Mitsumasa Fukuda

β-hydroxybutyrate alters GABA-transaminase activity in cultured astrocytes

The ketogenic diet has long been recognized as an effective treatment for medically refractory epilepsy. Despite nearly a century of use, the mechanisms underlying its clinical efficacy remain unknown. One of the proposed hypotheses for its anti-epileptic actions involves increased GABA concentration in the brain due to ketone bodies that become elevated with a ketogenic diet. In recent years, the notion that astrocytes could play a role in the evolution of abnormal cortical excitability in chronic neurological disorders, such as epilepsy, has received renewed attention. The present study examined the effects of beta-hydroxybutyrate, a ketone body, on GABA metabolism in rat primary cultured astrocytes. When beta-hydroxybutyrate was added to culture medium, GABA-transaminase (GABA-T) mRNA expression was significantly suppressed in time- and dose-dependent manners. GABA-T enzymatic activity in beta-hydroxybutyrate-treated astrocytes was also suppressed, in accordance with its gene expression. These effects were evident after 3 days of culture, which might coincide with depleted intracellular glycogen. GABA transporter, GAT-1, gene expression was strongly suppressed in cultured astrocytes after 5 days of culture with beta-hydroxybutyrate, although other type of GABA transporters did not display significant changes. These results suggest that beta-hydroxybutyrate induced by ketogenic diet may increase GABA concentration in the epileptic brain by suppressing astrocytic GABA degradation, leading to antiepileptic effects.

Interleukin-6 attenuates hyperthermia-induced seizures in developing rats

Previous studies indicated that several cytokines influenced the seizure propensity in convulsive disorders and were the cause of encephalopathies in childhood. We studied the role of one inflammatory cytokine, interleukin-6 (IL-6), in hyperthermia-induced seizures in developing rats. Twenty-four male Lewis rats (23-28 days old) were divided into three groups (n=8/IL-6 (500 ng), IL-6 (50 ng), and saline control groups). We applied human recombinant IL-6 intra-nasally to developing rats 1h before seizures induced by moist heated air (50 degrees C). The seizure latency was defined as the time from hyperthermia onset until the appearance of continuous seizure discharges on electroencephalography (EEG), and the seizure duration as the duration of continuous spike and wave discharges on EEG. Five of the eight rats in the IL-6 (500 ng) group, two in the IL-6 (50 ng) group, and one in the control group exhibited no seizure discharges during the 360 s heating period. In these cases, the seizure latency time was regarded as 360 s and the seizure duration time as 0 s. The median seizure latency for the IL-6 (500 ng) group, 360 s (range: 256-360), was significantly longer than that for the control one, 249 (121-360) (P<0.05). The seizure duration for the IL-6 (500 ng) group, 0 s (0-20), was significantly shorter than that for the control one, 33 (0-76) (P<0.025). Also, the adenosine receptor antagonist, aminophylline, prevented these effects of IL-6 on hyperthermia-induced seizures. These results indicate that IL-6 plays an anti-convulsive role through the adenosine system in hyperthermia-induced seizures, which might be relevant as to human febrile seizures.

Nonketotic Hyperglycinemia: Proposal of a Diagnostic and Treatment Strategy

Early myoclonic encephalopathy presents neonatally with fragmented myoclonus and a suppression-burst electroencephalography pattern. We describe a newborn boy with early myoclonic encephalopathy caused by nonketotic hyperglycinemia. He presented with severe hypotonia, progressive apneic episodes, and erratic myoclonus. Screening of deletions in GLDC, using the multiplex ligation-dependent probe amplification method, and a (13)C breath test confirmed the diagnosis of nonketotic hyperglycinemia. Treatment with the N-methyl-d-aspartate receptor antagonist ketamine exerted dramatic suppressive effects on his seizures, and ameliorated his clinical status.

Clinical study of epilepsy with severe febrile seizures and seizures induced by hot water bath.

