Douglas A. Eagles

A ketogenic diet increases the resistance to pentylenetetrazole-induced seizures in the rat

Summary: Purpose: The purpose of this study was to test the hypothesis that a ketogenic diet would increase the resistance of rats to pentylenetetrazole (PTZ)‐induced seizures and to understand the relation of ketonemia to seizure resistance.

Seizure resistance is dependent upon age and calorie restriction in rats fed a ketogenic diet

The present study was designed to evaluate the effects of age on the efficacy of the ketogenic diet in suppressing seizures evoked by tail-vein infusion of pentylenetetrazole (PTZ). Male rats of various ages were divided into three groups and fed one of three diets: (1) a calorie-restricted ketogenic diet, (2) a calorie-restricted normal (rodent chow) diet, or (3) a normal diet, ad libitum. After animals had been on experimental or control diets for more than 20 days, seizure threshold and blood levels of beta-hydroxybutyrate (beta-OHB) were determined. Animals fed a ketogenic diet exhibited significant elevations in levels of beta-OHB and seizure resistance compared to animals fed either a calorie-restricted normal diet or a normal diet, ad libitum. The levels of beta-OHB and seizure resistance were greatest for young pups. A surprising finding was that young animals fed a calorie-restricted rodent chow diet exhibited a significantly increased resistance to seizures compared to those fed the same diet, ad libitum. Results presented here demonstrate that the ketogenic diet produces the highest levels of ketonemia and seizure threshold in young animals. Collectively, these data suggest that age and caloric restriction are important considerations for implementing the ketogenic diet.

Higher ketogenic diet ratios confer protection from seizures without neurotoxicity

The present study was designed to establish a dose-response relationship for the efficacy of the ketogenic diet (KD). Sprague-Dawley rats were fed ketogenic diets containing varying ratios of fats; (carbohydrates + proteins) whereas control animals were fed rodent chow. Unless otherwise indicated, all animals were fed calorie-restricted, isocaloric diets beginning at P37 and ketonemia, seizure threshold and neurotoxic effects were determined. Despite being provided isocaloric quantities, animals fed lower ketogenic ratios gained weight relative to those fed diets having greater proportions of fats. A significantly increased metabolic rate was noted for animals fed a high-fat diet, suggesting a basis for the weight differences. Results also showed that the animals fed calorie-restricted high-fat diets exhibited significant ketonemia and protection from pentylenetetrazole (PTZ)-induced seizures. There were no detectable neurotoxic effects for any diet group. For animals of the same age, there was no correlation between beta-hydroxybutyrate (beta-OHB) and seizure threshold. These findings suggest that beta-OHB is not directly involved in the anticonvulsant mechanism of the diet. Also, data presented here show that the conventional 4:1 ketogenic diet does not confer the greatest level of seizure protection. We conclude that a 6:1 ketogenic diet, which shows no evidence of neurotoxicity, may be maximally efficacious in rats.

An anticonvulsant profile of the ketogenic diet in the rat.

The present study was designed to evaluate the anticonvulsant effects of a high-fat ketogenic diet (KD) in rats. Animals were maintained on one of four experimental diets: (1) calorie-restricted ketogenic (KCR); (2) calorie-restricted normal (NCR); (3) ad libitum ketogenic (KAL); or (4) ad libitum normal (NAL). The calorie-restricted diets were fed in quantities such that they were calorically equivalent. All animals began diet treatment at age P37 and each was subjected to one of five chemically-induced seizure tests: bicuculline (BIC; s.c.), picrotoxin (PIC; s.c.), kainate (KA, i.p. or s.c.) and gamma-butyrolactone (GBL, i.p.), strychnine (s.c.). Bipolar epidural electrodes were implanted under ketamine/xylazine anesthesia to permit recording the spike and wave discharges (SWD) characteristic of electroencephalograms during absence seizures. Ketonemia was assayed by measuring blood levels of beta-hydroxybutyrate (BHB) spectrophotometrically prior to induction of seizures in each experiment. Animals fed ketogenic diets (i.e. either calorie restricted or ad libitum) exhibited greater blood levels of BHB compared to control groups. Seizure results show that treatment with a KD: (1) reduced the incidence of bicuculline-induced convulsions; (2) diminished the number of picrotoxin-induced seizures (KCR group only); (3) increased latency to GBL-induced SWD and reduced both the number and duration of SWD; but (4) conferred no protection from strychnine-induced seizures; and (5) made KA-induced seizures more severe. Together these results indicate a spectrum of anticonvulsant action for the KD in rats that includes threshold seizures induced via GABA receptors (BIC, PIC, GBL) but not those induced at glycine (strychnine) or the KA-subclass of glutamate receptors. Uniquely, the KD is the only treatment described that protects against both convulsive and non-convulsive (absence) seizures in rats.

