Soo-Young Choi

Epileptogenic roles of astroglial death and regeneration in the dentate gyrus of experimental temporal lobe epilepsy

Recent studies have demonstrated that blockade of neuronal death in the hippocampus cannot prevent epileptogenesis in various epileptic models. These reports indicate that neurodegeneration alone is insufficient to cause epilepsy, and that the role of astrocytes in epileptogenesis should be reconsidered. Therefore, the present study was designed to elucidate whether altered morphological organization or the functionalities of astrocytes induced by status epilepticus (SE) is responsible for epileptogenesis. Glial responses (reactive microgliosis followed by astroglial death) in the dentate gyrus induced by pilocarpine‐induced SE were found to precede neuronal damage and these alterations were closely related to abnormal neurotransmission related to altered vesicular glutamate and GABA transporter expressions, and mossy fiber sprouting in the dentate gyrus. In addition, newly generated astrocytes showed down‐regulated expressions of glutamine synthase, glutamate dehydrogenase, and glial GABA transporter. Taken together, our findings suggest that glial responses after SE may contribute to epileptogenesis and the acquisition of the properties of the epileptic hippocampus. Thus, we believe that it is worth considering new therapeutic approaches to epileptogenesis involving targeting the inactivation of microglia and protecting against astroglial loss.

Spatiotemporal characteristics of astroglial death in the rat hippocampo‐entorhinal complex following pilocarpine‐induced status epilepticus

Recently we reported that astroglial loss and subsequent gliogenesis in the dentate gyrus play a role in epileptogenesis following pilocarpine‐induced status epilepticus (SE). In the present study we investigated whether astroglial damages in the hippocampo‐entorhinal complex following SE are relevant to pathological or electrophysiological properties of temporal lobe epilepsy. Astroglial loss/damage was observed in the entorhinal cortex and the CA1 region at 4 weeks and 8 weeks after SE, respectively. These astroglial responses in the hippocampo‐entorhinal cortex were accompanied by hyperexcitability of the CA1 region (impairment of paired‐pulse inhibition and increase in excitability ratio). Unlike the dentate gyrus and the entorhinal cortex, CA1 astroglial damage was protected by conventional anti‐epileptic drugs. α‐Aminoadipic acid (a specific astroglial toxin) infusion into the entorhinal cortex induced astroglial damage and changed the electrophysiological properties in the CA1 region. Astroglial regeneration in the dentate gyrus and the stratum oriens of the CA1 region was found to originate from gliogenesis, while that in the entorhinal cortex and stratum radiatum of the CA1 region originated from in situ proliferation. These findings suggest that regional specific astroglial death/regeneration patterns may play an important role in the pathogenesis of temporal lobe epilepsy. J. Comp. Neurol. 511:581–598, 2008.

Levetiracetam inhibits interleukin-1β inflammatory responses in the hippocampus and piriform cortex of epileptic rats

Levetiracetam (LEV, 2S-(oxo-1-pyrrolidinyl)butanamide, Keppra, UCB Pharma) is a new anti-epileptic drug used to treat certain types of seizures in epilepsy patients. However, the pharmacodynamics of LEV is still controversial. Recently, interleukin-1 beta (IL-1 beta) has been reported to involve in epileptic phenomena. Therefore, we investigated the effects of LEV on IL-1 beta system in the hippocampus and piriform cortex of chronic epileptic rats. As compared to controls, typical reactive astrogliosis and microgliosis were observed in the hippocampus and piriform cortex of epileptic animals. In addition, both reactive astrocytes and reactive microglia showed strong IL-1 beta and interleukin-1 receptor subtype 1 (IL-1R1) immunoreactivities. LEV reduced reactive gliosis and expression levels of IL-1 beta system in the hippocampus and the piriform cortex, while valproic acid did not. These findings suggest that the LEV may have, at least in part, anti-inflammatory effect, particularly against IL-1 beta system in neuroglia within epileptic brains.

