Figure 4.

QHRD106 decreased neuronal and microglial HMGB1 translocation in vivo via B2R

(A and B) Immunofluorescence of HMGB1 (red) in neuronal cells (NeuN⁺; green) in the mouse ischemic penumbra 1 and 3 days after stroke. Arrows point to the location of HMGB1. The low-magnification illustration in the white box is displayed in the high-magnification view in the right column. DAPI staining is shown in blue.

(C and D) Quantification of the percentage of neurons with HMGB1 cytoplasmic translocation in total neurons of the ischemic penumbra 1 and 3 days after stroke.

(E and F) Immunofluorescence of HMGB1 (red) in microglial cells (Iba-1⁺; green) in the mouse ischemic penumbra 1 and 3 days after stroke. The arrows point to the location of HMGB1. The low-magnification illustration in the white box is displayed in the high-magnification view in the right column. The microglia in the yellow box were selected for 3D reconstruction. DAPI staining is shown in blue.

(G) Quantification of the percentage of microglia with HMGB1 cytoplasmic translocation in total microglial cells of the ischemic penumbra 3 days after stroke.

(H–J) Confocal images were converted to binary images and then skeletonized. Microglial morphological analysis, including calculations of the average number of branches, average number of junctions, and average branch length, was performed with the Analyze Skeleton plugin of ImageJ.

(K) Quantification of the mean fluorescence intensity of Iba-1 1 and 3 days after stroke.

All data are presented as the mean ± SEM, n = 3/group. Statistical analyses were performed by one-way or two-way ANOVA with Tukey’s post hoc test. ∗p < 0.05, ∗∗p < 0.01 vs. the saline group; #p < 0.05, ##p < 0.01 vs. the QHRD106 group, “ns” indicates no significance (p > 0.05).