Fig. 2

Extracellular histones are not cytotoxic to mouse brain endothelial cells or native cerebrovascular endothelium. a Cytotoxic effect of histones (10, 25, 50, 100, and 250 μg/mL) on brain endothelial cells assessed by PI uptake and analyzed by flow cytometry after 3 h treatment. (Top) Histograms showing percentage of total PI uptake in brain endothelial cells exposed to different histone concentrations as representative from one experiment. (Bottom) Summary data showing histone-induced cytotoxicity expressed as percentage cell viability relative to vehicle-treated cells after exposure of histones. Mean ± S.E.M. of 6–8 experiments. b TEER measurements of brain endothelial cell monolayers treated with vehicle (saline) or histones (10, 25, 50, and 100 μg/mL) in the presence of the pan caspase inhibitor ZVAD-fmk (100 μM; 1 h treatment). Mean ± S.E.M. Treatments were done in triplicate with three independent biological replicates. c Representative traces illustrating lumen diameter responses in a cannulated posterior cerebral artery pressurized to 60 mmHg isolated from a saline and a histone-injected mouse. Endothelial-dependent dilations were assessed by SK/IK channel activation using NS309 (0.1–1 μM). d Summary data showing percent dilation to NS309 relative to passive (0 calcium) in arteries isolated from saline- and histone-treated mice. Data are presented as means ± S.E.M