Discussion
The major findings in the present study are that TNF-α signaling showed cellular specific responses of NF-κB phosphorylation in the PC following SE, which may be related to vasogenic edema formation followed by neutrophil infiltration. BBB disruption has been reported in experimental and human epilepsy [12,13,15,16,28]. Leakage of serum-derived components into the extracellular space is associated with hyperexcitability and seizure onset [12,13,15,16,28]. Furthermore, dysfunction of the BBB leads to epileptogenesis and contributes to progression of epilepsy [12,13,15,16,28]. In the present study, TNF-α immunoreactivity was obviously observed in microglia in the PC following SE. TNF receptor expressions were also up-regulated in astrocytes (TNFp55R and TNFp75R) and endothelial cells (TNFp75R). Furthermore, blockade of TNF-α signaling by sTNFp55 infusion effectively (but not completely) reduced volumes of SE-induced vasogenic edema and neuronal damage in the PC. These findings indicate that TNF-α may participate in astroglial and endothelial responses to SE, which are relevant to SE-induced vasogenic edema formation [5-8]. Indeed, TNF-α signaling increases BBB permeability in various experimental disease models [29]. In the present study, sTNFp55 infusion could not completely prevent SE-induced vasogenic edema and neuronal damage in the PC. Therefore, our findings suggest that TNF-α signaling may not be a unique upstream event in vasogenic edema development.
p65 phosphorylation of NF-κB enhances its transactivation potential, and p65 phosphorylation occurs in either the cytoplasm or the nucleus [30]. In the present study, p65-Thr435 immunoreactivity was detected in endothelial cells, and its immunoreactivity showed an inverse correlation to the degree of SMI-71 expression. SMI-71, an endothelial barrier antigen, is a protein expressed by endothelial cells of rat BBB [31]. Under pathological conditions, SMI-71 expression is lost in endothelial cells [5,7,8,30,32]. Acute phases of the above pathological conditions are accompanied by opening of the BBB and development of vasogenic edema [33]. Indeed, neutralization of SMI-71 in vivo leads to widening of intercellular junctions between endothelial cells and swelling of perivascular astrocytic processes [34], although SMI-71 is not localized at endothelial cell junctions [35-38]. In the present study, SMI-71 immunoreactivity was significantly reduced in blood vessels 1 day after SE when vasogenic edema and neuronal damage were observed. Furthermore, sTNFp55R infusion effectively prevented SE-induced SMI-71 down-regulation. With respect to the phosphorylation of p65-Thr435 by TNF-α [39], our findings indicate that TNF-α-mediated p65-Thr435 phosphorylation in endothelial cells may play an important role in vasogenic edema induction via SMI-71 degradation or its posttranslational dysfunction influencing BBB permeability.
In our previous studies [5,8], dystrophin (an actin-binding protein [40]) immunoreactivity was detected in blood vessels and in astrocytic perivascular end-feet, and was down-regulated 12 hrs after SE prior to the appearance of vasogenic edema and down-regulation of SMI-71 immunoreactivity. With respect to this previous report, changes in SMI immunoreactivity would be causes/results of interaction between endothelial cells and perivascular astrocytes. In the present study, p65-Ser276, p65-Ser311, p65-Ser529, and p65-Ser536 phosphorylation was observed in astrocytes following SE. Furthermore, sTNFp55R infusion effectively inhibited p65-Ser276 and p65-Ser311phosphorylation in astrocytes following SE. Therefore, it is likely that enhanced p65-Ser276 and p65-Ser311 phosphorylation may be involved in TNF-α-mediated BBB disruption. However, sTNFp55R infusion could not prevent p65-Ser529 and p65-Ser536 phosphorylations from SE insults. Since p65-Ser529 and p65-Ser536 are phosphorylated by TNF-α and IL-1β [41], it is likely that IL-1β-mediated p65-Ser529/Ser536 phosphorylation may also play a role in SE-induced vasogenic edema. Therefore, our findings indicate that both TNF-α and IL-1β may be synergists to play either a direct (by endothelial cells) or indirect (by astrocytes) role in the maintenance of BBB permeability.
Neutrophil infiltration into brain parenchyma is transiently observed during the acute phase of SE (4 - 36 hr after SE) and disappears thereafter [20]. SE rapidly increases synthesis and release of chemokines in various areas of the rodent brain [42]. Among them, MIP-2 is required for efficient neutrophil or lymphocyte recruitment to brain parenchyma [43]. In our previous study [20], neutrophil infiltration in the frontoparietal cortex was regulated by P2X7 receptor-mediated MIP-2 expression. In the PC, however, neither a P2X7 receptor agonist/antagonist nor IL-1Ra (an IL-1β antagonist) infusion could not affect leukocyte infiltration. In the present study, sTNFp55R infusion effectively inhibited neutrophil infiltration in the PC by reducing vasogenic edema formation in a MIP-2-independent manner. With respect to the present and our previous reports, it is therefore likely that vasogenic edema induced by TNF-α can induce neutrophil infiltration and press injury to evoke neuronal-astroglial loss in the PC, unlike other brain regions.
In conclusion, our findings reveal that impairments of endothelial cell function via TNF-α mediated p65-Thr 435 NF-κB phosphorylation may be involved in SE-induced vasogenic edema, which is relevant to neutrophil infiltration and neuronal-astroglial loss.