Caloric restriction augments brain glutamic acid decarboxylase-65 and -67 expression

The ketogenic diet is a very low‐carbohydrate, high‐fat diet used to treat refractory epilepsy. We hypothesized that this diet may act by increasing expression of glutamic acid decarboxylase (GAD), the rate‐limiting enzyme in γ‐aminobutyric acid (GABA) synthesis. Thus, we evaluated brain GAD levels in a well‐established, seizure‐suppressing, rodent model of the ketogenic diet. Because the diet is most effective when administered with a modest (∼10%) calorie restriction, we studied three groups of animals: rats fed ad libitum standard rat chow (Ad lib‐Std); calorie‐restricted standard chow (CR‐Std); and an isocaloric, calorie‐restricted ketogenic diet (CR‐Ket). We found that GAD67 mRNA was significantly increased in the inferior and superior colliculi and cerebellar cortex in both CR diet groups compared with control (e.g., by 45% in the superior colliculus and by 71% in the cerebellar cortex; P < .001). GAD65 mRNA was selectively increased in the superior colliculus and temporal cortex in both CR‐Std and CR‐Ket diet groups compared with ad lib controls. The only apparent CR‐Ket‐specific effect was a 30% increase in GAD67 mRNA in the striatum (P = .03). Enhanced GAD immunoreactivity was detected in parallel with the mRNA changes. These data clearly show that calorie restriction increases brain GAD65 and ‐67 expression in several brain regions, independent of ketogenic effects. These observations may explain why caloric restriction improves the efficacy of the ketogenic diet in treating epilepsy and suggest that diet modification might be useful in treatment of a number of brain disorders characterized by impaired GAD or GABA activity.

Calorie restriction of a high-carbohydrate diet elevates the threshold of PTZ-induced seizures to values equal to those seen with a ketogenic diet

The purpose of this study was to evaluate the contributions of ketonemia, caloric restriction, and carbohydrates to seizure protection in rats fed selected diets. Male Sprague-Dawley rats were fed experimental diets of two basic types, one high in carbohydrates and restricted to 90, 65, or 50% of the normal daily caloric requirement and the other a normal rodent chow diet restricted to 90 or 65% of the daily caloric requirement. After consuming their respective diets for 20 days, animals were subjected to tail-vein infusion of pentylenetetrazole (PTZ) to determine seizure threshold, taken as the dose required to evoke the first clonic reaction. Seizure thresholds were compared to those of rats fed control diets of either normal rodent chow fed ad libitum or a standard high-fat (ketogenic) diet calorie-restricted to 90% of daily caloric requirement, all animals age- and weight-matched at the time of diet onset. All diets were balanced for vitamins and minerals and contained at least 10% protein (by weight). Seizure threshold and ketonemia were elevated in both experimental diets in approximate proportion to the degree of calorie restriction. Animals fed the most severely restricted high-carbohydrate diet (50%) had seizure thresholds equal to those fed the ketogenic diet but had significantly lower ketonemia.

A ketogenic diet has different effects upon seizures induced by maximal electroshock and by pentylenetetrazole infusion

The purpose of these experiments was to determine whether a ketogenic diet previously shown to elevate seizure threshold also reduced seizure severity. Seizure threshold was tested by intravenous infusion of pentylenetetrazole (PTZ) whereas seizure severity was determined from measuring the hindlimb extension to flexion (E/F) ratio after seizures were evoked by maximal electroshock stimulation (MES). Surprisingly, seizures evoked by MES were more severe in animals fed a calorie-restricted ketogenic diet. Controls fed an isocaloric, calorie-restricted normal diet also exhibited more severe seizures than did animals fed the same diet ad libitum. When seizure threshold was evaluated in the same animals, those animals fed a calorie-restricted ketogenic diet exhibited a significant increase in seizure resistance compared to animals fed a ketogenic diet ad libitum, a calorie-restricted normal diet or a normal diet ad libitum. These findings suggest that both the amount and type of food affect seizures in rats and show that diet-related seizure protection depends upon the method by which seizures are provoked.

