Introduction

Hyperbaric oxygen (HBO 2 ) is an FDA‐approved medical therapy for at least 14 conditions including air/gas embolism, decompression sickness, carbon monoxide poisoning, and diabetic wounds, among others. HBO 2 involves breathing pressurized oxygen which results in increased dissolved oxygen in the blood plasma (Neuman and Thom 2008). This increased systemic partial pressure of oxygen (hyperoxia) helps deliver elevated levels of O 2 to various tissues. However, hyperoxia can also generate excessive oxygen‐free radicals which can damage cellular components through oxidative stress, potentially leading to significant tissue injury. Consequently, prolonged HBO 2 ‐induced hyperoxia has been shown to increase the risk, severity, and frequency of seizures (Clark and Thom 1997). This condition, which is a result of elevated tissue partial pressures of O 2 , is known as central nervous system oxygen toxicity (CNS‐OT), and occurs when breathing 100% O 2 at barometric pressures (Pb) >2.4 atmospheres absolute (ATA), and poses a limitation on the use of HBO 2 as a therapy (HBOT), and other applications of hyperbaric oxygen, such as recreational and technical divers using elevated O 2 partial pressures.

HBO 2 has additional applications within the field of research as a reliable, reproducible, and reversible stimulus for producing generalized tonic‐clonic seizures, proving useful for studying the physiological changes that occur during seizures, as well as for assessing the efficacy of various anti‐seizure methods in animal models. Rodent studies have shown that the development of hyperoxia‐induced seizures can be delayed by food deprivation. In a previous study, researchers demonstrated that fasting (24–36 h) could postpone the onset of seizures from HBO 2 by up to 300% (Bitterman et al. 1997). Throughout episodes of fasting, or with strict adherence to a ketogenic diet (KD), the body limits glucose availability, which suppresses insulin signaling and mobilizes free fatty acids (FFA) for fuel from adipose tissue stores (Cahill 2006). Adipose‐derived FFAs are generally impermeable to the blood brain barrier (BBB), however; hepatic ketogenesis converts FFA into ketone bodies, β‐hydroxybutyrate (βHB), and acetoacetate (AcAc). Acetone is also produced in small amounts due to spontaneous decarboxylation of AcAc. Under normal conditions, the concentration of systemic βHB is very low (≤0.1 mmol/L) and accounts for <3% of total cerebral metabolism (Hawkins et al. 1971). Conversely, during extended periods of fasting or strict KD adherence, ketone bodies accumulate in the blood (up to ~5–6 mmol/L) and cross the BBB via monocarboxylic acid transporters (MCT1‐4), allowing for their utilization as fuel by the brain (Prins 2007). Under conditions of fasting it has been reported that >60% of brain energy metabolism is derived from ketone bodies βHB and AcAc (Cahill 2006).

Factors that increase oxidative stress may disrupt metabolic control in the brain, and ketone bodies may restore metabolic homeostasis through a broad array of biochemical, molecular, and cellular changes (Yao et al. 2011; Simeone et al. 2018). Multiple benefits have been linked to the metabolic adaptations associated with fasting‐induced ketosis, including decreased production of reactive oxygen species (ROS), improved mitochondrial function, reduction in inflammation, and expression of brain‐derived neurotrophic factor (BDNF) (Maalouf et al. 2009; Marosi et al. 2016). The clinical efficacy of the KD has been validated in several animal models of epilepsy. The KD increases the threshold for seizures induced by amygdala kindling and GABA antagonists (such as pentylenetetrazole), and delays the development of seizures in EL mice, SD rats, Frings audiogenic seizure‐susceptible, and flurothyl‐treated mice (Hori et al. 1997; Bough and Eagles 1999; Todorova et al. 2000; Mantis et al. 2004; Bough and Rho 2007; Rho and Sankar 2008). In rodents with kainic acid‐induced seizures, the KD reduces the risk of developing epilepsy, and the severity of the symptoms. These effects may be explained by the reduction in hippocampal excitability and decreased supragranular mossy fiber sprouting (Muller‐Schwarze et al. 1999; Noh et al. 2003; Xu et al. 2006). The improvement observed with ketone utilization is similar to that observed with therapeutic doses of anti‐epileptic drugs (AEDs) (Bitterman and Katz 1987; Tzuk‐Shina et al. 1991) and novel anticonvulsants that inhibit excitatory glutamatergic neurotransmission (Chavko et al. 1998).

