Caffeine exacerbates seizure-induced death via postictal hypoxia

Sudden unexpected death in epilepsy (SUDEP) is the leading epilepsy-related cause of premature mortality in people with intractable epilepsy, who are 27 times more likely to die than the general population. Impairment of the central control of breathing following a seizure has been identified as a putative cause of death, but the mechanisms underlying this seizure-induced breathing failure are largely unknown. Our laboratory has advanced a vascular theory of postictal behavioural dysfunction, including SUDEP. We have recently reported that seizure-induced death occurs after seizures invade brainstem breathing centres which then leads to local hypoxia causing breathing failure and death. Here we investigated the effects of caffeine and two adenosine receptors in two models of seizure-induced death. We recorded local oxygen levels in brainstem breathing centres as well as time to cessation of breathing and cardiac activity relative to seizure activity. The administration of the non-selective A1/A2A antagonist caffeine or the selective A1 agonist N6-cyclopentyladenosine reveals a detrimental effect on postictal hypoxia, providing support for caffeine modulating cerebral vasculature leading to brainstem hypoxia and cessation of breathing. Conversely, A2A activation with CGS-21680 was found to increase the lifespan of mice in both our models of seizure-induced death.

simultaneously recording brain tissue oxygen levels in the hippocampus and brainstem breathing centers.Acutely, using intrahippocampal kainic acid and chronically using Kcna1 −/− mice, we found that caffeine significantly accelerated time to death in both models, indicating that caffeine consumption could be a modifiable risk factor in SUDEP pathophysiology.

Results
Acute caffeine, theophylline and N6 accelerate time to death whereas CGS-21680 delays it.Having previously established an acute model of seizure-induced death in mice 14 , we sought to screen caffeine and other drugs of interest to determine their relationship to SUDEP (Fig. 1).We first sought to determine if adenosine receptor modulation had an effect on time to seizure-induced death.Here we tested agonists and antagonists on the two primary adenosine receptors expressed in the brain: A 1 and A 2A .After intrahippocampal administration of the chemoconvulsant kainic acid in C57BL/6J mice, all mice were monitored using cardiorespiratory physiology and brain tissue oxygen to establish time to death defined as terminal apnea detected by diaphragmatic EMG 17 .All mice displayed several bouts of bilateral tonic-clonic seizures with intervening behavioral recovery.On average, mice that received vehicle died of seizure-induced terminal apnea 49.78 ± 1.30 min after intrahippocampal administration of kainic acid (Fig. 2).Mice that were treated with caffeine a non-selective A 1 / A 2A antagonist died sooner than vehicle mice with a mean time to death of 27.21 ± 2.65 min.We then examined theophylline, a metabolite of caffeine and a non-selective A 1 /A 2A antagonist.Theophylline displayed a similar temporal profile to caffeine, having their time to death accelerated with a mean time to death of 30.32 ± 3.67 min.To determine if caffeine was interacting with the A 1 receptor to influence time to death, we used the selective A 1 agonist N6-cyclopentyladenosine (N6).Mice that were treated with N6 had their time to death accelerated with a mean time to death of 35.95 ± 4.27 min.We next determined if caffeine's influence on time to death was mediated by the A 2A receptor.To this end we used CGS-21680, a selective A 2A agonist.Mice that received CGS-21680 displayed increased survival compared to vehicle, caffeine and N6 treated mice with a mean time to death of 114.0 ± 5.72 min.Our data provide evidence that caffeine accelerated time to death likely independent of the A 1 receptor as the use of the A 1 agonist N6 mirrored the effects of caffeine, a non-selective antagonist.Using CGS-21680 as a probative tool, caffeine is likely interacting at the A 2A receptor to accelerate time to death from hypoxia.