We followed five cases with severe febrile seizures (FS) with early onset and seizures induced by hot water bath, indicating severe myoclonic epilepsy in infancy (SME) or the peripheral form of SME. These cases, however, had far better clinical courses than that of SME or the peripheral form of SME. These cases were characterized by: (a) severe FS with early onset and seizures induced by hot water bath; (b) a lack of myoclonic seizures; (c) no psychomotor deterioration; (d) no underlying disorders or signs of brain insult on neurologic and laboratory examination; (e) relatively successful seizure control; (f) none of the severe EEG abnormalities seen in SME and the peripheral form of SME; and (g) a positive family history of convulsive disorders.

Interleukin-10 is associated with resistance to febrile seizures: Genetic association and experimental animal studies

Purpose:  Febrile seizures (FS) are the most common form of childhood convulsions. Many reports have shown that a proinflammatory cytokine, interleukin‐1 (IL‐1) β, may have a facilitatory effect on the development of FS. We have previously shown that the IL1B ‐511C/T single nucleotide polymorphism (SNP) is associated with simple FS of sporadic occurrence. The balance between pro‐ and antiinflammatory cytokines influences the regulation of infections and could, therefore, play a role in the pathogenesis of FS. Here, to determine whether pro‐ and antiinflammatory cytokine genes are responsible for the susceptibility to FS, we have performed an association study on functional SNPs of cytokine genes in FS patients and controls.

The pathogenic role of the NMDA receptor in hyperthermia-induced seizures in developing rats

Hyperthermia-induced seizures (HS) in rats have been used as a model of febrile seizures. Activation of the N-methyl-D-aspartate (NMDA) receptor by increased extracellular glutamate (Glu) in the cortex during hyperthermia may be involved in the induction of HS and HS kindling. To confirm this hypothesis, the effects of a potent blocker of the NMDA receptor, MK-801, on the threshold and pattern of HS were evaluated. The threshold temperatures for rats given 0.1 (low dose) and 0.5 (high dose) mg/kg MK-801 (i.p.) for the first time were 41.6 degrees C (39.7-42) (median, range) and 42.0 (41.2-42.0), respectively, which were significantly higher than the 40.5 (39.4-41.2) for rats without MK-801 administration (P < 0.01). The recurrent occurrence of HS suppressed the increase in the threshold temperature with age, and changed the seizure from partial to generalized seizures (HS kindling), whereas these effects of recurrent HS on the threshold and pattern of HS were inhibited by the high dose (0.5 mg/kg) of MK-801. MK-801 blocks HS and HS kindling. The activation of the NMDA receptor during hyperthermia plays an important role in the induction of HS and HS kindling.

The influence of blood gas changes on hyperthermia-induced seizures in developing rats

Fever induces seizures in infants with febrile convulsions or epilepsy. Hyperpnea induced by fever may contribute to the induction of these seizures. In order to examine this possibility, we evaluated the effect of changes in arterial blood gas tension on hyperthermia-induced seizures in developing rats. Electrical seizure discharges were induced by application of infra-red rays on the skull of rats under mechanical ventilation with different respiratory conditions. There was positive correlation between pCO(2) and the seizure threshold (ST) defined as a latency from the start of hyperthermia to the occurrence of seizures: ST (seconds, s) = 2.36 pCO(2) + 0.05 (R(2) = 0.80, P < 0.001). Seizure duration (SD) was longer at lower pCO(2) level: 18 (6-33) (median, range) s at pCO(2) ranging from 23 to 26 mmHg vs. 0 (0-7) s at pCO(2) ranging from 35 to 57 mmHg (P < 0.01). Hypoxia significantly increased ST: 84 (61-100) s at P0(2) ranging from 53 to 76 mmHg vs. 60 (51-72) s at P0(2) ranging from 87 to 131 mmHg (P < 0.01). Hyperoxia prolonged SD: 27 (10-30) s at P02 ranging from 100 to 170 mmHg vs. 9 (0-23) at P0(2) ranging from 53 to 93 mmHg (P < 0.02). Hypocarbia caused by fever-induced hyperpnea probably contributes to the generation of fever-induced seizures.

