PMC:3312845 / 18171-28469 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3312845","sourcedb":"PMC","sourceid":"3312845","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3312845","text":"Results\n\nTNF-α, and TNF receptor expression\nIn non-SE induced animals of the saline-infused groups, TNF-α immunoreactivity was weakly detected in PC neurons (data not shown). In 12 hr-post SE animals of the saline-infused group, most of the activated microglia showed strong TNF-α immunoreactivity (Figure 1A). This expression pattern was maintained up to 1 week after SE. In non-SE-induced animals of the saline-infused groups, TNFp55R and TNFp75R immunoreactivities were also weakly observed in astrocytes (data not shown). In 12 hr-post SE animals of the saline-infused group, TNFp55R immunoreactivity was observed in astrocytes (Figure 1B). Unlike TNFp55R, TNFp75R immunoreactivity was detected in endothelial cells as well as astrocytes (Figures 1C-E). One day to 1 week after SE, both TNFp55R and TNFp75R immunoreactivities were significantly reduced in astrocytes, not in endothelial cells, due to massive astroglial loss (data not shown) [5,7,8,20].\nFigure 1 Expression of TNF-α and TNF receptor in the PC 12 hr-post SE. (A) TNF-α immunoreactivity in Ox-42-positive microglia (arrows). (B) TNFp55R expression in astrocytes (arrows). (C-D) TNFp75R expression in astrocytes as well as endothelial cells (arrows). Bar = 25 (A-D) μm.\n\nEffect of sTNFp55R infusion on SE-induced serum-protein extravasation and neuronal damage\nIn our previous [5,7,8,20] and preliminary data, vasogenic edema and neuronal damage were noticeable at 1 day and 3 days after SE, respectively. Therefore, we determined that 3 days after SE was the best time point to evaluate the effect of sTNFp55R infusion on both vasogenic edema and neuronal damages induced by SE. In saline-treated animals, the PC was stained diffusely with anti-rat IgG (Figure 2A). The volume of vasogenic edema was 17.1 ± 1.5 mm3 (Figure 2E). The number of FJB-positive neurons in the PC was 236,145 ± 49,469 (Figure 2B). In sTNFp55R-treated animals, SE-induced vasogenic edema was attenuated to 9.8 ± 0.7 mm3 (Figures 2C and 2E). In addition, the number of FJB-positive neurons in the PC was 89,138 ± 5,698 (Figures 2D-E). Thus, sTNFp55R infusion attenuated SE-induced vasogenic edema and neuronal damage compared to saline-infused animals (p \u003c 0.05).\nFigure 2 Effect of sTNFp55R infusion on SE-induced serum-protein extravasation and neuronal damage. (A-D) Serum-protein extravasation and FJB-positive neuronal damages in the PC 3 days after SE. Compared to saline-infused animals, serum-protein extravasation and FJB-positive neuronal damage is markedly ameliorated in sTNFp55R-infused animals. Bars = 400 (A and C) and 50 (B and D) μm. (E) Quantitative analyses of serum-protein extravasation and FJB-positive neuronal damage in the PC 3 days after SE (mean ± S.E.M). Significant differences from saline-treated animals, *p \u003c 0.05.\n\nNF-κB phosphorylation\nIt 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 \u003c 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 \u003c 0.05, Figure 5B).\nFigure 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.\nFigure 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 \u003c 0.05. (B) Quantitative analysis of SMI-71 expression 1 day after SE (mean ± S.E.M). Significant differences from saline-infused animals, *p \u003c 0.05. (C) Linear regression analysis between p65-Thr435 phosphorylation and SMI-71 in the PC following SE.\nFigure 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.\n\nSMI-71 expression\nPreviously, we reported that SMI-71 (an endothelial barrier antigen) immunoreactivity decreased in the PC 1 day after SE [5]. Similarly, in 1 day-post SE animals of the saline-infused group, loss of SMI-71 immunoreactivity was detected in layer III/IV of the PC as compared to non-SE animals (Figures 4B and 5C-E, p \u003c 0.05). Thus, loss of SMI-71 immunoreactivity correlated with volume of vasogenic edema following SE. This reduction in SMI-71 was accompanied by increased p65-Thr435 phosphorylation (Figures 5C-D). Therefore, the degree of SMI-71 immunoreactivity was inversely correlated to p65-Thr435 phosphorylation with a linear coefficient of correlation of -0.6324 (p \u003c 0.05; Figure 4C). In addition, sTNFp55R infusion effectively alleviated p65-Thr435 phosphorylation and preserved SMI-71 immunoreactivity in endothelial cells following SE, as compared to saline infusion (p \u003c 0.05; Figures 4A-B, 5D and 5F).