P2X7 receptor regulates leukocyte infiltrations in rat frontoparietal cortex following status epilepticus

<p>Abstract</p> <p>Background</p> <p>In the present study, we investigated the roles of P2X7 receptor in recruitment and infiltration of neutrophil during epileptogenesis in rat epilepsy models.</p> <p>Methods</p> <p>Status epilepticus (SE) was induced by pilocarpine in rats that were intracerebroventricularly infused with either saline, 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP), or IL-1Ra (interleukin 1 receptor antagonist) prior to SE induction. Thereafter, we performed immunohistochemical studies for myeloperoxidase (MPO), CD68, interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2).</p> <p>Results</p> <p>In saline-infused animals, neutrophils and monocytes were observed in frontoparietal cortex (FPC) at 1 day and 2 days after SE, respectively. In BzATP-infused animals, infiltrations of neutrophils and monocytes into the FPC were detected at 12 hr and 1 day after SE, respectively. In OxATP-infused animals, neutrophils and monocytes infiltrated into the FPC at 1 day and 2 days after SE, respectively. However, the numbers of both classes of leukocytes were significantly lower than those observed in the saline-infused group. In piriform cortex (PC), massive leukocyte infiltration was detected in layers III/IV of saline-infused animals at 1-4 days after induction of SE. BzATP or OxATP infusion did not affect neutrophil infiltration in the PC. In addition, P2X7 receptor-mediated MCP-1 (released from microglia)/MIP-2 (released from astrocytes) regulation was related to SE-induced leukocyte infiltration in an IL-1β-independent manner.</p> <p>Conclusions</p> <p>Our findings suggest that selective regulation of P2X7 receptor-mediated neutrophil infiltration may provide new therapeutic approaches to SE or epilepsy.</p

Effects of creatine and β-guanidinopropionic acid and alterations in creatine transporter and creatine kinases expression in acute seizure and chronic epilepsy models

<p>Abstract</p> <p>Background</p> <p>In order to confirm the roles of creatine (Cr) in epilepsy, we investigated the anti-convulsive effects of Cr, creatine transporter (CRT) and creatine kinases (CKs) against chemical-induced acute seizure activity and chronic epileptic seizure activity.</p> <p>Results</p> <p>Two hr after pilocarpine (PILO)-seizure induction, ubiquitous mitochondrial CK (uMtCK) immunoreactivity was unaltered as compared to control level. However, brain-type cytoplasm CK (BCK) immunoreactivity was decreased to 70% of control level. CRT immunoreactivity was decreased to 60% of control level. Following Cr or Tat-CK treatment, uMtCK or CRT immunoreactivity was unaffected, while BCK immunoreactivity in Cr treated group was increased to 3.6-fold of control levels. β-Guanidinopropionic acid (GPA, a competitive CRT inhibitor) reduced BCK and CRT expression. In addition, Cr and tat-BCK treatment delayed the beginning of seizure activity after PILO injection. However, GPA treatment induced spontaneous seizure activity without PILO treatment. In chronic epilepsy rats, both uMtCK and CRT immunoreactivities were reduced in the hippocampus. In contrast, BCK immunoreactivity was similar to that observed in control animals. Cr-, GPA and tat-BCK treatment could not change EEG.</p> <p>Conclusion</p> <p>Cr/CK circuit may play an important role in sustaining or exacerbating acute seizure activity, but not chronic epileptic discharge.</p

Tumor necrosis factor-α-mediated threonine 435 phosphorylation of p65 nuclear factor-κB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