Acute caffeine reduces baseline brainstem oxygen. A previous study examining rat hippocampal
oxygen levels demonstrated that caffeine significantly reduces baseline oxygen and exacerbated seizure induced hypoxia 18 .Here we monitored brain tissue oxygenation in the hippocampus (HPC) and the pre-Bötzinger complex (PBC), a respiratory nucleus located in the ventral medulla responsible for the generation of rhythmic inspiratory breathing movements in C57BL/6J mice 24 .We measured oxygen levels at three time points prior to intrahippocampal kainic acid infusion to examine the influence of adenosinergic agonists and antagonists on physiological oxygen values in the HPC and PBC of the brainstem (Fig. 3).We examined baseline oxygen levels at pre-injection of drug or vehicle (T-1), 10-min post-injection of drug or vehicle (T-2) and 30-min Acute caffeine administration accelerates time to terminal hypoxia, apnea and death.We have demonstrated that caffeine significantly reduces baseline hippocampal and brainstem oxygen while CGS-21680 significantly increases its levels.Our next step was to establish the sequence of events that occur with cardiorespiratory function and oxygen following the administration of either caffeine or CGS-21680.Following the intrahippocampal administration of kainic acid in C57BL/6J mice, the partial pressure of oxygen (pO 2 ) remained above 10 mmHg.Oxygen levels at or below this threshold are considered severely hypoxic and are www.nature.com/scientificreports/associated poor clinical outcomes leading to increased morbidity and mortality 25 .Vehicle treated mice displayed the following sequence of events leading to death: terminal electrographic seizure in the PBC, with behavioural bilateral tonic-clonic seizure, terminal hypoxia in the HPC, followed terminal hypoxia in the PBC with concomitant terminal apnea occurring once pO 2 dropped below 4.8 ± 1.84 mmHg and finally followed by asystole several minutes later.Mice that were treated with caffeine displayed the same sequence of events, however, it was temporally accelerated with apnea occurring once pO 2 dropped bellow 5.1 ± 2.18 mmHg.Mice that were pretreated with CGS-21680, displayed significantly longer latency to time of death compared to vehicle and caffeine mice (Fig. 4).

Chronic adenosine receptor modulation influences time to death in Kcna1
−/− mice.Our acute intrahippocampal kainic acid experiments indicated that a single dose of caffeine or its metabolite significantly accelerated time to death and CGS-21680 reduced time to death.Here we determined if the observations made in the acute experiments could be replicated chronically using Kcna1 −/− mice which displays frequent self-generated seizures beginning in early life through to adulthood with high incidence of premature seizure-induced death 26 .On postnatal day (P) 30 mice were randomly assigned to either caffeine-in-water or water-only groups.
The median survival time was significantly shorter in mice treated with caffeine (n = 9) compared to the wateronly (n = 9) group (P37 vs. P42, respectively).Our acute experiments also indicated that a single dose of the A 2A www.nature.com/scientificreports/agonist, CGS-21680, significantly extended life.To test whether chronic CGS-21680 could also produce significant life extension, we administered this drug chronically.Due the solubility limitations, we administered CGS-21680 intracerebroventricularly via an osmotic minipump.The median survival time was significantly longer in mice treated with CGS-21680 (n = 9) compared to the water only (n = 9) group (P51 vs. P40 respectively) (Fig. 5).These data clearly demonstrate that chronic ingestion of caffeine may be detrimental in those with epilepsy, increasing the likelihood of premature mortality.Conversely, the A 2A agonist CGS-21680 was shown to be neuroprotective.

