Mice were subjected to 1 hour of tMCAO followed by 23 hours of reperfusion or sham surgery. Plasma was isolated and brains were analyzed for ischemic stroke brain damage by TTC staining 24 hours after stroke onset. Upon TTC staining, live brain tissue will stain red, while dead brain tissue will remain white (outlined with black dotted line). (A–D) Immediately after stroke onset, mice were injected with a platelet-depleting antibody or IgG control. Twenty-four hours later, plasma HMGB1 (A) and MPO-DNA complexes (B) were assessed as well as brain infarct volume (C and D). n = 6 for sham mice; n = 9 for groups subjected to stroke. (E–G) Immediately after stroke onset, mice were injected with a platelet-depleting antibody. One hour later, either recombinant HMGB1 (rHMGB1) or vehicle was administered. Twenty-four hours after stroke induction, plasma MPO-DNA complexes (E) were assessed as well as brain infarct volume (F and G). n = 7–9 per group. (H–K) Twenty-four hours before stroke induction, neutrophils were depleted by i.p. injection of neutrophil-depleting antibodies. Control mice were injected with an IgG control antibody. Twenty-four hours later, plasma HMGB1 (H) and MPO-DNA complexes (I) were assessed as well as brain infarct volume (J and K). n = 7–8 per group. (L–N) Twenty-four hours before stroke induction, neutrophils were depleted by i.p. injection of neutrophil-depleting antibodies. One hour after stroke onset, either rHMGB1 or vehicle was administered. Twenty-four hours after stroke induction, plasma MPO-DNA complexes (L) were assessed as well as brain infarct volume (M and N). n = 6–7 per group. Groups were compared by ordinary 1-way ANOVA (A, B, and D) or unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001.