Hea Jin Ryu

Astroglial loss and edema formation in the rat piriform cortex and hippocampus following pilocarpine‐induced status epilepticus

In the present study we analyzed aquaporin‐4 (AQP4) immunoreactivity in the piriform cortex (PC) and the hippocampus of pilocarpine‐induced rat epilepsy model to elucidate the roles of AQP4 in brain edema following status epilepticus (SE). In non‐SE‐induced animals, AQP4 immunoreactivity was diffusely detected in the PC and the hippocampus. AQP4 immunoreactivity was mainly observed in the endfeet of astrocytes. Following SE the AQP4‐deleted area was clearly detected in the PC, not in the hippocampus. Decreases in dystrophin and α‐syntrophin immunoreactivities were followed by reduction in AQP4 immunoreactivity. These alterations were accompanied by the development of vasogenic edema and the astroglial loss in the PC. In addition, acetazolamide (an AQP4 inhibitor) treatment exacerbated vasogenic edema and astroglial loss both in the PC and in the hippocampus. These findings suggest that SE may induce impairments of astroglial AQP4 functions via disruption of the dystrophin/α‐syntrophin complex that worsen vasogenic edema. Subsequently, vasogenic edema results in extensive astroglial loss that may aggravate vasogenic edema. J. Comp. Neurol. 518:4612–4628, 2010.

Differential expressions of aquaporin subtypes in astroglia in the hippocampus of chronic epileptic rats.

In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus.

P2X7 receptor differentially modulates astroglial apoptosis and clasmatodendrosis in the rat brain following status epilepticus.

Recently, it has been reported that astroglial loss/dysfunction plays a role in epileptogenesis. In addition, astroglial loss is accompanied by up‐regulation of P2X7 receptor expression in microglia. Therefore, we investigated whether P2X7 receptor is involved in astroglial damages induced by status epilepticus (SE). In the present study, astroglial loss showed the regional‐specific manner and the differential responses to P2X7 receptor functions. Both OxATP and brilliant blue G (P2X7 receptor antagonists) infusion prevented apoptotic astroglial loss in the molecular layer of the dentate gyrus and the frontoparietal cortex, while it promoted clasmatodendrosis in the CA1 region as compared to saline treatment. In contrast, BzATP (a P2X7 receptor agonist) treatment exacerbated apoptotic astroglial loss in the molecular layer of the dentate gyrus and the frontoparietal cortex, but alleviated SE‐induced astroglial swelling in the CA1 region. Astroglial loss in the piriform cortex was not affected by P2X7 receptor agonist‐ or antagonist‐infusion. These findings suggest that P2X7 receptor function differently modulates SE‐induced astroglial loss in distinct brain regions.

Decrease in dystrophin expression prior to disruption of brain-blood barrier within the rat piriform cortex following status epilepticus.

Increased permeability of the brain-blood barrier (BBB) in the piriform cortex (PC) has been reported in various animal models of temporal lobe epilepsy. Since BBB disruption induced by epileptogenic insult has not fully clarified, we attempted to determine whether changes in BBB-related molecules are associated with vasogenic edema in the PC. One day after status epilepticus (SE), PC neurons and astrocytes showed a pyknotic nucleus and shrunken cytoplasm accompanied by vasogenic edema. At this time point, SMI-71 (an endothelial barrier antigen) immunoreactivity had decreased in the PC. Prior to vasogenic edema formation (12 h after SE), dystrophin immunoreactivity disappeared within astrocytes, while the change in glial fibrillary acidic protein immunoreactivity was negligible. However, glucose transporter-1 (an endothelial cell marker) had increased at 12 h after SE. These findings indicate that dysfunction of dystrophin induced by SE may result in endothelial and astroglial damage with BBB breakdown and increase vascular permeability, leading to vasogenic edema that is involved in pathogenesis of epileptogenesis.

The protective effects of interleukin-18 and interferon-γ on neuronal damages in the rat hippocampus following status epilepticus.