Discussion
Self-limited seizures in both rodents and people with epilepsy result in vasoconstriction, hypoperfusion and hypoxia [12][13][14] .Postictal hypoxia was hypothesised to lead to SUDEP 16 .George and colleagues (2023) recently tested this neurovascular based hypothesis of SUDEP and found that seizure activity in brainstem breathing centres www.nature.com/scientificreports/led to severe hypoxia and subsequent breathing failure 17 .They also identified two drug targets, cyclooxygenase-2 and L-type calcium channels, that when blocked prevented postictal hypoxia for a period of time and extended life in their models of seizure-induced death.Caffeine and other drugs that modulate adenosine receptors were previously found to increase the length of postictal hypoxia 18 .In the present study we observed that caffeine significantly lowers baseline oxygen in the hippocampus and brainstem and accelerates time to death.We provide evidence against caffeine's involvement of the A 1 receptor by using the selective A 2A agonist CGS-21680 which was able to increase baseline oxygen and decrease time to death.Our study is the first to show an association between caffeine and postictal hypoxia in SUDEP pathophysiology.We demonstrated that caffeine accelerated time to death in both our acute and chronic models of seizureinduced death.This study made the critical observation that acute dosing of caffeine significantly reduced baseline physiological oxygen in both the hippocampus and pre-Bötzinger complex.The neurovascular theory of SUDEP posits that seizures that spread to the brainstem lead to hypoxia of breathing centres and subsequent breathing failure.The pre-seizure reduction in oxygen seen in response to caffeine can serve to increase the likelihood of fatal outcomes as it sets the stage for death.The cerebral vasoconstrictive effects of caffeine are well established [27][28][29] .Caffeine exerts its effect as a vasoactive substance on a wide range of molecular targets 30 .Primarily, it acts as a non-selective antagonist of the A 1 and A 2A adenosine receptors.We first isolated the receptor subtype responsible for promoting caffeine's effect on postictal hypoxia and early death in our models.We first examined A 1 activation using the selective A 1 agonist N6-Cyclopentyladenosine (N6).In our hands, N6 showed a similar effect to caffeine by accelerating time to death.We used the A 2A selective agonist CGS-21680 which reduced time to death in both our acute and chronic models.Based on these receptor pharmacology results, we have evidence that caffeine is likely interacting with A 2A receptors to produce its vascular effects.www.nature.com/scientificreports/Contrary to our findings, a previous study in mice showed that acute caffeine administration increased survival in a model of seizure-induced death 31 .In their study, to identify if increased adenosine was responsible for SUDEP, caffeine was administered after seizure onset following systemic kainic acid administration.Their study however did not quantify physiological parameters such as respiratory and cardiac physiology which limits the ability to draw conclusions regarding the cause of death.Further, the caffeine dose of 40 mg/kg exceeds the equivalent dose consumed by a typical human, reducing clinical translatability 32 .
The present study utilized two previously published models of seizure-induced death: acute intrahippocampal kainic acid which causes several bouts of bilateral tonic-clonic seizures with a resultant terminal seizure, and chronic Kcna1 −/− mice that display self-generated bilateral tonic-clonic seizures early in life with a high incidence of seizure-induced death 17 .These models were chosen as they recapitulate core features of SUDEP in humans, including young age, high seizure burden, and bilateral tonic-clonic seizures.As a function of these all-or-none seizure-induced death models, all mice died regardless of treatment as these drugs were administered to assess how they modulate time to death.We recorded several physiological parameters to assess the sequence of events leading to death.Breathing in this study was measured using a diaphragmatic EMG which records phrenic nerve activity.The EMG method determines cessation of diaphragmatic contraction and does not measure ventilation.Therefore, we cannot rule out a potential peripheral obstruction such as a laryngospasm.This model also produces several bouts of recurrent seizures which can lead to metabolic derangements and contribute to premature death.In our chronic study, Kcna1 −/− mice that received caffeine or CGS-21680 did not have their seizures recorded which limits our ability to assess whether latency to death was mediated by anti-seizure effects.
The intrahippocampal kainic acid model provides a unique tool to examine the physiological alterations that occur during and after a seizure, leading to mortality.High doses of kainic acid can evoke status epilepticus (SE) 33 which precludes a diagnosis of SUDEP.However, lowering the dose can produce non-status models 34,35 .Previous reports have established cerebral oxygen markers of SE in rodent models.Using oxygen sensitive probes, it has been demonstrated that SE produces robust and long-lasting hyperoxia which correlates with increased cerebral blood flow 36,37 .This is also consistent with other reports that show sustained cerebral hyperperfusion following kainic acid-induced SE 38 and pilocarpine-induced SE 39,40 .During recordings in this study, all mice had at least two behavioral seizures followed by postictal hypoxia and severe hypoxia prior to death, which is not associated with SE.
Brain function depends critically on moment-to-moment regulation of oxygen supply by the bloodstream to meet changing metabolic needs as seen during seizures.In fact, seizures significantly increase metabolic rate and oxygen consumption by mitochondria, severely limiting the availability of oxygen within the system 41 .This metabolic reduction in oxygen could contribute to seizure-induced mortality if it involved the brainstem and was temporally correlated to postictal vasoconstriction.Recently, Villa and colleagues (2023) demonstrated that postictal mitochondrial dysfunction and the unchecked conversion of oxygen to reactive oxygen species also contributes to postictal severe hypoxia 42 .Further they found that using a mild mitochondrial uncoupler which prevents the conversion of oxygen to reactive oxygen species ameliorated postictal hypoxia and provide a potential therapeutic treatment to reduce the risk of SUDEP 42 .
