HMGB1 is released by microglia following injury. (A) Representative images of mixed neuro-glial cell cultures at 7 days in vitro: control (top panel) and scratch injury (bottom panel-site of injury indicated by red lines). (B) Mixed neuro-glial cell cultures consisted of microglia (IB4+ cells), astrocytes (GFAP+ cells), and neurons (Tuj1+ cells). (C) Representative images for nuclear (top panel) and cytoplasmic pattern (bottom panel) of HMGB1 staining (Red). (D) Demonstrates a hyperacute increase in the proportion of microglial (IB4+) cells with nuclear-cytoplasmic HMGB1 translocation at 4hrs post injury with relative to control. This did not reach statistical significance in Tuj1+ or GFAP+ cells. (E) ELISA assays revealed significant increases in extracellular HMGB1 concentration at 6 h post-injury. Data represent mean ± standard error based on a sample that represents at least 10 wells per condition from three different experiments. For comparisons between two conditions, two-tailed student’s T-test was used, and for multiple different conditions, a two‐way ANOVA and one-way ANOVA with Dunnett's multiple comparison test was used. p values of < 0.05 were considered significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). cNSPCs = rat cortical neural stem cell progenitors; DIV = days in vitro.