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, 7 (1), 5496

Non-ketogenic Combination of Nutritional Strategies Provides Robust Protection Against Seizures

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Non-ketogenic Combination of Nutritional Strategies Provides Robust Protection Against Seizures

Glenn Dallérac et al. Sci Rep.

Abstract

Epilepsy is a neurological condition that affects 1% of the world population. Conventional treatments of epilepsy use drugs targeting neuronal excitability, inhibitory or excitatory transmission. Yet, one third of patients presents an intractable form of epilepsy and fails to respond to pharmacological anti-epileptic strategies. The ketogenic diet is a well-established non-pharmacological treatment that has been proven to be effective in reducing seizure frequency in the pharmaco-resistant patients. This dietary solution is however extremely restrictive and can be associated with complications caused by the high [fat]:[carbohydrate + protein] ratio. Recent advances suggest that the traditional 4:1 ratio of the ketogenic diet is not a requisite for its therapeutic effect. We show here that combining nutritional strategies targeting specific amino-acids, carbohydrates and fatty acids with a low [fat]:[proteins + carbohydrates] ratio also reduces excitatory drive and protects against seizures to the same extent as the ketogenic diet. Similarly, the morphological and molecular correlates of temporal lobe seizures were reduced in animals fed with the combined diet. These results provide evidence that low-fat dietary strategies more palatable than the ketogenic diet could be useful in epilepsy.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Novel diet combining nutritional strategies reducing the ratio of [fat]:[proteins + carbohydrates]. (A) combined diet composition of the total caloric supply. (B) proportion of macronutrients caloric supply for SD, CD, and KD. (C) monitoring mice weight every third day upon 3 weeks of dietary treatment revealed no change in body mass.
Figure 2
Figure 2
CD treatment reduces excitatory synaptic transmission. (A) Hippocampal slices prepared from mice fed with KD or CD displayed reduced basal synaptic transmission compared to control SD fed animals (SD controls, n = 14; CD, n = 7; KD, n = 6; p < 0.001). Calibration bars: 10 ms, 0.5 mV. (B) Analysis of blood ketone bodies concentrations revealed a significant increase in both β-hydroxybutyrate (βHB; p < 0.001) and acetoacetate (ACA; p < 0.001) in the KD group only (n = 10). Markers of glycolysis (glucose, p < 0.01; lactate, p < 0.05; pyruvate, p < 0.05) were also significantly reduced in mice supplied with KD, indicating that this diet only induces high level of ketosis. The only change triggered by CD was a significant reduction in lactate (p < 0.05). (C) Analysis of the fibre volley amplitude as a function of stimulation intensity revealed a significant reduction in presynaptic excitability of CD and KD fed mice as compared to controls fed SD (CD: p < 0.001, KD: p < 0.01). (D) CD and KD dietary treatment also resulted in a clear enhancement of paired-pulse facilitation (SD, n = 5; KD, n = 6, p < 0.05; CD, n = 7, p < 0.001), which indicates a decreased probability of release. Example traces show facilitation at 50 ms ISI. Calibration bars: 10 ms, 0.5 mV. (E) The low-affinity competitive antagonist γ-d-glutamylglycine (γ-DGG), at a non-saturating concentration (0.5 mM) at which its potency depends on glutamate concentration decreased neurotransmission to a greater extent in CD and KD fed mice compared to SD controls (SD, n = 9; KD, n = 6, p < 0.001; CD, n = 7, p < 0.05), indicating lower glutamate synaptic concentrations in the KD and CD groups. Calibration bars: 10 ms, 0.5 mV. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3
Figure 3
CD treatment decreases susceptibility to seizures. (A,B) Injection of pentylenetetrazol (PTZ; 50 mg/ml) i.p. promptly resulted in paroxysmal events and status epilepticus in control SD fed mice. In mice receiving KD or CD treatment, the ictus (example indicated by a black bar) was triggered significantly later (SD, n = 7; KD, n = 5, p < 0.01; CD, n = 8, p < 0.001), indicating a similar protective effect of both diets. (A,C) Corroborating this finding, the frequency of interictal events (example shown by an arrow) was found to be significantly reduced in both KD and CD fed mice compared to SD controls (all p < 0.05). Calibration bars: 10 s, 500 µV. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
CD treatment reduces chronic seizure occurrence. (A) The KA mouse model of chronic epilepsy. KA (20 mM) was injected unilaterally by mean of a cannula connected to a precision pump into the intra-hippocampal region. (B) Mice under SD were left 3 weeks to develop chronic seizures and were then fed with KD or CD, controls were left on SD. (C,D) EEG detected seizures were drastically decreased in mice treated with KD (n = 8, p < 0.01) and CD (n = 13, p < 0.001) compared with SD controls (n = 10). (C,E) EEG detected seizures showed similar durations between SD, KD and CD mice. Calibration bars: 10 min, 200 µV. **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
CD treatment rescues cytological and molecular correlates of chronic epilepsy. (A) Tissue obtained from the recording site of KA mice was collected for immunoblotting. (B,C) Expression of GFAP was significantly reduced in mice treated with KD and CD (n = 4 per group, all p < 0.05). (D) KA mice were prepared for histological analysis of hippocampal area CA1, CA3 and DG. This article was published in The mouse brain in stereotaxic coordinates, Paxinos, G & Franklin, K, p.98, Copyright Elsevier Academic Press, 2001. This figure is not covered by the CC BY licence. Elsevier. All rights reserved, used with permission. (E,F) Both CD and KD (CD, n = 5; KD, n = 5) fed mice showed protection against dispersion of the granule cell layer (g.c.l.) compared to SD fed mice (SD, n = 6) and mice that did not receive KA (CT, n = 3).*p < 0.05. (GJ) CD and KD fed mice were also protected against cell loss in CA1 and CA3 compared to SD animals. **p < 0.01, ***p < 0.001. (h) hilus, m.l.: molecular layer, s.l.: stratum lucidum, s.o.: stratum oriens, s.p.: stratum pyramidale, s.r.: stratum radiatum.

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