Acute sleep deprivation enhances susceptibility to the migraine substrate cortical spreading depolarization

Migraine is a multifactorial neurovascular disorder characterized by recurrent episodes of headache. One third of patients experience transient neurological symptoms associated with their attacks, the so-called migraine aura. Migraine is among the most common neurological diseases, affecting approximately 20% of the adult population [1]. The socioeconomic impact is high [2, 3], and the World Health Organization recognized migraine as a major public health problem by ranking it as #2 among all diseases causing disability [4] and as #1 in under-50-year-old humans of both genders [5]. Migraine attacks may increase in frequency over time, and approximately 2.5% of patients with episodic migraine develop chronic migraine [6]. The transition to more frequent attack patterns is influenced by genetic predisposition, co-morbid conditions, life events and lifestyle [7].

Sleep disorders are well known risk factors for migraine chronification [7, 8] and changes in wake-sleep patterns such as sleep deprivation are common migraine triggers [9]. Compared to a few decades ago, adults sleep less, and sleeping as little as possible is often seen as an admirable behavior. Sleep loss may result from total sleep deprivation (such as shift workers might experience), chronic sleep restriction (due to work, medical conditions or lifestyle), or sleep disruption (in sleep disorders such as sleep apnea or restless legs syndrome) [10]. Sleep disorders are frequently observed in specific headache diagnoses (e.g., migraine, tension-type headache, cluster headache) [11] and other nonspecific headache patterns (i.e., chronic daily headache, hypnic headache, “awakening” or morning headache) [12]. In migraineurs, sleep disorders are well known predictive factors of progression from episodic to chronic forms [13]. In contrast, a targeted behavioral sleep intervention can lead to an improvement in headache frequency and reversion from chronic to episodic migraine [14]. The sleep deprivation-induced increase in adenosine levels might be an underlying mechanism, because adenosine levels are elevated during migraine attacks, and administration of adenosine can precipitate migraine attacks [15].

Cortical spreading depolarization (CSD), discovered by Leão in 1944 [16], is considered the electrophysiological correlate of migraine aura, and a possible trigger of migraine headache [17]. CSD is an intense propagating depolarization of neuronal and glial membranes. Evoked when the local extracellular potassium concentration [K⁺]_e exceeds a critical threshold, CSD causes a loss of membrane resistance and massive ionic disturbances with cell swelling. Genetic and environmental factors important for the clinical manifestation of migraine have been shown to modulate cortical excitability, including the potential to elevate [K⁺]_e and glutamate to levels sufficient to initiate and facilitate CSD. For example, mutations causing the migraine-associated syndromes familial hemiplegic migraine (FHM) or cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) enhance CSD susceptibility [18, 19]. Similarly, gonadal hormones modulate CSD susceptibility, to explain the female preponderance in migraine [18, 20]. In the setting of sleep deprivation, adenosine overload with overstimulation of A1 receptors is a possible mechanism modulating neuronal activity and CSD, because adenosine A₁ receptor activation contributes to the persistent secondary phase of Leão’s cortical spreading depression [21, 22].

Mean arterial blood pressure, arterial pCO₂ and pH were within normal physiological range and did not differ among groups (Table 1).

We investigated the effect of sleep deprivation on CSD susceptibility, the electrophysiological correlate of migraine aura. Our experiments show that acute sleep deprivation for both 6 and 12 h, dose-dependently, increases CSD susceptibility in rats, as evidenced by a reduced electrical threshold for CSD induction in the 12 h sleep deprived group, and an increased frequency of CSD upon continuous topical application of KCl in both 12 h and 6 h sleep deprived groups. In order to maximize reliability and relevance of our findings, CSD susceptibility was tested using two well-established independent but complementary experimental paradigms (KCl application and electrical stimulation) that provided coherent results.

Clinically, sleep deprivation or excessive sleep, as well as other sleep disturbances are among to the most common attack triggers reported by patients with primary headaches (e.g. migraine without aura [25, 29], migraine with aura [30], familial hemiplegic migraine [31], tension-type headache [32, 33]). Conversely, sleep is associated with the resolution or relief of migraine attacks [34, 35]. Over half of reported migraine attacks are followed by daytime sleepiness and migraineurs often choose to sleep looking for relief from their headache [35]. Similarly, in animals, CSD is followed by an increase of NREM sleep phase duration, suggesting an increased need for sleep after an attack [36]. Patients with chronic migraine report shorter nightly sleep times than those with episodic migraine, and are more likely to exhibit difficulties falling asleep, staying asleep, are more prone to develop sleep triggered headache, and choose to sleep because of headache [35, 37]. The quality of sleep is decreased in adults with migraine [38, 39] and over half of them report difficulty initiating and maintaining sleep, at least occasionally [35]. Short sleepers, who routinely sleep 6 h or less per night, exhibit more severe headache patterns and are more likely to develop morning headaches on awakening than individuals who sleep longer [35]. Taken together, it appears there is a relationship between migraine and sleep disturbance, with a possible cause-effect relationship not being entirely clear at this point.

