Background
It has been demonstrated that exogenous ketone (ketogenic) supplements, such as ketone ester (KE), not only increase the level of ketone bodies (e.g., β-hydroxybutyrate/βHB) [
1‐
5], but also maintain blood levels of ketone bodies in both animals and humans [
2,
3,
6]. Ketone bodies, such as βHB, enter into the brain through blood-brain barrier and provide fuel to brain cells [
7,
8] improving cell energy metabolism (e.g., enhance mitochondrial ATP synthesis) [
9]. Moreover, ketone supplement-induced ketosis can suppress neuronal excitability [
7,
10,
11], modulate functions of ion channels and neurotransmitter systems (e.g., increase GABA and adenosine levels) [
7,
12‐
14] and influence inflammatory processes (e.g., decrease the concentration and expression of proinflammatory cytokines) [
15]. It was suggested that these effects of ketosis may have therapeutic potential in the treatment of several central nervous system (CNS) diseases, such as Alzheimer’s disease, Parkinson’s disease, epilepsy and psychiatric disorders (e.g., anxiety, schizophrenia and depression) [
1,
3,
8,
16]. It was also demonstrated that exogenous ketone supplements, such as KE and ketone salt (KS) are relatively well-tolerated without (or with minimal) adverse effects [
1,
2,
6,
16,
17]. However, exact mechanism(s) of action of exogenous ketone supplement-generated ketosis on CNS diseases and other pathophysiological and physiological processes are largely unknown.
It was suggested that ketosis may modulate sleep and sleep-like effects [
18‐
22]. Indeed, it has been demonstrated recently that nutritional ketosis (evoked by exogenous ketone supplements, such as KE) delayed the onset of inhalational anesthetics isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether)- induced anesthesia (immobility) [
23] (light phase of anesthesia: loss of consciousness without movement, which was defined as ‘immobility’) [
24]. Nevertheless, mechanism of action of ketosis-induced changes in isoflurane-evoked anesthesia remains unknown. It was suggested that changes, for example, in functioning of different ion channels (e.g., K
ATP channels), neurotransmitter systems (e.g., GABAergic and adenosinergic system) and mitochondria (e.g., mitochondrial respiration) may have a role in ketone supplement-evoked effects on isoflurane-generated anesthesia [
19,
23,
25‐
27]. However, it has also been demonstrated that ketosis (evoked by exogenous ketone supplements) [
1,
2,
4,
5] may increase adenosine level in the brain [
14] and adenosine may have a role not only in the sleep [
28], but also the generation of sleep-like effects [
29,
30]. Therefore, in this study, we examined the effect of ketone supplement KE, KS and their mix (KEKS), as well as mix of KS and KE with medium chain triglyceride (MCT) oil (KSMCT and KEMCT, respectively) on isoflurane-induced onset of anesthesia (latency to immobility). Animals (Wistar Albino Glaxo Rijswijk/WAG/Rij rats) were fed with standard diet (SD) and were gavaged with different ketogenic supplements for 1 week (2.5 g/kg/day). After the last supplement gavage we recorded the time until onset of immobility (under 3% isoflurane). In the second part of the study, the potential role of adenosine receptors in the nutritional ketosis-evoked effects on isoflurane-induced onset of anesthesia (immobility) was investigated. We used a specific adenosine A1 receptor (A1R) antagonist DPCPX (1,3-dipropyl-8-cyclopentylxanthine) (intraperitoneally/i.p. 0.2 mg/kg) and a selective adenosine A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (i.p. 0.5 mg/kg) alone as well as in combination with KEKS (2.5 g/kg/day, gavage).
This study is the continuation of our previous study on genetically absence epileptic WAG/Rij rat strain (a well-investigated model of human absence epilepsy) [
31], in which it was demonstrated that exogenous ketone supplements (such as KE) delayed the onset of isoflurane-induced anesthesia (increased the time required before immobility) [
23]. These effects may be clinically relevant because administration of exogenous ketone supplements-induced ketosis are more and more widely used as a metabolic therapy in the treatment of different CNS diseases, such as epilepsy or other seizure disorders [
2,
8,
32‐
35]. Consequently, in order to implement a safe and successful anesthesia, potential effects of ketosis on the latency to anesthesia might need to be considered when epileptic patients are undergoing anesthetic procedures. For this reason, this study was performed on WAG/Rij rats, to better understand the ketone supplement-evoked effects on isoflurane-generated onset of anesthesia and its mechanism of action under epileptic condition.
In this study we hypothesized that adenosine receptor inhibition may modulate the exogenous ketone supplement-evoked delay in the latency to onset of immobility.
Discussion
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 [
23], but also KSMCT, KEKS and KEMCT are able to increase both the blood level of βHB (ketosis) and number of seconds required before induction of anesthesia (immobility).
Although isoflurane has been used in patients for nearly 50 years [
19], 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 [
42‐
44]. It was hypothesized, that anesthetics, such as isoflurane may induce anesthesia through common endogenous arousal neural circuitry/sleep pathways [
44,
45].