Interleukin-1β enhances susceptibility to hyperthermia-induced seizures in developing rats

Cytokines have been shown to influence susceptibility to febrile seizures and epilepsy. In this study, the role of interleukin-1beta (IL-1beta) was examined in developing rats. IL-1beta and interleukin-1 receptor antagonist (IL-1ra) were administered to developing rats, and seizures were induced by moist warm air. Twenty male Lewis rats (21-23 days old) were divided into two groups (IL-1beta and saline control groups) and two holes were made in the skull for EEG electrodes. We applied human recombinant IL-1beta intra-nasally 1h before seizures induced by moist warm air. The brain temperature at the appearance of seizure discharges on EEG, and the latency time from the hyperthermia onset until the appearance of seizure discharges on EEG were measured. And the same study using IL-1ra was performed. The median brain temperature for the IL-1beta group, 42.6 degrees C (range: 41.8-43.0), was significantly lower than that for the control, 42.9 (42.3-43.4) (P=0.043). The brain temperature for the IL-1ra group, 43.3 (42.8-43.7), was significantly higher than that for the control, 42.9 (42.2-43.5) (P=0.011), and the latency time for the IL-1ra group, 398s (270-561), was significantly longer than that for the control, 325 (252-462) (P=0.035). These results demonstrate that IL-1beta promotes hyperthermia-induced seizures in developing rats.

Adenosine A1 receptor blockage mediates theophylline-associated seizures.

Theophylline‐associated seizures (TAS) often progress to prolonged or treatment‐resistant convulsions. Theophylline is a nonselective adenosine receptor antagonist. Adenosine is an endogenous anticonvulsant that can terminate seizures. Fever and young age have been reported to be risk factors for TAS. To elucidate the mechanism of TAS, we investigated the effect of theophylline and adenosine receptor ligands on hyperthermia‐induced seizures in juvenile rats. The treatment dose of theophylline or control saline was injected intraperitoneally 1 h before hyperthermia‐induced seizures. The seizure threshold in the theophylline group was significantly lower and seizure duration was significantly longer than those in the control group. The addition of a selective adenosine A1 receptor agonist and an adenosine kinase inhibitor completely counteracted the effects of theophylline. Moreover, a selective A1 antagonist caused a significantly longer seizure duration compared with the control. These findings suggest that blockage of the adenosine A1 receptor is the main cause of TAS.

Activation of central adenosine A2A receptors lowers the seizure threshold of hyperthermia-induced seizure in childhood rats

Adenosine is a potent neuromodulator in the central nervous system (CNS). The functional deterioration of adenosine A(1) receptors in the CNS was reported to cause a failure of termination of seizures and to a lower seizure threshold of hyperthermia-induced seizures (HS) in childhood rats, which may contribute to adenosine-related convulsive disorders such as theophylline-associated seizures in childhood patients. In contrast to the inhibitory effect of adenosine A(1) receptors, the function of adenosine A(2A) receptors remains controversial. To clarify the function of adenosine A(2A) receptors in childhood convulsive disorders associated to hyperthermia, we investigated the in vivo interaction between adenosine A(2A) receptors and their ligands in HS in childhood rats. Adenosine selective A(2A) receptor ligands were injected intraperitoneally before HS. We measured brain temperature at the onset of seizures and the mortality rate after HS. We found that brain temperature at seizure onset was significantly higher in the A(2A) receptor antagonist group compared with that in the control group (p<0.05), and there was no significant difference in mortality among the groups. In contrast, brain temperature at seizure onset was significantly lower in the A(2A) receptor agonist group compared with that in the control group (p<0.05), and mortality was significantly higher in the A(2A) agonist group compared with that in the control group (p<0.001). The activation of the adenosine A(2A) receptor might enhance seizures associated to hyperthermia in the childhood human brain, and be involved in the pathogenesis of sudden unexpected death in epilepsy (SUDEP) in childhood patients with convulsive disorders.