A ketogenic diet decreases sevoflurane-induced burst suppression in rats

Abstract

Background: The brain requires a continuous fuel supply to support cognition and can get energy from glucose and ketones. Dysregulated brain metabolism is thought to contribute to perioperative neurocognitive disorders and anesthesia-induced burst suppression. Therefore, we investigated the relationship between brain metabolites and neurophysiology during the behavioral states of sleep and anesthesia under a standard diet (SD) or a ketogenic diet (KD).

Methods: We measured prefrontal cortex glucose, lactate, and electroencephalogram in Fischer344 rats during spontaneous sleep/wake followed by 3 % sevoflurane anesthesia. Nine rats were fed a KD and 8 rats a SD. To assess the role of adenosine receptor-mediated ketone activity on burst suppression, 5 additional rats on the KD received an intraperitoneal injection of vehicle or the adenosine A1 receptor antagonist, DPCPX, before 3 % sevoflurane.

Results: Sevoflurane induced larger fluctuations in glucose (p < 0.001) and lactate (p = 0.015) concentrations compared to sleep as measured by the standard deviation (glucose 0.085 mM and lactate 0.16 mM in sleep/wake and 0.25 mM and 0.41 mM during sevoflurane respectively). Changes in glucose and lactate were closely tied to electrophysiological oscillations. Animals on the KD had reduced burst suppression ratio (mean 10 % in KD vs 30 % in SD) (p = 0.007) as well as increased time to loss of movement (mean 14 min in KD vs 8 min in SD) (p = 0.003) compared to SD. DPCPX in KD rats showed a trend to increased burst suppression, reduced the time to start of burst suppression (45 min in KD+vehicle to 37 min KD+DPCPX) (p = 0.007), and increased duration of burst suppression (49 min in KD+vehicle to 90 min in KD+DPCPX) (p = 0.046) compared to KD+vehicle.

Conclusions: It is thought that anesthesia-induced burst suppression reflects an underlying deficiency in brain energy. Accordingly, we found that upregulating ketones, which increase available brain ATP levels, delayed anesthetic induction and decreased burst suppression consistent with the idea that the underlying metabolic state of the brain influences an anesthetic's effect on the brain. These findings suggest that metabolic interventions could be useful therapeutic targets to modulate brain activity during sleep and anesthesia. Future studies will examine whether ketones can reduce the cognitive symptoms associated with postoperative delirium.

Keywords: anesthesia; delirium; ketones; lactate; metabolism; sleep.

Fig. 1.

Prefrontal cortex (PFC) glucose and lactate levels vary with the sleep wake cycle in rat (representative trace from an animal on the ketogenic diet (KD)). A) Experiment timeline on the day of the experiment. B) Hypnogram. C) Extracellular glucose (blue) and lactate (red) concentration in the PFC. D) Multitaper spectrogram of the PFC EEG. E) PFC EEG voltage trace. F) EMG voltage trace.

Fig. 2.

Prefrontal cortex (PFC) glucose and lactate levels have a biphasic relationship with sevoflurane anesthesia (representative trace from a different animal on the ketogenic diet (KD)). Lactate increases during periods of high alpha and theta power associated with induction and emergence phases of anesthesia. Glucose increases during high delta power and burst suppression phases. A) Experimental timeline on the day of the experiment. B) Sevoflurane concentration in the chamber. C) Extracellular PFC glucose (blue) and lactate (red) concentration. D) Multitaper spectrogram of the PFC EEG. E) PFC EEG voltage trace. F) EMG voltage trace.

Fig. 3.

Extracellular lactate levels vary across behavioral state and are higher during sevoflurane anesthesia compared to sleep and wakefulness. A) Spectrogram of the EEG, hypnogram of the scored state, example lactate model fit for sleep/wake where the black line indicates the lactate concentration measured from the biosensor and the orange line indicates the modeled lactate concentration. Beta coefficients from the lactate model show the magnitude of the lactate increase from NREM to wake/REM sleep. B) Example lactate model fit for corresponding sevoflurane condition. C) Beta coefficients associated with lactate changes during Wake/REM, induction, and emergence compared to NREM/deep anesthesia lactate levels were statistically different using the Kruskal-Wallis one way analysis of variance (p = 0.001). A multiple-comparison corrected post hoc test found lactate to be lower during Wake/REM compared to induction and emergence. (Wake/REM vs induction: p = 0.003, Wake/REM vs emergence: p = 0.007). D) Lactate model concentrations for NREM sleep and deep anesthesia are significantly different (p < 0.001). The dynamic range in mM of glucose (E) and lactate (F) is greater during sevoflurane than sleep/wake (Glucose, paired t-test: p = 0.0002; Lactate, paired t-test: p = 0.015). Box plots show the median plus the lower quartile Q1 and upper quartile Q3 where the whiskers are the (non-outlier) minimum and maximum values.

