Analysis of brain microglial/leukocyte responses after anti- TNF therapy. (A) Gating strategy: FSC/SSC was used to define leukocytes, monocytes and granulocytes. Singlet cells were identified using FSC-A/FSC-H, and only live cells were included. Dot plots showing CD45highGr1+ granulocytes,, CD45dimCD11b+ microglia, and CD45highCD11b+Gr1- macrophages (B) Microglial numbers were increased at six and 24 hours in all groups compared to the respective non-lesioned mice. Furthermore, the number of microglia was significantly increased in XPro1595-treated mice compared to saline-treated mice at 24 hours. MFI for CD45 in microglia was significantly increased at 24 hours in XPro1595- and etanercept-treated mice compared to saline-treated mice. Analysis of CD11b+CD45high leukocytes showed an increase in the number of infiltrating leukocytes in all groups at 24 hours compared to non-lesioned mice. No change in MFI for CD45 in leukocytes was observed (four to six per group). (C) CD11b-immunostained sections six hours, 24 hours and five days after pMCAO (shown for saline-treated mice). Scale bar: 100 μm. cc: corpus callosum, IF: infarct. (D) Anti-TNF therapy did not affect brain CD11b and iNOS mRNA levels. Brain IL-1β mRNA levels were found increase at 24 hours in etanercept-treated mice, compared to six hours and five days and to saline- and XPro1595-treated mice. Arg1 mRNA levels were found to increase in saline-treated mice at 24 hours compared to six hours and five days and in XPro1595-treated mice at 24 hours compared to five days. IL-10 mRNA levels were found to decrease in XPro1595-treated mice 24 hours and five days compared to six hours (one-way ANOVA, four to six per group). (*P <0.05, **P <001). Arg1, arginase 1; ANOVA, analysis of variance; FSC, forward scatter; IL, interleukin; iNOS, inducible nitric oxide synthase; MFI, mean fluorescent intensity; pMCAO, permanent middle cerebral artery occlusion; SSC, side scatter; TNF, tumor necrosis factor; qPCR, quantitative polymerase chain reaction.