Fig 1: URMC-099 modulates the phenotype of activated microglia in EAE hippocampus. A, Staining for Iba1 in hippocampal area CA1 shows ramified microglia in sham-immunized mice, and microglia with shorter, thicker processes in EAE. B, Iba1-positive cells in the hippocampi of both sham and EAE mice coexpress microglia-specific marker Tmem119 along their processes and to a more variable extent in the cell body (upper panels). Iba1-positive, Tmem119-negative cells consistent with monocyte-derived macrophages could be found in the pia during EAE (inset), but not in or around the hippocampus. Staining for Ly-6B, expressed on neutrophils and some recently-generated macrophages, is sparse in sham and EAE hippocampi (lower panels, arrowheads), although clusters occurred sporadically in the pia and other area of some EAE brains (inset). Scale bars: 20 µm. s.o., CA1 stratum oriens; pyr, pyramidal layer; s.r., stratum radiatum. C, Quantification of microglial images shows increased Iba1 intensity and reduction in the ratio of surface area to volume in EAE microglia regardless of treatment with URMC-099 or CLFB1134. Microglial expression of lysosomal marker CD68 is increased in many hippocampi from vehicle-treated EAE mice but not in mice treated with URMC-099 or CLFB1134; n = 10 male mice (sham-vehicle), n = 6 male mice (EAE-vehicle and EAE-099), n = 7 male mice (EAE-1134). D, E, Western blottings and band densitometry for markers of inflammation and microglial phenotype in hippocampal protein extracts. Markers associated with proinflammatory microglial activation tended to increase in vehicle-treated EAE hippocampi and were decreased by URMC-099, with significant changes in iNOS (inducible nitric oxide synthase) and immunoglobulin receptor FC?R1/CD64 (lower single band), and a non-significant trend toward increased pro-IL1ß. Proinflammatory marker CD86 does not significantly increase in vehicle-treated EAE but is decreased by URMC-099 treatment. Arg1 and IL-10, canonical markers of anti-inflammatory alternative activation, are not significantly changed in EAE or with URMC-099. Consistent with immunostaining results, Iba1 is up-regulated in vehicle-treated EAE hippocampi and is further increased with URMC-099 treatment. Complement component C1q follows a similar pattern; n = 10 male mice (sham-vehicle), n = 7 male mice (EAE-vehicle), n = 11 male mice (EAE-099); one representative blot with three lanes per condition is depicted; *p < 0.05, **p < 0.01, one-way ANOVA with Sidak (iNOS, FC?R1/CD64, CD86) or Tukey (Iba1, SA/Vol, C1q) post hoc test.