Consistent with these epidemiological findings, increasing evidence suggests that sleep deprivation has adverse effects on several pathways involved in headache, including cortical excitability, neurotransmission and neurogenesis [40]. Sleep disturbance impairs endogenous pain-inhibitory function and increases spontaneous pain, in particular headache [41]. Lack of sleep has been shown to increase cortical excitability, as a possible mechanism to explain our findings of an increased CSD susceptibility in sleep-deprived rats. For example, 4 h of sleep deprivation has been shown to enhance intrinsic cortical activity and excitability in rats, possibly through a Ca²⁺-dependent mechanism [42]. Similarly, in healthy humans using single pulse transcranial magnetic stimulation, it has been observed that sleep deprivation enhances cortical excitability with reduction of short intracortical inhibition [43]. As a possible underlying mechanism, sleep deprivation has been shown to increase levels of the excitatory neurotransmitter glutamate in rat cerebral cortex [44]. Moreover, sleep deprivation induces an increase in the expression of glutamate receptors in rat hippocampus, which is involved in increased susceptibility of short and long-term depression in that area [45]. Glutamatergic mechanisms have been implicated in CSD susceptibility [46], and, thus, an increase in glutamatergic neurotransmission could explain the sleep deprivation-induced increase in CSD susceptibility. In addition, lack of sleep may enhance CSD susceptibility by inhibiting the Na⁺/K⁺-ATPase in the setting of energy failure secondary to oxidative stress. The surge in adenosine triphosphate (ATP) levels, the energy currency of brain cells, occurs when neuronal activity is reduced, as during sleep. The levels of phosphorylated AMP-activated protein kinase (P-AMPK), important for cellular energy sensing and regulation, are lower during the sleep-induced ATP surge than during wake or sleep deprivation [47]. Accordingly, prolonged sleep deprivation significantly decreases the activity of anti-oxidative enzymes such as superoxide dismutase and glutathione peroxidase that regulate the level of reactive oxygen species [48] and has been shown to increase lipid peroxidation in the rat hippocampus, thalamus and hypothalamus, leading to a higher oxidative stress in these brain regions compared to others [49].

Another potential link between migraine, sleep deprivation and CSD is altered neurotransmitter dynamics. For example, adenosine has been implicated in migraine pathophysiology, because adenosine levels were shown to be elevated during migraine attacks and administration of adenosine can precipitate migraine attacks [15]. Moreover, the adenosine A2A receptor gene haplotype was found to be associated with migraine with aura [50]. Adenosine has been shown to be elevated after CSD in brain slices under conditions likely to trigger CSD in vivo, and adenosine receptor activation has been shown to be involved in the prolonged depression of synaptic transmission after CSD [51]. At the same time, a role for adenosine in sleep regulation has been suggested by studies showing a progressive increase in extracellular adenosine in the basal forebrain during prolonged wakefulness [52], possibly explaining the potential CSD-mediated negative effect of sleep deprivation on migraine.

The high prevalence of sleep disturbances in migraineurs could also depend in part on a dysregulation of the cyclic secretion of melatonin. During a migraine attack, the plasma levels of melatonin are decreased [53], and evidence suggests that administering melatonin to migraine sufferers relieves pain and decreases headache recurrence in some cases [54]. Accordingly, melatonin slows down retinal spreading depression [55] and can attenuate the process of trigeminovascular nociception induced by CSD in rats [56]. In addition, serotoninergic processes have been implicated in mediating the migraine promoting effect of sleep deprivation. Serotonin is a neuromodulator with a pivotal role in regulating several central nervous system activities, including pain threshold and sleep induction. Sleep deprivation causes an enhancement of serotonergic neurotransmission in the brain, as suggested in animal studies. In sleep-deprived rats, elevated serotonin levels have been measured in the hippocampus [57] and in serotonergic raphe nuclei [58], which, with their widespread cortical projections, are part of the monoaminergic wake promoting system. Accordingly, human in vivo studies showed that plasma levels of serotonin and its precursor tryptophan exhibit a significant increase during sleep deprivation compared to sleep [59] and that a single night of total sleep deprivation causes significant increases of serotonin 2A receptor binding potentials in a variety of cortical regions [60]. Future experiments could aim to clarify the role of melatonin and serotonin in mediating the effect of sleep deprivation on CSD, for example by testing if melatonin supplementation or anti-serotonergic drugs diminish or abrogate the sleep deprivation-induced increase in CSD susceptibility.

