The link between brain acidosis, breathing and seizures: a novel mechanism of action for the ketogenic diet in a model of infantile spasms

Abstract

Infantile spasms (IS) syndrome is a catastrophic, epileptic encephalopathy of infancy that is often refractory to current antiepileptic therapies. The ketogenic diet (KD) has emerged as an alternative treatment for patients with medically intractable epilepsy, though the prospective validity and mechanism of action for IS remains largely unexplored. We investigated the KD's efficacy as well as its mechanism of action in a rodent model of intractable IS. The spasms were induced using the triple-hit paradigm and the animals were then artificially reared and put on either the KD (4:1 fats: carbohydrate + protein) or a control milk diet (CM; 1.7:1). ³¹Phosphorus magnetic resonance spectroscopy (³¹P MRS) and head-out plethysmography were examined in conjunction with continuous video-EEG behavioural recordings in lesioned animals and sham-operated controls. The KD resulted in a peripheral ketosis observed both in the blood and urine. The KD led to a robust reduction in the frequency of spasms observed, with approximately a 1.5-fold increase in the rate of survival. Intriguingly, the KD resulted in an intracerebral acidosis as measured with ³¹P MRS. In addition, the respiratory profile of the lesioned rats on the KD was significantly altered with slower, deeper and longer breathing, resulting in decreased levels of expired CO₂. Sodium bicarbonate supplementation, acting as a pH buffer, partially reversed the KD's protective effects on spasm frequency. There were no differences in the mitochondrial respiratory profiles in the liver and brain frontal cortex measured between the groups, supporting the notion that the effects of the KD on breathing are not entirely due to changes in intermediary metabolism. Together, our results indicate that the KD produces its anticonvulsant effects through changes in respiration leading to intracerebral acidosis. These findings provide a novel understanding of the mechanisms underlying the anti-seizure effects of the KD in IS. Further research is required to determine whether the effects of the KD on breathing and intracerebral acid-base balance are seen in other paediatric models of epilepsy.

Keywords: acidosis; epilepsy; plethysmography; respiration; spectroscopy.

Graphical Abstract

Figure 1

The rat pups in the model of infantile spasms (IS) were successfully reared on the control milk diet (CM) and ketogenic diet (KD), using the pup-in-a-cup setup. (A) A representative schematic of the experimental protocols. (B) The growth trajectories of all groups were similar until P10, where control-KD (C-KD; n = 9) and lesioned-KD (L-KD; n = 9) animals had lower body weights compared to untreated controls (control-CM; C-CM; n = 8). Body weights were not different between lesioned-CM (L-CM; n = 8) and C-CM or between L-KD and C-KD. (C, D) The KD resulted in elevated BHB levels both in the blood (C) and urine (D) at P7 and P12. The data were analysed with two-way repeated-measures ANOVA followed by Holm-Sidak comparison and are presented as (B) mean ± SEM and (C, D) box and whisker plots (median with 5th and 95th percentile). *P < 0.05, **P < 0.01, ***P < 0.001. DOX, doxorubicin; LPS, lipopolysaccharide; MRS, magnetic resonance spectroscopy; PCPA, p-chlorophenylalanine.

Figure 2

The ketogenic diet (KD) reduced behavioural spasms and interictal spikes in lesioned rats. (A) Lesioned-KD (L-KD; n = 9) rats had significantly lower levels of behavioural spasms from P5 to P12 compared to lesioned rats fed the control milk diet (L-CM; n = 9). Behavioural spasms in L-KD rats were abolished by P10, while spasms in L-CM rats were not abolished until P12. Behavioural spasms were not observed in control-CM (C-CM; n = 9) or control-KD (C-KD; n = 9) rats. (B) EEG activity was recorded in a subset of animals. Interictal spike frequency was highest in L-CM rats (n = 5) throughout the recording period, but the KD significantly reduced this lesion-induced increase in spike frequency in L-KD rats (n = 5) from P5 to P12. The spike frequency decreased sharply from P5 to P6 in both C-KD (n = 4) and C-CM (n = 4) rats and was sustained at this low level for the remainder of the experimental period with a transient increase in spiking activity in C-CM rats (n = 4) at P9 and P10. (C) A representative example from the spike detection software showing 3 SD threshold from the background signal for spike detection. Each circle indicates the identification of one spike on the raw EEG signal. (A, B) The data were analysed with two-way repeated measures ANOVA followed by Holm-Sidak comparison. The data are presented as mean ± SEM. *P < 0.05, ***P < 0.001.

