Fig. 4

HMGB1-IL1-β-IL1R mediates the anti-neurogenic effect of the sclerotic hippocampal microenvironment. A Representative images of immunocytochemistry for cellular localization of HMGB1 (red) and DAPI (blue) in 2D hippocampal monolayer cultures. Top panel depicts cytoplasmic pattern of HMGB1 staining whilst the bottom one shows a nuclear localisation of HMGB1 (Scale bar 10 μm). B Treatment with 10 ng/ml IL-1β significantly induced a cytoplasmic phenotype, which can be blocked with IL-1Ra. C Secretion of HMGB1 from 3D hippocampal cell cultures as measured by ELISA significantly decreased with IL-1Ra treatment at 14 and 17 DIV. D No difference in cell death between controls and IL-1Ra-treated 3D cell cultures as measured by LDH assay for the duration of the experiment. E Blocking HMGB1 signalling in 3D hippocampal cultures increased neuronal cell density and proportion by 32 ± 10% (p < 0.05) and 26 ± 1% p < 0.0001 respectively (n = 3). In 2D hippocampal monolayers, addition of Box-A completely blocked the anti-neurogenic effect of IL-1β. Values are means ± SE. Comparisons made using student’s t test for single comparisons (E) and one (B, F) or two-way ANOVA (C, D)with Bonferroni post-hoc for multiple comparisons with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, considered significant. Each experiment included at least 12 3D cultures (discs) from 3 different patients