The endogenous nucleoside adenosine plays an important role in several physiological processes 43 .In the central nervous system, adenosine imparts an inhibitory tone on synaptic activity and is a potent modulator cerebral blood flow and tissue oxygenation [43][44][45] .Adenosine levels in the brain increase during pathological states such as ischemia, hypoxia, and seizures 46,47 and may play a role in seizure termination.Despite its antiseizure properties, excessive adenosine is postulated to exert detrimental effects in the brainstem 48,49 .The adenosine hypothesis of SUDEP states that seizure-induced increase of adenosine in the brainstem leads to inhibition of breathing circuits via the activation of adenosine receptors 31,48,49 .However, contrary to this hypothesis, we show that activation of the A 2A receptor is beneficial by extending life.The endogenous ligand for the A 2A receptor is adenosine.Among the adenosine receptors, A 2A is the primary receptor responsible for the vasodilatory effects of adenosine.
We also demonstrate that the A 2A agonist CGS-21680 both increased baseline hippocampal and brainstem oxygen and prolonged time to death.Activation of the A 2A receptor mediates vasodilation in a variety of vascular beds including cerebral vasculature [50][51][52][53][54] .This work aligns with previous studies showing a robust dilatory effect of CGS-21680 50 .Ngai and colleagues (2001) found that CGS-21680 induced significant dose-dependent vasodilation of cerebral arterioles 55 .Further, using the selective A 2A antagonist ZM-241385, they were able to inhibit the dilatory response to both CGS-21680 and adenosine.They concluded that the A 2A receptor was responsible for the adenosine-induced dilatory effect they saw in rat cerebral arterioles.
Caffeine has been shown to display vasoactive properties through a number of molecular systems, including adenosine 30 .Adenosine's actions are mediated by specific cell surface receptors coupled to G proteins classified as A 1 , A 2A , A 2B , and A 3 56 .Caffeine acts as a competitive antagonist of the A 1 and A 2A receptors.However, caffeine also influences cerebrovascular dynamics through mechanisms independent of adenosine receptors.Caffeine can liberate calcium from intracellular stores and modulate calcium channels and increase prostaglandin E2 (PGE2) synthesis [57][58][59] .Previous studies have found that seizure-induced vasoconstriction is mediated by vasoactive prostanoids, such as PGE2 12,16 .They found that ibuprofen, a cyclooxygenase-2 blocker prevented postictal vasoconstriction, hypoperfusion and brain tissue hypoxia.They also showed that ibuprofen did not influence seizure duration, indicating that its effects were attributable to the neurovascular phenomenon and not a seizurealtering mechanism 17 .Caffeine's ability to increase the production of PGE2 coupled with seizure induced PGE2 activation could potentially culminate to produce fatal outcomes when it occurs in the brainstem and may have occurred in our models.
Sudden unexpected death in epilepsy continues to be a major cause of death in people with drug resistant epilepsy 60,61 .Our data support a detrimental effect of caffeine on SUDEP pathophysiology in two models of seizure-induced death.In our hands acute administration of caffeine significantly decreased oxygen in both the hippocampus and brainstem and accelerated time to death.The ubiquitous nature of caffeine consumption makes this finding of considerable clinical significance.Caffeine consumption is an easily modifiable risk factor for SUDEP and reveals a promising strategy to reduce death.Additionally, the present study points to the utility of A 2A agonists as a promising tool to reduce SUDEP risk.

Methods
Animals.Animal care and use procedures were carried out in accordance with standards set by the Canadian Council for Animal Care and ARRIVE guidelines.Experimental protocols were approved by the Health Sciences Animal Care Committee at the University of Calgary (AC20-0170).Male C57BL/6J mice aged 7 weeks (P50) and weighing 25-32 g were obtained from Charles River Laboratories (Wilmington, MA, USA).Male Kcna1 −/− mice on a C3HeB/FeJ background were obtained from heterozygous breeding colonies established at the University of Calgary.Animals were housed in a temperature-controlled environment with ad libitum access to food and water.All procedures complied with ARRIVE guidelines.