Figure 3

The ketogenic diet (KD) has neuroprotective effects and is associated with increased survival. (A) A representative image of a 20 µm coronal section of the brain from a lesioned rat fed the control milk diet- (L-CM; upper image) and a lesioned rat fed the KD (L-KD; lower image) rat depicting the lesion induced on the right hemisphere of the brain in the model of IS. (R) = right brain hemisphere; (L) = left brain hemisphere. Scale bar = 1500 µm. (B) The right/left brain hemispheric ratio was not different between the control-CM (C-CM; n = 7) and control-KD (C-KD; n = 8) rats but was significantly reduced in the lesioned rats. The KD significantly increased the interhemispheric ratio in L-KD (n = 9) compared to L-CM (n = 9) rats. There was no difference in the interhemispheric ratio between L-KD and lesioned rats on the KD supplemented with sodium bicarbonate (L-KD-HCO3; n = 9). L-KD-HCO3 rats also displayed an increased interhemispheric ratio compared to L-CM. (C) The survival rate was lowest in lesioned rats, but KD treatment significantly prolonged survival in these animals. The data were analysed using one-way ANOVA followed by Holm-Sidak comparison (B), and Kaplan-Meier survival analysis (C). (B) The data are presented as box and whisker plots (median with 5th and 95th percentile). *P < 0.05, ***P < 0.001.

Figure 4

The ketogenic diet (KD) improved the neurodevelopmental profile in lesioned rats. (A) Representative tracings of the tracked open field activity of lesioned and sham-operated control rats at postnatal day (P) 10. (B) The total distance travelled in the tracked open field activity was lowest in lesioned rats given the control milk-diet (L-CM; n = 8). The KD treatment significantly improved motor activity in lesioned rats (L-KD; n = 8) compared to control-CM (C-CM; n = 6) and control-KD (C-KD). Motor activity was not different between C-CM and C-KD (n = 6). (C) An ultrasonic vocalization (USV) schematic illustrating the detection of ultrasonic syllables (I) and (II) based on a set detection threshold. X-axis = time; Y-axis = frequency. (D) USV calls were significantly reduced in L-CM rats compared to all other groups at P7 and P11. The data were analysed using one-way ANOVA (B) and two-way repeated measures ANOVA (D) followed by Holm-Sidak comparison. The data are presented as box and whisker plots (median with 5th and 95th percentile). **P < 0.01, ***P < 0.001.

Figure 5

The ketogenic diet (KD) induced intracerebral acidosis which had anticonvulsant effects. (A) Representative image of the ³¹P magnetic resonance spectroscopy (MRS) peaks obtained from postnatal day 7 lesioned rats on the control milk diet (L-CM; red) and lesioned rats on the KD (L-KD; black) rats. Note the chemical shift in inorganic phosphate (Pi) in the L-KD animal. (B) Intracerebral pH, as determined from the chemical shift in (Pi) and phosphocreatine (PCr) peaks on the ³¹P MRS, was lower in L-KD (n = 7) rats compared to L-CM (n = 6), control-CM (n = 5) and control-KD (n = 5) animals. (C) Pre-treatment of lesioned rats exposed to the KD (L-KD) with sodium bicarbonate (n = 8) to reverse intracerebral acidosis, resulted in a higher spasm frequency compared to L-KD, but lower spasm frequency compared to L-CM. The data were analysed using one-way ANOVA followed by Holm-Sidak comparison. The data are presented as box and whisker plots (median with 5th and 95th percentile). ***P < 0.001.

Figure 6

The ketogenic diet (KD) altered the respiratory profile of lesioned rats. (A) Representative breathing tracings for lesioned rats fed the control milk diet- (L-CM) and KD (L-KD) at postnatal day 7. Compared L-CM (n = 8), L-KD (n = 9) rats displayed a (B) lower respiration rate (RR), (C) higher tidal volume (VT), (D) an increase in total duration of breath (TTot), (E) no changes in minute ventilation (F) a decreased level of expired CO₂ (VCO₂), (G) increase in ventilatory efficiency slope (VE/VCO₂). The increase in TTot (D) and decrease in VCO₂ (F) in L-KD rats was also observed compared to the controls. No ventilatory changes were observed in control-CM (n = 8) and control-KD (n = 7) animals. The data were analysed using one-way ANOVA followed by Holm-Sidak comparison. The data are presented as box and whisker plots (median with 5th and 95th percentile). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

The ketogenic diet (KD) had no effect on mitochondrial respiration in the liver and the frontal cortex. (A) A schematic of the mitochondrial respiration protocol. No differences were observed in basal respiration, total mitochondrial respiration, maximal respiration or ATP-linked mitochondrial respiration in the liver (B-E) or frontal cortex (F-I) between all control and treatment groups. Control-Control milk diet (C-CM; n = 6), control-KD (C-KD; n = 6); lesioned-CM (L-CM; n = 7); lesioned-KD (L-KD; n = 7). The data were analysed using one-way ANOVA followed by Holm-Sidak comparison. The data are presented as box and whisker plots (median with 5th and 95th percentile).