In this study we demonstrated that inhibition of A1Rs completely abolished the KEKS-evoked delay in isoflurane-induced anesthesia (immobility) in WAG/Rij rats. Moreover, we extended our previous results showing that not only gavage of KE and KS , but also KSMCT, KEKS and KEMCT are able to increase both the blood level of βHB and number of seconds required before anesthetic induction (immobility).
Although isoflurane has been used in patients for nearly 50 years , its mechanism of action remains largely unknown. In spite of that both behavioral and physiological differences in functioning of sleep and general anesthetics-induced sleep-like state were demonstrated (e.g., general anesthesia is not able to appear spontaneously), it was suggested that several brain areas, such as cerebral cortex and the hypothalamic nucleus ventrolateral preoptic area may participate in both processes [37-39]. It was hypothesized, that anesthetics, such as isoflurane may induce anesthesia through common endogenous arousal neural circuitry/sleep pathways [39,40].
Administration of exogenous ketone supplements by gavage and subsequent metabolism [17,41,42] increases levels of ketone bodies in the blood stream (ketosis) [1,2,4,5]. Ketone bodies, such as βHB may enter into the brain through blood brain barrier and modulate different physiological and pathophysiological processes, such as sleep or seizures [7,8,12]. As ketosis (βHB) increases adenosine level  in the brain tissue and adenosine has a role in the sleep generation [27,28], enhanced level of βHB generated by ketone supplements may modulate naturally occurring sleep. Indeed, exogenous ketone supplement-generated ketosis may evoke a decrease in total sleep time through ventrolateral preoptic area [20,21,39]. Moreover, it has been demonstrated that level and metabolism of both ketone bodies [7,18,43], as well as adenosine and expression of adenosine receptors  are regionally different in the brain, which strengthen the modulatory role of ketone bodies and adenosine in processes such as sleep and sleep-like states. Ketosis-evoked increase in extracellular adenosine levels may change neuronal activity in different brain areas [22,44] implicated in sleep/sleep-like effects by its receptors. Increased level of adenosine was demonstrated during waking whereas adenosine concentration decreased during sleep in the brain . Adenosine agonists induced sleep/electroencephalographic slow-wave activity, but adenosine receptor antagonists (e.g., a non-selective antagonist of adenosine receptors caffeine) reversed effects of adenosine on the sleep . Moreover, adenosine accumulates under, for example, sleep deprivation and may have a role in the anesthetic action of isoflurane [27,39]: theophylline (a non-selective antagonist of adenosine receptors) reversed the cerebral effects of isoflurane in dogs (e.g., EEG has been changed from a sleep pattern to an awake pattern)  and caffeine accelerated emergence from isoflurane-evoked anesthesia in humans . Moreover, enhanced activity of A1Rs (e.g., by an A1R agonist N-sulfophenyl adenosine) may cause increase in anesthesia recovery time  and isoflurane may activate A1Rs . It has been demonstrated that receptors of adenosine, such as inhibitory A1Rs and excitatory A2ARs are expressed brain areas implicated in the generation of sleep and sleep-like effects, such as ventrolateral/lateral preoptic area and basal forebrain . Thus, adenosine may be a link between the anesthetic actions of isoflurane and sleep regulation as an endogenous sleep factor. Both A1Rs and A2ARs are implicated in sleep generation, but A2ARs are considered more important in sleep regulation : increased activity of A2ARs, for example, in ventrolateral/lateral preoptic area may induce sleep through sleep-active/promoting neurons [50,51]. However, our results suggest that A2ARs did not modulate the effect of exogenous ketone supplement-evoked ketosis on isoflurane-induced anesthesia (latency to immobility) (Fig. 1G). It was also demonstrated that inhibition or disinhibition by A1Rs (e.g., in wake-promoting neurons of basal forebrain or sleep-active neurons of ventrolateral preoptic area, respectively) may induce sleep [28,52,53]. Nevertheless, A1Rs may also promote wakefulness by inhibition of sleep-active neurons in lateral preoptic area . Based on these results, we can hypothesize that adenosinergic system may modulate the influence of exogenous ketone supplement-generated ketosis on the onset of isoflurane-induced immobility by inhibition of sleep active neurons (possibly through A1Rs), which processes lead to delay in the anesthetic effects of isoflurane. Moreover, modulatory effects of adenosine receptor antagonists and an A1R agonist on isoflurane-induced anesthetic effects and on emergence from anesthesia [29,47,48] were also demonstrated. Thus, it is possible that exogenous ketone supplement-induced ketosis not only delay the onset of isoflurane induced anesthesia (immobility), but also modulate the time required for recovery from anesthesia. However, further studies are needed to determine the exact effect and mechanism(s) of action of exogenous ketone supplement-evoked ketosis on isoflurane-generated anesthetic effects.
It has been demonstrated that gavage of exogenous ketone supplements, such as KSMCT for 7 days not only increases the number of seconds required before isoflurane-induced anesthetic induction (the time until immobility) (Fig. 1D) , but also generates decrease in both anxiety level on elevated plus maze  and absence epileptic activity  in WAG/Rij rats. These effects may be in correlation with enhanced level of βHB (Fig. 1E and F) [23,31,54]. Moreover, it was showed that inhibition of A1Rs may abolish the anti-anesthetic (Fig. 1G), antiepileptic  and anxiolytic  effects of exogenous ketone supplements, suggesting that adenosinergic system may modulate the ketone supplements (ketosis) induced influences in the CNS. Indeed, it was proposed that adenosinergic system (mainly through A1Rs) has a role in the modulation of sleep and sleep-like effects [27-29], different types of epilepsies [55-57] and anxiety [58-60]. However, new studies are needed to reveal the likely (at least partly) common mechanism(s), as well as interactions of adenosine receptors and adenosine receptor-evoked changes (e.g., in ion channels, signal transduction, metabolic processes) in different brain areas involved in sleep/sleep-like effects, epilepsy and anxiety, by which ketone supplements could exert its above mentioned influences.
One limitation of our study is that we used the WAG/Rij rat strain exclusively to investigate the effect of ketone supplementation on isoflurane-induced anesthesia. In addition, during this study we narrowed our focus on the influence of ketone supplement-evoked effects to the adenosinergic system. Nevertheless, this WAG/Rij rat strain is extensively used for investigation of different drugs on CNS diseases [1,61-64], and the present study further supports our previous experiments  on the role of the adenosinergic system. It has been suggested that the ketosis-evoked increase in adenosine levels  can modulate influence of ketone supplements not only on different CNS diseases [8,31,54], but also sleep and sleep-like effects [20,21,27-29] via adenosinergic system (likely through A1Rs). Consequently, we propose that the adenosinergic system may be one of main neurotransmitter systems by which ketone supplements can exert their influence on isoflurane-induced anesthesia.