Gene expression in the epileptic (EL) mouse hippocampus.

The neuropathology of hippocampal seizure foci in human temporal lobe epilepsy (TLE) and several animal models of epilepsy reveal extensive neuronal loss along with astrocyte and microglial activation. Studies of these models have advanced hypotheses that propose both pathological changes are essential for seizure generation. However, some seizure foci in human TLE show an extreme loss of neurons in all hippocampal fields, giving weight to hypotheses that favor neuroglia as major players. The epileptic (EL) mouse is a seizure model in which there is no observable neuron loss but associated proliferation of microglia and astrocytes and provides a good model to study the role of activated neuroglia in the presence of an apparently normal population of neurons. While many studies have been carried out on the EL mouse, there is a paucity of studies on the molecular changes in the EL mouse hippocampus, which may provide insight on the role of neuroglia in epileptogenesis. In this paper we have applied high throughput gene expression analysis to identify the molecular changes in the hippocampus that may explain the pathological processes. We have observed several classes of genes whose expression levels are changed. It is hypothesized that the upregulation of heat shock proteins (HSP70, HSP72, FOSL2 (HSP40), and their molecular chaperones BAG3 and DNAJB5 along with the down regulated gene MALAT1 may contribute to the neuroprotection observed. The increased expression of BDNF along with immediate early gene expression (FosB, JunB, ERG4, NR4A1, NR4A2, FBXO3) and the down regulation of GABRD, DBP and MALAT1 it is hypothesized may contribute to the hyperexcitability of the hippocampal neurons in this model. Activated astrocytes and microglia may also contribute to excitability pathomechanisms. Activated astrocytes in the ELS mouse are deficient in glutamine synthetase and thus reduce the clearance of extracellular glutamate. Activated microglia which may be associated with C1Q and MHC class I molecules we propose may mediate a process of selective removal of defective GABAergic synapses through a process akin to trogocytosis that may reduce neuronal inhibition and favor hyperexcitability.

The EL mouse: a natural model of autism and epilepsy.

PURPOSE: Autism is a multifactorial disorder that involves impairments in social interactions and communication, as well as restricted and repetitive behaviors. About 30% of individuals with autism develop epilepsy by adulthood. The EL mouse has long been studied as a natural model of multifactorial idiopathic generalized epilepsy with complex partial seizures. Because epilepsy is a comorbid trait of autism, we evaluated the EL mouse for behaviors associated with autism. METHODS: We compared the behavior of EL mice to age-matched control DDY mice, a genetically related nonepileptic strain. The mice were compared in the open field and in the light-dark compartment tests to measure activity, exploratory behavior, and restricted and repetitive behaviors. The social transmission of food preference test was employed to evaluate social communication. Home-cage behavior was also evaluated in EL and DDY mice as a measure of repetitive activity. KEY FINDINGS: We found that EL mice displayed several behavioral abnormalities characteristic of autism. Impairments in social interaction and restricted patterns of interest were evident in EL mice. Activity, exploratory behavior, and restricted behavior were significantly greater in EL mice than in DDY mice. EL mice exhibited impairment in the social transmission of food preference assay. In addition, a stereotypic myoclonic jumping behavior was observed in EL mice, but was not seen in DDY mice. It is of interest to note that seizure activity within 24 h of testing exacerbated the autistic behavioral abnormalities found in EL mice. SIGNIFICANCE: These findings suggest that the EL mouse expresses behavioral abnormalities similar to those seen in persons with autism. We propose that the EL mouse can be utilized as a natural model of autism and epilepsy.

Brain lipid analysis in mice with Rett syndrome.

Rett syndrome (RS) is an X-linked neurodevelopmental disorder mostly involving mutations in the gene for methyl-CpG-binding protein 2 (MECP2). Ganglioside abnormalities were previously found in cerebrum and cerebellum in RS patients. We evaluated total lipid distribution in cerebrum/brainstem, hippocampus, and cerebellum in male mice carrying either the Mecp2 (tm1.1Bird) knockout mutation or the Mecp2 (308/y) deletion mutation. The concentration of the neuronal enriched ganglioside GD1a was significantly lower in the cerebrum/brainstem of Mecp2 (tm1.1Bird) mice than in that of age matched controls, but was not reduced in the Mecp2 (308/y) mice. No other differences in brain lipid content, including myelin-enriched cerebrosides, were detected in mice with either type of Mecp2 mutation. These findings indicate that the poor motor performance previously reported in the RS mutant mice is not associated with major brain lipid abnormalities and that most previous brain lipid abnormalities observed in RS patients were not observed in the Mecp2 (tm1.1Bird) or the Mecp2 (308/y) RS mice.