\n\nNeutrophil infiltration\nRecent studies have reported that neutrophils infiltrate the brain under certain pathological conditions [26]. Indeed, we have reported massive neutrophil infiltration in layer III/IV of the PC 1 day after SE [20]. In the present study, 1 day-post-SE animals of the saline-infused group showed infiltration of MPO-positive neutrophils into the PC. Similarly, 1 day-post-SE animals of the sTNFp55R-infused group showed neutrophil infiltration into the PC 1 day after SE (Figure 6A). The number of neutrophils/area in the PC region (including the vasogenic edema region and the non-vasogenic edema region) of sTNFp55R-infused animals was significantly lower than that of the saline-infused group (Figure 6D, p \u003c 0.05). However, there was no difference in neutrophil infiltration per unit area of vasogenic edema between the saline- and sTNFp55R-infused groups (Figure 6D). Furthermore, neutrophil infiltration showed a direct proportion to the area of vasogenic edema, with a linear coefficient of correlation of 0.8631 (p \u003c 0.05, Figure 6E). Therefore, our findings indicate that SE-induced neutrophil infiltration into the PC may be correlated to TNF-α-mediated vasogenic edema formation.\nFigure 6 Effect of sTNFp55R infusion on neutrophil infiltration and MIP-2 expression following SE. (A) Neutrophil infiltration in vasogenic edema lesion 1 day after SE. (B) MIP-2 expression in the PC 1 day after SE. (C) Astroglial expression of MIP-2 (arrows). Bars = 12.5 (A and C) and 150 (B) μm. (D) Quantitative analysis of neutrophil infiltration 1 day after SE (mean ± S.E.M). Significant differences from saline-infused animals, *p \u003c 0.05. (E) Linear regression analysis between the number of infiltrated neutrophils/area in the vasogenic edema region and the area of vasogenic edema in the PC. (F) Quantitative analysis of the number of MIP-2 positive cells per the unit area of vasogenic edema 1 day after SE (mean ± S.E.M). There is no difference in the number of MIP-2-positive cells per unit area of vasogenic edema between sTNFp55R-infused animals and saline-infused animals. (G) Linear regression analysis between the number of MIP-2 positive cells per unit area in vasogenic edema region and the area of vasogenic edema in the PC.\n\nMIP-2 expression\nMIP-2 is a powerful chemokine that contributes to recruitment of neutrophils [27]. MIP-2 is undetectable or present at low levels under physiological conditions, and shows transient increases under pathological conditions via TNF-α and/or interleukin-1β (IL-1β)-dependent mechanisms [14]. Thus, it would be plausible that TNF-α-mediated MIP-2 expression may provoke SE-induced neutrophil infiltrations. To confirm this hypothesis, we investigated MIP-2 expression in the PC. Consistent with our previous study [20], some MIP-2-positive astrocytes were observed in the core and periphery of the vasogenic edema lesions, but not in the non-vasogenic edema region (Figure 6B and 6C). Although the number of MIP-2 positive cells per unit area in the PC region of sTNFp55R-infused animals was significantly lower than that of the saline-infused group due to reduction of the area of vasogenic edema, there was no difference in the number of MIP-2 positive cells per unit area of vasogenic edema between sTNFp55R-infused animals and saline-infused animals (Figure 6F). Furthermore, the number of MIP-2-positive cells showed a direct proportion to the unit area of vasogenic edema with a linear coefficient of correlation of 0.682 (p \u003c 0.05, Figure 6G). Therefore, together with reduction in neutrophil infiltration in the PC region of sTNFp55R-infused animals, our findings provide evidence that TNF-α may regulate SE-induced neutrophil infiltration at least in the PC via vasogenic edema formation and not via direct TNF-α-mediated MIP-2 expression in astrocytes.","divisions":[{"label":"title","span":{"begin":0,"end":7}},{"label":"sec","span":{"begin":9,"end":1238}},{"label":"title","span":{"begin":9,"end":43}},{"label":"p","span":{"begin":44,"end":957}},{"label":"figure caption","span":{"begin":958,"end":1238}},{"label":"p","span":{"begin":968,"end":1238}},{"label":"sec","span":{"begin":1240,"end":2791}},{"label":"title","span":{"begin":1240,"end":1329}},{"label":"p","span":{"begin":1330,"end":2207}},{"label":"figure 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