The effect of anti- TNF therapy on the number of granulocytes in the infarct. (A) Representative photomicrographs of TB-stained brain sections from saline-, XPro1595-, and etanercept-treated mice allowed to survive for 24 hours after focal cerebral ischemia, demonstrating infiltration of polymorphonucleated cells into the ischemic infarct. Co-localization of polymorphonucleated cells in TB-stained sections with a granulocyte (Gr1) marker was verified using immunohistochemistry in saline-treated mice allowed to survive for 24 hours. Scale bars: left 30 μm and right 10 μm. (B) Estimation of the number of infiltrating polymorphonucleated cells per mm2 within the infarct showed a significantly increased number of cells in saline-treated mice (58.6 ± 14.1 granulocytes/mm2) compared to XPro1595-treated mice (17.4 ± 3.0 granulocytes/mm2) 24 hours after ischemia (*P <0.05, one-way ANOVA, followed by Bonferroni post-hoc test; four to eight per group). (C) Flow cytometry analysis of the number of infiltrating CD11b+CD45highGr1+ cells in the ipsilateral neocortex of non-lesioned control mice and mice six and 24 hours after pMCAO showed a significant increase in the total number of infiltrating granulocytes in saline-, XPro1595- and etanercept-treated mice at 24 hours compared to non-lesioned control mice. No difference between treatment groups was observed (four to six per group). ANOVA, analysis of variance; h, hours; pMCAO, permanent middle cerebral artery occlusion; TB, toluidine blue; TNF, tumor necrosis factor.