Improvement in motor and exploratory behavior in Rett syndrome mice with restricted ketogenic and standard diets.

Rett syndrome (RTT) is a rare X-linked autistic-spectrum neurological disorder associated with impaired energy metabolism, seizure susceptibility, progressive social behavioral regression, and motor impairment primarily in young girls. The objective of this study was to examine the influence of restricted diets, including a ketogenic diet (KD) and a standard rodent chow diet (SD), on behavior in male Mecp2(308/y) mice, a model of RTT. The KD is a high-fat, low-carbohydrate diet that has anticonvulsant efficacy in children with intractable epilepsy and may be therapeutic in children with RTT. Following an 11-day pretrial period, adult wild-type and mutant Rett mice were separated into groups that were fed either an SD in unrestricted or restricted amounts or a ketogenic diet (KetoCal) in restricted amounts for a total of 30 days. The restricted diets were administered to reduce mouse body weight by 20-23% compared to the body weight of each mouse before the initiation of the diet. All mice were subjected to a battery of behavioral tests to determine the influence of the diet on the RTT phenotype. We found that performance in tests of motor behavior and anxiety was significantly worse in male RTT mice compared to wild-type mice and that restriction of either the KD or the SD improved motor behavior and reduced anxiety. We conclude that although both restricted diets increased the tendency of Rett mice to explore a novel environment, the beneficial effects of the KD were due more to calorie restriction than to the composition of the diet. Our findings suggest that calorically restricted diets could be effective in reducing the anxiety and in improving motor behavior in girls with RTT.

Glucose reduces the anticonvulsant effects of the ketogenic diet in EL mice.

The ketogenic diet (KD) is known to be anticonvulsant and anti-epileptogenic. While the mechanism behind this therapeutic benefit is unclear, a reduction of circulating glucose levels through calorie restriction (CR) has been implicated. Foods or drinks that elevate blood glucose are known to compromise the therapeutic benefit of the KD in some children with epilepsy. We therefore evaluated the effect of a calorie restricted KD (KD-R) with supplementation of glucose in the drinking water of EL mice, a natural model of idiopathic generalized epilepsy, prior to seizure testing to assess the effect of glucose on seizure generation. Mice were fed either a standard diet or the KD unrestricted (SD-UR and KD-UR, respectively), or the KD restricted (KD-R). d-Glucose (25 mM) was supplemented in the drinking water of KD-R fed mice for 0.5h or for 2.5h prior to seizure testing. Each restricted mouse served as its own body weight control to achieve a 15-18% body weight reduction. Seizure susceptibility, body weights, and plasma glucose and ß-hydroxybutyrate levels were measured over a nine-week treatment period. Body weights and glucose levels remained high over the testing period in both the SD-UR and the KD-UR groups, but were significantly reduced in all R-fed groups. A significant increase in ß-hydroxybutyrate levels was observed in all KD groups. Seizure susceptibility remained highest in the SD-UR group, was slightly reduced in the KD-UR group, and was significantly reduced after three weeks in all R-fed groups. Supplementation of glucose prior to seizure testing resulted in a decrease of seizure threshold for R-fed mice, but did not alter bodyweight or circulating glucose levels. The KD has both an anticonvulsant and antiepileptogenic effect in EL mice. Here we confirm that CR enhances the anticonvulsant action of the KD in EL mice. Additionally, we show for the first time that supplementation of glucose decreases the anticonvulsant action of the KD, which further supports the hypothesis that CR works through transitioning metabolism from glucose to ketone utilization for energy.

The calorically restricted ketogenic diet, an effective alternative therapy for malignant brain cancer.