Effects of GABAergic transmissions on the immunoreactivities of calcium binding proteins in the gerbil hippocampus.

Although reduced calcium binding protein (CBP) immunoreactivities in the epileptic hippocampus have been well established, it has been controversial that these changes may directly indicate neuronal degeneration. In the present study, therefore, we investigated CBP expressions in the gerbil hippocampus following treatment with γ‐aminobutyric acid (GABA) receptor antagonists in order to assess whether altered CBP expressions are the result of either abnormal excitation or indicative of neuronal damage/degeneration. Seizure‐sensitive (SS) gerbils showed a loss/decline of CBP immunoreactivities in some hippocampal neurons as compared with seizure‐resistant (SR) gerbils. In muscimol (GABAA receptor agonist) treated SS gerbils, expression levels of CBP were enhanced as compared with saline‐treated SS gerbils. Bicuculline (a GABAA receptor antagonist) treatment markedly reduced CBP immunoreactivities in hippocampal neurons of the SR gerbil. Baclofen (a GABAB receptor agonist) treatment increased CBP immunoreactivities in the hippocampus of SS gerbils, although its effect was lower than that of muscimol treatment. Moreover, phaclofen (GABAB receptor antagonist) treated SR gerbil showed reduction in calbindin D‐28K immunoreactivity, not parvalbumin immunoreactivity, in the hippocampus. These findings therefore suggest that reduced CBP immunoreactivities may be the consequence of abnormal discharge caused by loss of GABAergic inhibition rather than an indication of the neuronal damage/degeneration. J. Comp. Neurol. 485:153–164, 2005.

Bilateral enhancement of excitation via up-regulation of vesicular glutamate transporter subtype 1, not subtype 2, immunoreactivity in the unilateral hypoxic epilepsy model.

In the present study, the change of vesicular glutamate transporter (VGLUT) immunoreactivity on long-term impaired excitability in the hippocampus after recovery from unilateral hypoxic-ischemic insult was investigated in order to extend our understanding of the mechanism of epileptogenesis using unilateral hypoxic epilepsy models. Both the lesioned (submitted to ischemia) and the unlesioned hippocampi exhibited the frequent occurrence of interictal spikes and occasionally the sustained ictal discharges. However, paired-pulse inhibition was significantly reduced in the unlesioned dentate gyrus, not in the lesioned dentate gyrus. VGLUT1 immunoreactivity was significantly elevated in both hippocampi following hypoxic ischemia, although VGLUT2 immunodensity was unaltered. These findings suggest that the enhancement of VGLUT1 immunoreactivity in both hippocampi after unilateral hypoxic ischemia may contribute to the hyperexcitability, which may play an important role in the epileptogenesis (presumably accompanied by altered inhibitory transmission) after neurodegeneration.

Hyperthermic seizure induces persistent alteration in excitability of the dentate gyrus in immature rats

Febrile seizure (FS) is the most common type of seizure that occurs during early childhood. It has been proposed that atypical FS (prolonged, multiple, or lateralized) results in the development of recurrent complex partial seizures accompanied by Ammons horn sclerosis or mesial temporal sclerosis, which is the most common of the intractable epilepsy. To elucidate the characteristics of epileptogenesis or acquired epilepsy following FS, we performed prospective long-term studies using hyperthermia-induced seizure model. Rat pups (postnatal 11 day old) were induced to hyperthermia (41-43 degrees C in core temperature) by exposure to a 175 W mercury vapor lamp. Six-nine weeks after hyperthermic seizure, the dentate gyrus showed impairments of paired-pulse inhibitions and excitability ratio. In addition, newly generated granule cells and synaptogenesis were observed in this region. Ten-twelve weeks after hyperthermic seizure, animals (approximately 68%) showed electroencephalographic seizure activity with increased VGLUT-1 immunoreactivity in the dentate gyrus. Parvalbumin immunoreactivity was markedly reduced in the hilus. These findings indicate that in this model the epileptogenic changes in the dentate gyrus may be based on the persistent alterations in excitability via neurogenesis, synaptogenesis, and impaired GABA(B) receptor-mediated inhibition.