Recently, Kilic et al. reported that the enhanced susceptibility to CSD after sleep deprivation may be due to impaired K+ and glutamate clearance by astrocytes consequent to sleep deprivation-induced suppression of glycogen use and synaptic energy substrate deficiency [61]. The authors demonstrated that sleep deprivation for 6 h using the same “gentle handling method” decreased threshold for CSD induction and prolonged the duration of CSDs without changing the CSD propagation speed and frequency, which, on the contrary, had increased in our experiments. Furthermore, this study reported that the decreased CSD thresholds could be recovered via the use of an alternate energy supply (D-glucose or L-lactate superfusion), linking decreased CSD thresholds to an activity/energy mismatch resulting from prolonged wakefulness [61]. An alternate energy supply was able to revert the enhancement of CSD susceptibility and reduced clearance of K+ during synaptic activity. Differences between results of the Kilic study and ours could be attributed to species differences, because mice show a predominantly vasoconstrictive response to CSD, which is in contrast to the biphasic vascular response that is seen in humans or rats, used in our experiments. In addition, Kilic et al. administered urethane/xylazine as anesthetic, which has a different effect on cortical excitability than isoflurane, which was used in our study [62].

We also tested the effect of chronic sleep deprivation on CSD susceptibility in rats sustaining the loss of VLPO neurons, a previously validated model of chronic sleep restriction [26]. It is well established that the VLPO is a key sleep-promoting cell group in the hypothalamus, and, already in 1930, von Economo reported that lesions of this region cause prolonged insomnia in humans [63]. Neurons in the preoptic hypothalamus are important regulators of sleep onset and sleep maintenance [64]. VLPO is thought to play a major role in causing sleep by GABAA receptor-mediated hyperpolarization and inhibition of histaminergic neurons in the tuberomammillary nucleus [65], which are important for promoting wakefulness [66]. VLPO lesions induce long-lasting insomnia in rats, with a 60–70% decrease in delta power and a 50–60% decrease in NREM sleep time [26]. VLPO lesion experiments allowed us to study the effect of chronic partial sleep loss on CSD without continual stressful experimental interventions, to avoid a possible confounding impact of stress on CSD. We here show that CSD susceptibility is not altered 6 weeks or 12 weeks after VLPO lesioning, suggesting that the effect of acute sleep deprivation on CSD is not mediated via reduced NREM sleep, that acute total sleep deprivation has a different effect on cortical excitability than chronic partial sleep restriction, and/or that compensatory mechanisms cause adaptation of cortical excitability within the chronic setting.

Our study has several limitations, including the fact that animal sleep deprivation models may not allow direct extrapolation to patients. However, gentle handling is very effective at inducing total sleep deprivation as determined by electroencephalography [67] and seems to be a valid model of typical sleep deprivation in humans. While the gentle handling method is less stressful than other methods that induce sleep deprivation, it still may be a cause of stress. It is therefore possible that the pathways induced by sleep deprivation in our animal model are different from those that occur in humans who consciously decide to stay awake or delay sleep. However, we feel that acute stress may not be important for our study endpoint, CSD susceptibility, as acute stress has recently been shown to not affect CSD susceptibility in rodents [68]. In addition, we used an invasive method to induce and record CSD, which may have affected our endpoint of CSD susceptibility. However, assessment of CSD susceptibility with electrocortical recordings, and CSD induction with KCl and electrical stimuli are the most established methods, used in many other respected studies in the field [17‐19]. Another possible shortcoming is the fact that rats were anesthetized during CSD assessment, and it has been shown that anesthetics modify CSD threshold [62]. Future experiments could perform CSD recordings in the sleep-deprived awake non-anesthetized rat, using non-invasive techniques for CSD induction, such as optogenetics [69]. In addition, assessment of additional migraine surrogates might further underscore the relevance of our findings for migraine, such as monitoring of face grimacing [70], and measurement of blood levels of adenosine, melatonin or serotonin - factors that are important in migraine and might mediate the effect of sleep deprivation on CSD. Finally, future experiments that confirm our results in mutant mice carrying human migraine mutations, such as FHM, and/or in female rodents, could further underscore the clinical relevance of our findings.