Liver and brain TNF expression following anti- TNF therapy. (A) TNF mRNA+ cells six hours, 24 hours and five days after pMCAO (shown for saline-treated mice). Scale bar: 30 μm. TNF mRNA levels increased transiently at 24 hours (four to six per group). (B) A few TNF mRNA+ cells were located in the liver preferentially at six hours (shown for saline-treated mouse), but also to some extent in etanercept-treated mice at 24 hours. Scale bar: 30 μm. Liver TNF mRNA levels were decreased in saline-treated mice at 24 hours compared to six hours and in XPro1595-treated mice at 24 hours compared to six hours and five days (*P <0.05, **P <0.01, three to six per group). (C) TNF stained sections from mice that had survived six hours (inserts), 24 hours and five days. At 24 hours and five days TNF protein expression was localized to the infarct and peri-infarct and cells displayed microglial and leukocyte morphology. Scale bars = 200 μm (low magnifications) and 20 μm (high magnifications). (D) Western blots for TNF levels after pMCAO demonstrating reduced TNF levels at six hours in XPro1595- (27.9% ± 3.3%) and etanercept-treated (16.2% ± 2.3%) mice compared to saline-treated mice (100% ± 1.3%) (***P <0.001, one-way ANOVA, followed by Bonferroni post-hoc test). At 24 hours, TNF protein levels were comparable in saline- (100% ± 50.3%), XPro1595- (102.7% ± 3.4%) and etanercept-treated (93.2% ± 20.4%) mice. (E) Flow cytometry profiles gated on CD11b+CD45+ cells showing primarily TNF+ microglia at 24 hours after pMCAO. Only few TNF+ macrophages and TNF+ granulocytes were present at 24 hours (four to six per group, **P <0.01; ****P <0.0001). ANOVA, analysis of variance; d, days; FMO, fluorescence minus one; h, hours; pMCAO, permanent middle cerebral artery occlusion; rTNF, recombinant TNF; TNF, tumor necrosis factor; qPCR, quantitative polymerase chain reaction.