Based upon these results and other studies, nutritional ketosis has been shown to be an effective treatment option for patients with drug‐resistant seizure disorders (Freeman and Kossoff 2010). The clinical use of therapeutic ketosis is well documented in both children and adults (Klein et al. 2010). Despite multiple studies demonstrating the efficacy of fasting and ketosis in seizure reduction, the molecular mechanisms have remained largely unknown. However, a recent study using absence seizure‐prone Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats has proposed the involvement of the adenosinergic A1 receptor (AA1R) pathway (Kovács et al. 2017). An additional study suggests other stabilizing mediators could be involved, including polyunsaturated fatty acids, which modulate ion channels (Rogawski et al. 2016).

The anticonvulsant effects of the KD typically correlate with increased concentrations of ketone bodies in the blood, particularly AcAc and acetone (Bough and Rho 2007; Mcnally and Hartman 2012). However, when compared to prolonged fasting, the levels of blood ketones associated with the KD are often limited due to the difficulty of nearly complete carbohydrate restriction required (Cahill 2006). Ketone supplementation with esters of βHB or AcAc can induce a rapid and sustained therapeutic ketosis that mimics the effects of prolonged fasting or a rigid KD without dietary restriction (Desrochers et al. 1995; Brunengraber et al. 1997). Moreover, previous research has established that orally administered esters of βHB are tolerated and safe in rats (Clarke et al. 2012), as well as humans (Clarke et al. 2012). In 2013, D′Agostino et al. investigated whether a ketone diester [R,S‐1,3‐butanediol acetoacetate diester (BD‐AcAc 2, KE)] (Ciraolo et al. 1995; Desrochers et al. 1995; Puchowicz et al. 2000), could replicate fasting and the KD's ability to increase latency to seizure (LS). Using 3 months old SD rats, they showed that KE is effective in causing a rapid (<30 min) and sustained (> 4 h) elevation of AcAc (>3 mmol/L) and βHB (>3 mmol/L), and delayed LS by 574 ± 116% compared with control (water) due to the effect of AcAc and acetone, but not βHB alone (D'Agostino et al. 2007). During that study 5 ATA O 2 pressure was tested. Therefore, to make our results comparable we used the same conditions for the present study. In a more recent study, the ability of KE to elevate blood βHB rapidly (<30 min) and for a sustained period (8 h) was further demonstrated in SD rats (Kesl et al. 2016).

Medium‐chain triglyceride (MCT) oil contains fatty acids 8–10 carbons in length. Upon ingestion, these medium‐chain fatty acids are readily converted to βHB causing a rapid elevation in blood ketones. It has been demonstrated that co‐ingestion of MCTs along with other ketogenic supplements prolonged ketone elevation (Kesl et al. 2016). Additionally, MCTs have been previously shown to have antiseizure effects (Augustin et al. 2018), therefore in this study we also tested ketogenic agents combined with MCTs.

The potential for ketone supplementation to circumvent the dietary restriction associated with the KD to achieve therapeutic ketosis has been previously discussed (Ari et al. 2015; D'Agostino 2016). This strategy to induce nutritional ketosis would be favored by those unwilling or unable to follow a strict KD. From an operational perspective, the use of ketone supplementation offers practical advantages due to the rapid onset and ability to titrate the dosage and formulation for individual response, pharmacokinetic profile and associated mechanistic properties (Bough and Eagles 1999; Kovács et al. 2017).

The specific aim of this study was to expand upon previous research establishing the link between therapeutic ketosis and the resulting antiseizure effects. We hypothesized that ketogenic strategies that elevate both βHB and AcAc would have the greatest potential delaying CNS‐OT. Thus, we explored the effect of different dosages and combinations of exogenous ketone supplements on CNS‐OT seizures in an older age group of male SD rats (18 months), which is used to model middle age in humans.