Modulating Immune Responses in Stroke

Platelets were depleted by i.v. injection of 2 mg/kg platelet-depleting antibodies (R300, Emfret) immediately after stroke induction. Platelet counts were measured using a Hemavet hematology analyzer (Drew Scientific). Neutrophils were depleted by i.p. injection of 5 mg/kg anti–mouse Ly6G (1A8, BE0074-1, Bio X Cell) and 5 mg/kg anti–rat Kappa IgG (MAR 18.5, BE0122, Bio X Cell) (22 (link)), 24 hours before stroke induction. Neutrophil depletion was confirmed via flow cytometry using granularity and Ly6C staining (Supplemental Figure 10 and ref. 22 (link)). Recombinant disulfide HMGB1 (rHMGB1; HM-120, HMGBiotech) was administered at 0.5 mg/kg i.v. 1 hour after stroke onset. HMGB1 was blocked by injection of 15 mg/kg of BoxA (HM-014, HMGBiotech) immediately before stroke induction. Neonatal NET-inhibitory factor (nNIF) and its inactive, scrambled peptide control (SCR) were synthesized as previously described by the University of Utah DNA/Peptide Synthesis Core Facility and injected i.v. at a concentration of 10 mg/kg at the indicated time points. PAD4 was inhibited by i.v. injection of GSK-199 (17489, Cayman Chemical) at 30 mg/kg, and NETs were degraded by i.v. injection of DNase I (dornase alfa, University of Utah Pharmacy) at 2.5 mg/kg, immediately before stroke induction.