreparedfromCNSofnon-tgmice. Interestingly,ELISAanalysisof IL-6 levels in the hippocampus have revealed low levels andnodifferences between the IL-6 tg andnon-tg hippocampus,althoughhigher levelsandgenotypicdifferenceswerenoted in thecerebellum[52,56]. ThecerebellumistheCNSregionwiththehighestlevelofIL-6mRNAexpressioninthetransgenicmice, particularly in theBergmanglial [49]. Theseresultsmayindicate that IL-6producedbyhippocampal astrocytes invivo is rapidly releasedanddegraded.Othershavenoteddifficulty inmeasuring IL-6 levels inCNStissueusingcommercialELISAkits,whichmaymean that thereare technical issues toberesolved[57]. Inspiteof the lackofdifferences inmeasureable levelsof IL-6protein, increase expressionof IL-6 regulatedgenes (e.g., GFAP, eb22, Socs3) andelevated levels of STAT3and the activatedformofSTAT3(phosphoSTAT3), thedownstreampartnerof IL-6signal transductionthrough whichIL-6acts to increaseGFAP[58–60],wereobservedin theCNSof IL-6 tgmice. Theseresultsare consistentwithactionsofelevated levelsof IL-6 in the IL-6 tgCNS. Proteinmeasurements intheCNSofthetwoCCL2transgenic linesshowedthattheolderCCL2-tg SJLmiceexpresshigher levelsofCCL2in thehippocampusthan in theCCL2-tgmice. CCL2levels measuredbyELISAwere~1.3ng/mLat3–4monthsofageand~3.0ng/mLat7–9monthsofage in hippocampalhomogenate fromtheCCL2-tgSJLmice[61]. In theCCL2-tgmice,CCL2levelsmeasured byELISAwere~1.2 ng/mLat 3–5months of age and~1.5 ng/mLat 7–9 [58]. CCL2 levelswere ~0.2ng/mLinhippocampalhomogenates fromthenon-tgmice fromboththeCCL2-tgandCCL2-tg SJL lines. Studiesof supernatants fromastrocyteculturesprepared fromCNSofCCL2-tgSJLmice showedthatastrocytesconstitutivelysecrete largeamountsofCCL2(e.g.,~3.5ng/mL)[34]. ExpressionofCXCL-10 in theCNSofCXCL10-tgmicehasbeencharacterizedat themRNAlevel by in situhybridization [29]. Thehighest levelsofCXCL10mRNAwereobserved in thehippocampus, olfactorybulb,periventricularzone, corticalareas, cerebellum,andchoroidplexusof theCXCL10-tg CNS (mice 5–6months of age). Western blot studies confirmedhigh levels ofCXCL10protein in thehippocampus, and immunohistochemical stainingconfirmedexpressionofCXCL10protein in astrocytes [29]. NoCXCL10mRNAorprotein expressionwasobserved innon-tgmice. Levels of CXCL10protein in theCNSofCXCL10-tgmicehavenotbeenmeasuredbyELISA. Elevated levelsofneuroimmune factors are typically associatedwithpathological conditions, whereaslowlevelsappeartoexistunderphysiologicalconditions.However, therangeofproteinlevels expressedduringphysiologicalandpathophysiological conditionshasyet tobefullyelucidatedfor mostneuroimmunefactors.Althoughelevated levels IL-6,CCL2andCXCL-10mRNAand/orprotein havebeendocumented in theCNSof therespective transgenicmice, it isunknownifprotein levels for the three transgenic linesare functionallycomparable.However,mRNAorprotein levelswereshown tobewithin therangeassociatedwithexperimentally inducedpathophysiologicalconditions in the CNSof IL-6 tg [62],CXCL10-tg [29]andCCL2-tgSJLmice [34]. 4.2.Neuropathology Ingeneral,before3–6monthsofage, theheterozygousIL-6,CCL2,andCXCL10transgenicmice showrelatively littleneuropathology. In the IL-6 tgmice, the cerebellumshows thehighest levels of IL-6mRNAexpression in theCNSof the IL-6 tgmice andgreatest neuropathological changes, themostprominentbeingneovascularization[49,63].Age-dependentneuropathological changes in the cortex andhippocampusof the IL-6 tgmicewere evident in immunohistochemical studies of synapticandcellularproteins. Theneuropathological changes includedreducedimmunostainingfor thepresynapticproteinsynapsin I indicativeofsynapticdamage(cortex,12monthsofage), reduced immunostainingformicrotubuleassociatedprotein-2 (MAP-2) indicativeofdendriticdamage(cortex at 3 and12monthsof age), reduced immunostaining forparvalbumin, a calciumbindingprotein expressedbyinhibitory interneurons (hippocampusat3and12monthsofage),andeventual lossof the interneurons,andreducedimmunostainingforcalbindin,acalciumbindingproteinexpressedby inhibitory interneurons (cortex,12monthsofage) [49,54]. 5