Ischemia-related changes of adrenocorticotropic hormone immunoreactivity and its protective effect in the gerbil hippocampus after transient forebrain ischemia

In the present study, the temporal and spatial alterations of adrenocorticotropic hormone (ACTH) immunoreactivity in the gerbil hippocampus after 5 min transient forebrain ischemia were investigated as followed up 7 days after ischemic insult, and the effects of ACTH after ischemic insult were also investigated 4 days after ischemic insult. The ectopic expression of ACTH (1-24 fragments) immunoreactive neurons in the cornus ammonis 1 (CA1) region of hippocampus and hilar region of the dentate gyrus 1 day after the ischemic insult was observed. Judging from the double immunofluorescence study, these neurons contain GABA. Four days after ischemic insult, the ACTH immunoreactivity was localized in CA1 pyramidal cells and glia near the stratum pyramidale, which normally do not express ACTH. In addition, in the saline-treated groups, the percentage of the detected Cresyl Violet positive neurons was 11.2% compared with the sham-operated group 4 and 7 days after ischemic insult. In these groups, the OX-42 immunoreactive microglia were detected in the strata pyramidale, oriens and radiatum. However, in the Org2766 (analog of ACTH)-treated group, 57.8% neurons compared with the sham-operated group were stained with Cresyl Violet 4 and 7 days after ischemic insult. In these groups, the OX-42 immunoreactive microglia were significantly reduced in the stratum pyramidale. These results suggest that transient forebrain ischemia may provoke selective ectopic and enhanced expression of ACTH in the hippocampus, and further suggest that ACTH plays an important role in reducing the ischemic damage.

Up-regulated astroglial TWIK-related acid-sensitive K+ channel-1 (TASK-1) in the hippocampus of seizure-sensitive gerbils: A target of anti-epileptic drugs

In order to identify the modulation of TASK (TWIK-related Acid-Sensitive K(+)) channel expressions in epilepsy, we conducted a comparative analysis of TASK channel immunoreactivities in the hippocampus of seizure-resistant (SR) and seizure-sensitive (SS) gerbils. There was no difference of the TASK-1 and TASK-2 channel expressions in the hippocampi of young SR and SS gerbils (1-2 months old). In adult SS gerbil hippocampus, TASK-1 immunoreactivity in astrocytes was higher than that in adult SR gerbil hippocampus. After seizures, TASK-1 immunoreactivity was significantly down-regulated in astrocytes of the SS gerbil hippocampus. In addition, various anti-epileptic drugs selectively affect TASK-1 immunoreactivity in astrocytes of the SS gerbil hippocampus. Gabapentin, lamotrigine, topiramate and valproic acid reduced the number of TASK-1(+) astrocytes in the hippocampus to 10-25% of that in saline-treated SS adult gerbils, whereas carbamazepine and vigabatrin decreased to approximately 50%. Therefore, the present study demonstrates that up-regulated TASK-1 immunoreactivity in astrocytes may be involved in the seizure activity of SS adult gerbils and suggests that the astroglial TASK-1 channel may be a target for epilepsy therapeutics.

Inactivation of human glutamate dehydrogenase by aluminum.

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a kinact of 2.7 min-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP+, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 μM. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD+-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in α helices and β sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.

The combination of metallothionein and superoxide dismutase protects pancreatic β cells from oxidative damage

Reactive oxygen species are considered an important cause of the death of pancreatic β cells, thereby triggering the development of type 2 diabetes as well as failure of islet transplantation. The biological properties of metallothionein (MT) and superoxide dismutase (SOD) are likely to be related to their antioxidant and free‐radical scavenging abilities, but their access across biological membranes is limited.