Path analysis shows that increasing ketogenic ratio, but not beta-hydroxybutyrate, elevates seizure threshold in the Rat.

Previous work has identified several criteria that may be important in determining the efficacy of the ketogenic diet as a treatment for intractable epilepsy in children. The present study was designed to investigate the influence of four major variables on seizure threshold, i.e. ketogenic ratio, body weight, age at diet onset and β-hydroxybutyrate in rats. Path analysis was used to statistically model and quantify the causal relationships among variables. Results indicate that seizure threshold was significantly elevated with increasing ketogenic ratios (i.e. more fats vs. carbohydrates and proteins) and decreasing weight. Conversely, age at diet onset and plasma levels of β-OHB showed no causal relation to seizure resistance. These results suggest that the efficacy of the ketogenic diet is independent of the level of ketonemia but is markedly influenced by diet and growth.

The effects of chronic norepinephrine transporter inactivation on seizure susceptibility in mice

Epilepsy and depression are comorbid disorders, but the mechanisms underlying their relationship have not been identified. Traditionally, many antidepressants have been thought to increase seizure incidence, although this remains controversial, and it is unclear which medications should be used to treat individuals suffering from both epilepsy and depression. Since the neurotransmitter norepinephrine (NE) has both antidepressant and anticonvulsant properties, we speculated that NE transporter (NET) inhibitor antidepressants might be therapeutic candidates for comorbid individuals. To test this idea, we assessed the effects of chronic administration (via osmotic minipump) of the selective NET inhibitor reboxetine on flurothyl-induced seizures in mice. We found that reboxetine had both proconvulsant and anticonvulsant properties; it lowered both seizure threshold and maximal seizure severity. NET knockout (NET KO) mice essentially phenocopied the effects of reboxetine on flurothyl-induced seizures, and the trends were extended to pentylenetetrazole and maximal electroshock seizures (MES). Furthermore, reboxetine had no further effect in NET KO mice, demonstrating the specificity of reboxetine for the NET. We next tested the chronic and acute effects of other classes of antidepressants (desipramine, imipramine, sertraline, bupropion, and venlafaxine) on seizure susceptibility. Only venlafaxine was devoid of proconvulsant activity, and retained some anticonvulsant activity. These results suggest that chronic antidepressant drug treatment has both proconvulsant and anticonvulsant effects, and that venlafaxine is a good candidate for the treatment of epilepsy and depression comorbidity.

Localization and Synaptic Release of N‐acetylaspartyl‐glutamate in the Chick Retina and Optic Tectum

The neuropeptide, N‐acetylaspartylglutamate (NAAG), was identified in the chick retina (1.4 nmol/retina) by HPLC, radioimmunoassay and immunohistochemistry. This acidic dipeptide was found within retinal ganglion cell bodies and their neurites in the optic fibre layer of the retina. Substantial, but less intense, immunoreactivity was detected in many amacrine‐like cells in the inner nuclear layer and in multiple bands within the inner plexiform layer. In addition, NAAG immunoreactivity was observed in the optic fibre layer and in the neuropil of the superficial layers of the optic tectum, as well as in many cell bodies in the tectum. Using a newly developed, specific and highly sensitive (3 fmol/50 μI) radioimmunoassay for NAAG, peptide release was detected in isolated retinas upon depolarization with 55 mM extracellular potassium. This assay also permitted detection of peptide release from the optic tectum following stimulation of action potentials in retinal ganglion cell axons of the optic tract. Both of these release processes required the presence of extracellular calcium. Electrically stimulated release from the tectum was reversibly blocked by extracellular cadmium. These findings suggest that NAAG serves an extracellular function following depolarization‐induced release from retinal amacrine neurons and from ganglion cell axon endings in the chick optic tectum. These data support the hypothesis that NAAG functions in synaptic communication between neurons in the visual system.