BACKGROUND: Malignant brain cancer persists as a major disease of morbidity and mortality in adults and is the second leading cause of cancer death in children. Many current therapies for malignant brain tumors fail to provide long-term management because they ineffectively target tumor cells while negatively impacting the health and vitality of normal brain cells. In contrast to brain tumor cells, which lack metabolic flexibility and are largely dependent on glucose for growth and survival, normal brain cells can metabolize both glucose and ketone bodies for energy. This study evaluated the efficacy of KetoCal, a new nutritionally balanced high fat/low carbohydrate ketogenic diet for children with epilepsy, on the growth and vascularity of a malignant mouse astrocytoma (CT-2A) and a human malignant glioma (U87-MG). METHODS: Adult mice were implanted orthotopically with the malignant brain tumors and KetoCal was administered to the mice in either unrestricted amounts or in restricted amounts to reduce total caloric intake according to the manufacturers recommendation for children with refractory epilepsy. The effects KetoCal on tumor growth, vascularity, and mouse survival were compared with that of an unrestricted high carbohydrate standard diet. RESULTS: KetoCal administered in restricted amounts significantly decreased the intracerebral growth of the CT-2A and U87-MG tumors by about 65% and 35%, respectively, and significantly enhanced health and survival relative to that of the control groups receiving the standard low fat/high carbohydrate diet. The restricted KetoCal diet reduced plasma glucose levels while elevating plasma ketone body (beta-hydroxybutyrate) levels. Tumor microvessel density was less in the calorically restricted KetoCal groups than in the calorically unrestricted control groups. Moreover, gene expression for the mitochondrial enzymes, beta-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-ketoacid CoA transferase, was lower in the tumors than in the contralateral normal brain suggesting that these brain tumors have reduced ability to metabolize ketone bodies for energy. CONCLUSION: The results indicate that KetoCal has anti-tumor and anti-angiogenic effects in experimental mouse and human brain tumors when administered in restricted amounts. The therapeutic effect of KetoCal for brain cancer management was due largely to the reduction of total caloric content, which reduces circulating glucose required for rapid tumor growth. A dependency on glucose for energy together with defects in ketone body metabolism largely account for why the brain tumors grow minimally on either a ketogenic-restricted diet or on a standard-restricted diet. Genes for ketone body metabolism should be useful for screening brain tumors that could be targeted with calorically restricted high fat/low carbohydrate ketogenic diets. This preclinical study indicates that restricted KetoCal is a safe and effective diet therapy and should be considered as an alternative therapeutic option for malignant brain cancer.

Management of multifactorial idiopathic epilepsy in EL mice with caloric restriction and the ketogenic diet: role of glucose and ketone bodies.

BACKGROUND: The high fat, low carbohydrate ketogenic diet (KD) was developed as an alternative to fasting for seizure management. While the mechanisms by which fasting and the KD inhibit seizures remain speculative, alterations in brain energy metabolism are likely involved. We previously showed that caloric restriction (CR) inhibits seizure susceptibility by reducing blood glucose in the epileptic EL mouse, a natural model for human multifactorial idiopathic epilepsy. In this study, we compared the antiepileptic and anticonvulsant efficacy of the KD with that of CR in adult EL mice with active epilepsy. EL mice that experienced at least 15 recurrent complex partial seizures were fed either a standard diet unrestricted (SD-UR) or restricted (SD-R), and either a KD unrestricted (KD-UR) or restricted (KD-R). All mice were fasted for 14 hrs prior to diet initiation. A new experimental design was used where each mouse in the diet-restricted groups served as its own control to achieve a 20-23% body weight reduction. Seizure susceptibility, body weights, and the levels of plasma glucose and beta-hydroxybutyrate were measured once/week over a nine-week treatment period. RESULTS: Body weights and blood glucose levels remained high over the testing period in the SD-UR and the KD-UR groups, but were significantly (p < 0.001) reduced in the SD-R and KD-R groups. Plasma beta-hydroxybutyrate levels were significantly (p < 0.001) increased in the SD-R and KD-R groups compared to their respective UR groups. Seizure susceptibility remained high in both UR-fed groups throughout the study, but was significantly reduced after three weeks in both R-fed groups. CONCLUSIONS: The results indicate that seizure susceptibility in EL mice is dependent on plasma glucose levels and that seizure control is more associated with the amount than with the origin of dietary calories. Also, CR underlies the antiepileptic and anticonvulsant action of the KD in EL mice. A transition from glucose to ketone bodies for energy is predicted to manage EL epileptic seizures through multiple integrated changes of inhibitory and excitatory neural systems.