NF-κB phosphorylation
It is well established that TNF-α is one of the major stimuli toward phosphorylation of NF-κB. To confirm TNF-α-mediated signaling following SE, we performed an immunohistochemical study using five phospho-NF-κB antibodies. Compared to non-SE animals (data not shown), 12 hr-post SE animals of the saline-infused group showed p65-Ser276, p65-Ser311, p65-Ser529, and p65-Ser536 phosphorylation in astrocytes (not endothelial cells). sTNFp55R infusion effectively reduced p65-Ser276 and p65-Ser311 phosphorylation (p < 0.05, respectively), while it could not affect p65-Ser529 or p65-Ser536 phosphorylation (Figures 3 and 4A). In contrast, p65-Thr435 phosphorylation was increased in endothelial cells (not astrocytes) within the PC of saline-infused animals 12 hr after SE (Figure 5A). In addition, sTNFp55R infusion effectively alleviated SE-induced p65-Thr435 phosphorylation in endothelial cells, compared to saline infusion (p < 0.05, Figure 5B).
Figure 3  Effect of sTNFp55R infusion on NF-κB phosphorylation in astrocytes 12 hr after SE. In 12 hr-post SE animals of the saline-infused group (A, C, E and G), astrocytes show p65-Ser276 (A), p65-Ser311 (C), p65-Ser529 (E), and p65-Ser536 (G) phosphorylation (arrows). sTNFp55R infusion (B, D, F and H) effectively reduces p65-Ser276 (B) and p65-Ser311 (D) phosphorylation, while it does not affect p65-Ser529 (F) and p65-Ser536 (H) phosphorylation (arrows). Bar = 12.5 μm.
Figure 4  Quantitative analyses of the effect of sTNFp55R infusion on NF-κB phosphorylation and SMI-71 expression. (A) Quantitative analysis of NF-κB phosphorylation 12 hr after SE (mean ± S.E.M). Significant differences from saline-infused animals, *p < 0.05. (B) Quantitative analysis of SMI-71 expression 1 day after SE (mean ± S.E.M). Significant differences from saline-infused animals, *p < 0.05. (C) Linear regression analysis between p65-Thr435 phosphorylation and SMI-71 in the PC following SE.
Figure 5  Effect of sTNFp55R infusion on p65-Thr435 phosphorylation in endothelial cells following SE. (A-B) Inhibition of p65-Thr435 phosphorylation by sTNFp55R infusion 12 hr after SE. p65-Thr435 phosphorylation is rarely observed in astrocytes (arrows). (C) Endothelial p65-Thr435 phosphorylation in non-SE animals. (D-E) Endothelial p65-Thr435 phosphorylation in saline-infused animals 1 day after SE. p65-Thr435 phosphorylation is enhanced, while SMI-71 expression is reduced in GLUT-1-positive endothelial cells (arrows). (F-G) Endothelial p65-Thr435 phosphorylation in sTNFp55R-infused animal 1 day after SE. sTNFp55R infusion effectively reduces p65-Thr435 and preserves SMI-71 expression in GLUT-1-positive endothelial cells (arrows). Bars = 12.5 (A-D) and 25 (E-G) μm.