<p>Abstract</p> <p>Background</p> <p>Status epilepticus (SE) induces severe vasogenic edema in the piriform cortex (PC) accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF)-α in blood-brain barrier (BBB) disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE.</p> <p>Methods</p> <p>SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, and soluble TNF p55 receptor (sTNFp55R) prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunits.</p> <p>Results</p> <p>Following SE, most activated microglia showed strong TNF-α immunoreactivity. In addition, TNF p75 receptor expression was detected in endothelial cells as well as astrocytes. In addition, only p65-Thr435 phosphorylation was increased in endothelial cells accompanied by SMI-71 expression (an endothelial barrier antigen). Neutralization of TNF-α by soluble TNF p55 receptor (sTNFp55R) infusion attenuated SE-induced vasogenic edema and neuronal damages via inhibition of p65-Thr435 phosphorylation in endothelial cells. Furthermore, sTNFp55R infusion reduced SE-induced neutrophil infiltration in the PC.</p> <p>Conclusion</p> <p>These findings suggest that impairments of endothelial cell functions via TNF-α-mediated p65-Thr 485 NF-κB phosphorylation may be involved in SE-induced vasogenic edema. Subsequently, vasogenic edema results in extensive neutrophil infiltration and neuronal-astroglial loss.</p

P2X7 receptor activation ameliorates CA3 neuronal damage via a tumor necrosis factor-alpha-mediated pathway in the rat hippocampus following status epilepticus

Abstract Background The release of tumor necrosis factor-α (TNF-α) appears depend on the P2X7 receptor, a purinergic receptor. In the present study, we addressed the question of whether P2X7 receptor-mediated TNF-α regulation is involved in pathogenesis and outcome of status epilepticus (SE). Methods SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP), adenosine 5'-triphosphate-2',3'-dialdehyde (OxATP), A-438079, or A-740003 prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunit phosphorylations. Results Following SE, P2X7 receptor agonist (BzATP) infusion increased TNF-α immunoreactivity in dentate granule cells as compared with that in saline-infused animals. In addition, TNF-α immunoreactivity was readily apparent in the mossy fibers, while TNF-α immunoreactivity in CA1-3 pyramidal cells was unaltered. However, P2X7 receptor antagonist (OxATP-, A-438079, and A-740003) infusion reduced SE-induced TNF-α expression in dentate granule cells. In the CA3 region, BzATP infusion attenuated SE-induced neuronal damage, accompanied by enhancement of p65-Ser276 and p65-Ser311 NF-κB subunit phosphorylations. In contrast, OxATP-, A-438079, and A-740003 infusions increased SE-induced neuronal death. Soluble TNF p55 receptor (sTNFp55R), and cotreatment with BzATP and sTNFp55R infusion also increased SE-induced neuronal damage in CA3 region. However, OxATP-, sTNFp55R or BzATP+sTNFp55R infusions could not exacerbate SE-induced neuronal damages in the dentate gyrus and the CA1 region, as compared to BzATP infusion. Conclusions These findings suggest that TNF-α induction by P2X7 receptor activation may ameliorate SE-induced CA3 neuronal damage via enhancing NF-κB p65-Ser276 and p65-Ser311 phosphorylations

Status epilepticus induces vasogenic edema via tumor necrosis factor-α/ endothelin-1-mediated two different pathways.

Status epilepticus (SE) induces vasogenic edema in the piriform cortex with disruptions of the blood-brain barrier (BBB). However, the mechanisms of vasogenic edema formation following SE are still unknown. Here we investigated the endothelin B (ETB) receptor-mediated pathway of SE-induced vasogenic edema. Following SE, the release of tumor necrosis factor-α (TNF-α) stimulated endothelin-1 (ET-1) release and expression in neurons and endothelial cells. In addition, TNF-α-induced ET-1 increased BBB permeability via ETB receptor-mediated endothelial nitric oxide synthase (eNOS) activation in endothelial cells. ETB receptor activation also increased intracellular reactive oxygen species by NADPH oxidase production in astrocytes. These findings suggest that SE results in BBB dysfunctions via endothelial-astroglial interactions through the TNF-α-ET-1-eNOS/NADPH oxidase pathway, and that these ETB receptor-mediated interactions may be an effective therapeutic strategy for vasogenic edema in various neurological diseases

Tumor necrosis factor-alpha-mediated threonine 435 phosphorylation of p65 nuclear factor-kappaB subunit in endothelial cells induces vasogenic edema and neutrophil infiltration in the rat piriform cortex following status epilepticus