Diaphragmatic surgery.On the same day after brain implantation surgery, mice were maintained on 2% isoflurane mixed with 100% oxygen.Diluted lidocaine (0.5% 1:4 lidocaine, saline) was injected subcutaneously at the incision site on the dorsal segment of the ribs just below the ribcage.Electrode wires were implanted as described by Pagliardini and colleagues into the diaphragm to record phrenic nerve activity 62 .In brief, multi-stranded Teflon-coated stainless-steel electrode wire (#AS633, Cooner Wire) were passed through the diaphragm using a suture needle and secured in place by a knot.A second electrode wire was placed 1 mm away from the first to provide a bipolar recording.The electrode ends were then tunneled under the skin to an incision on the animal's dorsum, between the scapulae.The uninsulated ends of the electrode wires were then attached to male amphenol pins and secured in a 9-pin ABS socket (Ginder Scientific) that protruded from the back.The incision sites were sutured, and the mice recovered.Mice were administered injectable buprenorphine (0.05 mg/kg) every 12 h for 3 days post-operatively and were given 7 days to recover before beginning acute experiments.Chronic experiment surgery.Intracerebroventricular surgery.Male Kcna1 −/− mice on postnatal day 26, mice were anesthetized with 5% isoflurane and maintained on 2% isoflurane mixed with 100% oxygen.The skull was secured in a stereotaxic apparatus using ear and incisor bars.Diluted lidocaine (0.5% 1:4 lidocaine, saline) was injected subcutaneously over the incision site followed by a sagittal incision along the midline of the head.The incision was then extended over the dorsum of the mouse posterior to the scapulae.Haemostats were introduced subcutaneously to widen incision and produce dead space for pump insertion.A burr hole was made over the right lateral ventricle at these coordinates relative to bregma: (AP) + 0.3, (ML) − 1.0, (DV) − 3.0 30 .The infusion cannula was secured to the skull using Metabond Quick Adhesive (C&B) and dental acrylic.Mice were administered injectable buprenorphine (0.05 mg/kg) every 12 h for 3 days post-operatively and mice were allowed to recover for 3 days before starting the experiment on P30.The patency and placement of the cannula was verified at the end of the experiments using methylene blue dye (Sigma-Aldrich) administration.
Drug administration.Acute drug administration.After a 7-day recovery period from surgery, male C57BL/6J mice were administered either caffeine (10 mg/kg), theophylline (10 mg/kg), N6-Cyclopentyladenosine (N6) (1 mg/kg) or CGS-21680 (1 mg/kg) or vehicle DMSO for lipophilic drugs or saline for hydrophilic drugs) intraperitoneal (i.p) 30 min prior to infusion of kainic acid.Kainic acid (1.4 μg in 0.4 μL) was infused unilaterally through an intrahippocampal cannula into the right dorsal hippocampus at 0.1 μL/min through a 1 μL Hamilton syringe (Hamilton Robotics, Reno NV) and micro-syringe pump (Harvard Apparatus, model 55-2222, Canada).All drugs were purchased from Cayman Chemicals.In acute experiments, local field potential, EMG and brain pO 2 were recorded continuously and time to death following infusion was recorded.
Chronic caffeine in drinking water.Male Kcna1 −/− mice on a C3HeB/FeJ congenic background were used.These mice carry a deletion of the Kcna1 gene on chromosome 6 and are lacking functional voltage-gated Kv1.1 channels.Starting the experiment on postnatal day 30, male mice received either water (vehicle) or water with 0.5 mg/mL caffeine (Sigma-Aldrich).Mice were monitored every 12 h and date of death recorded.Caffeine was protected from light and replaced every 12 h to ensure photostability and solubility.

Figure 1 .
Figure 1.Acute experimental timeline.Male C57BL/6J mice on P50 underwent brain implantation surgery and diaphragmatic surgery (Day 0).Mice recovered for one week before beginning terminal experiment (Day 7).Mice were injected with either caffeine, theophylline, N6-Cyclopentyladenosine (N6) or CGS-21,680, 30 min prior to infusion of kainic acid.Physiological parameters including heart rate, respiratory rate, brain partial pressure of oxygen and local field potential were recorded until death.