Administration of exogenous ketone supplements by gavage and subsequent metabolism [
17,
46,
47] increases levels of ketone bodies in the blood stream (nutritional ketosis) [
1,
2,
4,
32]. 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 [
14] in the brain tissue and adenosine has a role in the sleep generation [
28,
29], 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,
44]. Moreover, it has been demonstrated that level and metabolism of both ketone bodies [
7,
18,
48], as well as adenosine and expression of adenosine receptors [
49] 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,
49] 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 [
50]. 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 [
51]. Moreover, adenosine accumulates under, for example, sleep deprivation and may have a role in the anesthetic action of isoflurane [
28,
44]: 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) [
30] and caffeine accelerated emergence from isoflurane-evoked anesthesia in humans [
52]. Moreover, enhanced activity of A1Rs (e.g., by an A1R agonist N-sulfophenyl adenosine) may cause increase in anesthesia recovery time [
53] and isoflurane may activate A1Rs [
54]. 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 [
29]. Thus, adenosine may be a link between the anesthetic actions of isoflurane and sleep regulation as an endogenous sleep factor.
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 [
29,
55,
56]. Nevertheless, A1Rs may also promote wakefulness by inhibition of sleep-active neurons in lateral preoptic area [
57] and in ventrolateral preoptic area [
58]. Consequently, we can hypothesize that adenosinergic system may modulate the influence of exogenous ketone supplements, such as KEKS, on the onset of isoflurane-induced anesthesia (immobility) by inhibition of sleep active neurons (possibly by ketosis and, as a consequence, through increase in adenosine level as well as its A1Rs) [
14,
23], which processes lead to delay in the anesthetic effects of isoflurane. Indeed, although the A1R antagonist DPCPX alone did not change the isoflurane-generated anesthetic effect (immobility), combined administration of DPCPX with KEKS completely abolished the KEKS-evoked increase in latency to immobility under isoflurane anesthesia (Fig.
3). Moreover, adenosine receptors may also modulate anesthesia recovery time [
52,
53]. Thus, it is possible that exogenous ketone supplements not only delay the onset of isoflurane-induced anesthesia (immobility) [
23] (Fig.
2a and
3), 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 supplements (ketosis) on isoflurane-generated anesthetic effects.
As it was demonstrated, not only A1Rs but also A2ARs are implicated in sleep generation, and A2ARs are considered more important in sleep regulation [
29]: increased activity of A2ARs, for example, in ventrolateral/lateral preoptic area may induce sleep through sleep-active/promoting neurons [
57,
59]. It has been demonstrated that A1Rs are abundantly expressed in the brain whereas A2AR expression is week in most of brain areas such as ventrolateral preoptic area [
29,
49,
60]. Thus, as it was demonstrated, effects of adenosine on both sleep [
29,
55‐
58] and processes of anesthesia may be brain region- and receptor-dependent. In addition, our knowledge relating to the exact role of adenosinergic system in modulation of both isoflurane-evoked anesthesia and connections between brain areas implicated in processes of anesthesia is far from complete. Consequently, it is possible that A1Rs are predominant whereas A2ARs are secondary (if any) in adenosine-evoked influences on anesthesia at least at this level of isoflurane-generated anesthesia (loss of consciousness without movement: immobility) in WAG/Rij rats. Indeed, our results suggest that A2ARs have no effect on isoflurane-generated anesthesia (immobility): neither isoflurane-induced anesthesia (latency to immobility) nor the effect of exogenous ketone supplement KEKS on isoflurane-induced anesthesia (latency to immobility) were modulated by the A2AR antagonist SCH 58261 (Fig.
3). Nevertheless, it cannot be excluded that this physiologically effective dose of A2AR antagonist SCH 58261 (0.5 mg/kg) may not have been adequate to investigate its influence on isoflurane-generated light phase of anesthesia (loss of consciousness without movement, immobility), but may modulate the later/deeper phase(s) of isoflurane-evoked anesthesia. However, more studies are needed to explain the exact role of adenosine and its receptors in isoflurane-induced anesthesia.
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.
2a) [
23], but also generates decrease in both anxiety level on elevated plus maze [
36] and absence epileptic activity [
32] in WAG/Rij rats. These effects may be in correlation with enhanced level of βHB [
23,
32,
36] (Fig.
2b and c). Moreover, it was showed that inhibition of A1Rs may abolish the anti-anesthetic (Fig.
3), antiepileptic [
32] and anxiolytic [
36] 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 (e.g., through A1Rs) has a role in the modulation of sleep and sleep-like effects [
28‐
30], different types of epilepsies [
61‐
63] and anxiety [
64‐
66]. 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,
67‐
71], and the present study further supports our previous experiments [
23] on the role of the adenosinergic system. It has been suggested that the ketosis/βHB-evoked increase in adenosine levels [
14] can modulate influence of ketone supplements not only on different CNS diseases [
8,
32,
36], but also sleep and sleep-like effects [
20,
21,
28‐
30] 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. However, to get comprehensive view on influence of ketone supplements on isoflurane-evoked anesthesia more studies are needed on other animal strains and humans, on changes not only in adenosinergic, but also other neuromodulatory/neurotransmitter systems (such as cholinergic, dopaminergic, and GABAergic system), and on other phases of anesthesia/emergence from anesthesia [
24,
53,
72] by administration of different/higher doses and types of ketone supplements. Further studies are also needed to reveal exact effects of different doses of drugs were used, such as DPCPX and SCH 58261, on isoflurane-generated anesthesia by administration of distinct methods (e.g., not only i.p., but also microinjections/microdialysis to specific brain areas, such as basal forebrain, as well as intravenous administration) [
57,
58,
73].
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