Fig. 4.

Rats on the ketogenic diet (KD) have less burst suppression than rats on the standard diet (SD) at the same level of sevoflurane anesthesia. Example multitaper spectrograms of the EEG are shown for a rat on the SD (A) or KD (B) under 3 % sevoflurane anesthesia (indicated by black bars) and the burst suppression ratio (BSR) overtime is shown below each spectrogram. C) Raw EEG voltage traces from the above example recordings at 50 minutes showing the differences in burst suppression between the two groups. D) Animals on the KD had a lower group mean BSR compared to animals on the SD (two sample t-test, p = 0.007). E) Animals on the KD took longer to lose movement from sevoflurane than animals on the SD as measured by EMG activity (two sample t-test, p = 0.003). F) There was no significant difference in time to return of movement measured from the end of sevoflurane. Box plots show the median plus the lower quartile Q1 and upper quartile Q3 where the whiskers are the minimum and maximum. (n = 8, SD; n = 9, KD).

Fig. 5.

Rats on the ketogenic diet (KD) have significantly reduced burst suppression compared to rats on the standard diet (SD) and DPCPX reverses the KDs reduced burst suppression. A) SD and KD burst suppression ratio bootstrapped 95 % confidence intervals. B) SD and KD bootstrapped difference, black bar along the bottom indicates time period where BSR is significantly different at alpha = 0.05. SD and KD BSR significantly different from minute 43.4–129.8. C) Adenosine A1 antagonist DPCPX counteracts the reduction in BSR due to the KD. D) Bootstrapped paired difference with 95 % confidence intervals. Of note, A/B experiments had a slower rise in sevoflurane whereas C/D experiments had a faster rise in sevoflurane based on the flow rate of oxygen delivering sevoflurane. KD (n = 9) and KD+DPCPX (n = 5) BSR significantly different from minute 8–35, and from 65.4 to 112.8.

Fig. 6.

The adenosine A1 receptor antagonist, DPCPX reverses the ketogenic diet-induced reduction in burst suppression. A,B) The average multitaper spectrogram during 3 % sevoflurane (indicated by the black bar) for animals on the ketogenic diet (A) and the same animals on the ketogenic diet given 0.2 mg/kg DPCPX in an intraperitoneal injection just before the start of sevoflurane anesthesia (B). C) The total mean burst suppression ratio was not significant between KD+vehicle and KD+DPCPX (pairwise comparison). D) DPCPX reverses the delay in burst suppression associated with the KD, p = 0.007. E) The time to the end of burst suppression as measured relative to the end of sevoflurane was not significant (where 0 min is the end of sevoflurane administration). Negative values indicate animals that stopped showing burst suppression before sevoflurane ended. F) DPCPX increased the duration of burst suppression compared to the ketogenic diet alone (paired t-test, p = 0.046, n = 9, KD+vehicle; n = 5 KD+DPCPX). G) 1) Under standard diet (SD) conditions, glucose primarily provides ATP resulting in a baseline level of extracellular ATP and adenosine. 2) Support for the hypometabolism theory of burst suppression: the ketogenic diet (KD) increases ATP so that the brain does not run out of energy as quickly resulting in less burst suppression. 3) Support for the hyperexcitability theory of burst suppression: KD increases intra and extracellular ATP which increases extracellular adenosine which binds to adenosine A1Rs which activates K_ATP channels and stabilizes the hyperexcitability of burst suppression. 4) DPCPX blocks adenosine A1Rs and reverses the KD-induced decrease in burst suppression back to a level more similar to the SD condition where baseline levels of adenosine are present.