To elucidate whether interleukin-18 (IL-18) or interferon-γ (IFN-γ) participates in neurodegeneartion, we investigated the changes in IL-18 and IFN-γ systems within the rat hippocampus following status epilepticus (SE). In non-SE induced animals, IL-18, IL-18 receptor α (IL-18Rα), IFN-γ and IFN-γ receptor α (IFN-γRα) immunoreactivity was not detected in the hippocampus. Following SE, IL-18 immunoreactivity was increased in CA1-3 pyramidal cells as well as dentate granule cells. IL-18 immunoreactivity was also up-regulated in astrocytes and microglia/macrophages. IL-18Rα immunoreactivity was detected in astrocytes and microglia/macrophages. IFN-γ immunoreactivity was detected only in astrocytes within all regions of the hippocampus. IFN-γRα immunoreactivity was increased in neurons as well as astrocytes. Intracerebroventricular infusions of recombinant rat IL-18 or IFN-γ alleviated SE-induced neuronal damages, while neutralization of IL-18, IFN-γ or their receptors aggravated them, as compared to saline-infused animals. These findings suggest that astroglial-mediated IFN-γ pathway in response to IL-18 induction may play an important role in alleviation of SE-induced neuronal damages.

Human PEP-1-ribosomal protein S3 protects against UV-induced skin cell death

The consequences of ultraviolet (UV) exposure are implicated in skin aging and cell death. The ribosomal protein S3 (rpS3) is one of the major proteins by which cells counteract the deleterious effects of UV and it plays a role in the repair of damaged DNA. In the present study, we investigated the protective effects of PEP‐1‐rpS3 fusion protein after UV‐induced cell injury. A human rpS3 gene was fused with PEP‐1 peptide in a bacterial expression vector to produce a genetic in‐frame PEP‐1‐rpS3 fusion protein. The expressed and purified fusion proteins were efficiently transduced into skin cells in a time‐ and dose‐dependent manner. Once inside the cells, transduced PEP‐1‐rpS3 fusion protein was stable for 48 h. We showed that transduced PEP‐1‐rpS3 fusion protein increased cell viability and dramatically reduced DNA lesions in the UV exposed skin cells. Immunohistochemical analysis revealed that PEP‐1‐rpS3 fusion protein efficiently penetrated the epidermis as well as the dermis of the subcutaneous layer when sprayed on animal skin. These results suggest that PEP‐1‐rpS3 fusion protein can be used in protein therapy for various disorders related to UV, including skin aging and cancer.

p65/RelA-Ser529 NF-κB Subunit Phosphorylation Induces Autophagic Astroglial Death (Clasmatodendrosis) Following Status Epilepticus

Clasmatodendrosis is an irreversible astroglial degenerative change, which includes extensive swelling and vacuolization of cell bodies and disintegrated and beaded processes. This study was designed to elucidate whether clasmatodendrosis may be one of the autophagy-related degeneration of astrocytes. In this study, clasmatodendritic astrocytes were observed only in the stratum radiatum in the CA1 region. Vacuoles in clasmatodendritic astrocytes showed LAMP-1 immunoreactivity. In addition, both LC3-II and Beclin-1 expression were detected in most of clasmatodendritic astrocytes as well as a few non-vacuolized astrocytes. Clasmatodendritic astrocytes also showed p65/RelA-Ser529 phosphorylation in the nuclei. The neutralization of TNF-α by sTNFp55R infusion reduced clasmatodendritic astrocytes with nuclear p65/RelA-Ser529 phosphorylation. Therefore, these findings suggest that clasmatodendrosis may be autophagic astroglial death in response to epileptic seizures through TNF-α-mediated p65/RelA-Ser529 phosphorylation.

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-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

BackgroundStatus 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.MethodsSE 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.ResultsFollowing 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.ConclusionThese 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.

The reverse roles of transient receptor potential canonical channel-3 and -6 in neuronal death following pilocarpine-induced status epilepticus.

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca2+, which is expressed in many cell types, including neurons. However, the alterations in TRPC receptor expressions in response to status epilepticus (SE) have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 and TRPC6 in neuronal death following SE. In non-SE animals, TRPC3 and TRPC6 immunoreactivity was abundantly detected in the dendrites of pyramidal cells and the cell bodies of dentate granule cells. Following SE, TRPC3 expression was significantly elevated in CA1-, CA3 pyramidal cells, and dentate granule cells, while TRPC6 expression was reduced in these regions. Pyrazole-3 (a TRPC3 inhibitor) effectively prevented up-regulation of neuronal TRPC3 expression induced by SE. Hyperforin (a TRPC6 activator) effectively prevented down-regulation of neuronal TRPC6 expression induced by SE. In addition, both Pyr3 and hyperforin effectively protected neuronal damages from SE. Therefore, the present study yields novel information regarding the role of TRPC3 and 6 in epileptogenic insults and suggests that TRPC 3 and 6 may be involved in neurodegeneration following SE.