Abstract Background Status epilepticus (SE) induces severe vasogenic edema in the piriform cortex (PC) accompanied by neuronal and astroglial damages. To elucidate the mechanism of SE-induced vasogenic edema, we investigated the roles of tumor necrosis factor (TNF)-α in blood-brain barrier (BBB) disruption during vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced SE. Methods SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline-, and soluble TNF p55 receptor (sTNFp55R) prior to SE induction. Thereafter, we performed Fluoro-Jade B staining and immunohistochemical studies for TNF-α and NF-κB subunits. Results Following SE, most activated microglia showed strong TNF-α immunoreactivity. In addition, TNF p75 receptor expression was detected in endothelial cells as well as astrocytes. In addition, only p65-Thr435 phosphorylation was increased in endothelial cells accompanied by SMI-71 expression (an endothelial barrier antigen). Neutralization of TNF-α by soluble TNF p55 receptor (sTNFp55R) infusion attenuated SE-induced vasogenic edema and neuronal damages via inhibition of p65-Thr435 phosphorylation in endothelial cells. Furthermore, sTNFp55R infusion reduced SE-induced neutrophil infiltration in the PC. Conclusion These findings suggest that impairments of endothelial cell functions via TNF-α-mediated p65-Thr 485 NF-κB phosphorylation may be involved in SE-induced vasogenic edema. Subsequently, vasogenic edema results in extensive neutrophil infiltration and neuronal-astroglial loss

ETB receptor-mediated reduction of dystrophin and AQP4 expression in astrocytes.

<p>(<b>A</b>–<b>C</b>) Effects of BQ788 and Cav1-peptide on dystrophin and AQP4 mRNA/protein expression levels 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (D–F) Effects of BQ788 and Cav-1 peptide on dystrophin and AQP4 expression in astrocytes 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. Scale bar: <b>D</b> and <b>E</b>, 12.5 μm.</p

SE-induced vasogenic edema formation via the ETB receptor-mediated eNOS pathway.

<p>(<b>A</b>–<b>B</b>) Effect of BQ788 on SMI-71 expression and ETB receptor expression 12 h after SE. (<b>C</b>) Effect of BQ788 on ET_B receptor mRNA expression 12 h after SE (means ± s.e.m., n = 5, respectively); paired Student’s t-test. (<b>D</b>) Nitrate/nitrite (NO products) concentration in the PC after SE (mean ± s.d., n = 5): *P < 0.05 versus basal level; paired Student’s t-test. (<b>E</b>–<b>G</b>) Effects of BQ788 and Cav1-peptide on eNOS mRNA/protein expression 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (H–J) Effects of of BQ788 and Cav1-peptide on eNOS, SMI-71 and NT expression 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (<b>K</b>–<b>N</b>) Quantification of vasogenic edema formation 3days after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus vehicle-treated animals; #P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. Scale bars: <b>A, B, H</b> and <b>I</b>, 25 μm; <b>K-M</b>, 400 μm.</p

The roles of TNF-α in SE-induced vasogenic edema in the PC.

<p>(<b>A</b>) The extracellular TNF-α concentration after SE (mean ± s.d., n = 5): *P < 0.05 versus the basal level; paired Student’s t-test. (<b>B</b> and <b>C</b>) Quantification of western blots for TNF-α protein expression and p65-Thr435 NFκB phosphorylation 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 by Student’s t-test. (<b>D</b>–<b>I</b>) Immunofluorescence data for TNFp75R, p65-Thr435 NFκB phosphorylation and SMI-71 12 h after SE. (<b>J</b>–<b>K</b>) Effects of TNFp55R and SN50 on p65-Thr435 NF-κB phosphorylation and SMI-71 immunoreactivity. (<b>L</b>–<b>M</b>) Quantification of the fluorescence intensities of SMI-71 expression and p65-Thr435 NFκB phosphorylation 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; one-way analysis of variance (ANOVA) followed by Tukey’s test. (<b>N</b>–<b>Q</b>) Quantification of vasogenic edema attenuation by sTNFp55R and SN50 3 days after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus vehicle treated animals by one-way ANOVA followed by Tukey’s test. Scale bars: <b>D–K</b>, 25 μm; <b>O-Q</b>, 400 μm.</p