- Email: [email protected]
Sleep disruption increases seizure susceptibility: Behavioral and EEG evaluation of an experimental model of sleep apnea
Physiology & Behavior 155 (2016) 188–194
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Sleep disruption increases seizure susceptibility: Behavioral and EEG evaluation of an experimental model of sleep apnea Dragan Hrnčić a, Željko Grubač a, Aleksandra Rašić-Marković a, Nikola Šutulović a, Veselinka Šušić b, Jelica Bjekić-Macut c, Olivera Stanojlović a,⁎ a b c
Laboratory of Neurophysiology, Institute of Medical Physiology “Richard Burian”, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia Serbian Academy of Sciences and Arts, 11000 Belgrade, Serbia CHC Bežanijska Kosa, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia
H I G H L I G H T S • • • • •
We investigated the effects of sleep disruption on seizure susceptibility of rats. We used experimental model of lindane-induced refractory seizures. Sleep disruption frequency resembled that in patients with severe sleep apnea. Sleep disruption potentiated both behavioral and EEG epileptic activity in rat. This could explain the occurrence of epileptic symptoms in sleep apnea patients.
a r t i c l e
i n f o
Article history: Received 11 June 2015 Received in revised form 14 December 2015 Accepted 15 December 2015 Available online 17 December 2015 Keywords: Sleep disruption Epilepsy Convulsive behavior EEG Rats
a b s t r a c t Sleep disruption accompanies sleep apnea as one of its major symptoms. Obstructive sleep apnea is particularly common in patients with refractory epilepsy, but causing factors underlying this are far from being resolved. Therefore, translational studies regarding this issue are important. Our aim was to investigate the effects of sleep disruption on seizure susceptibility of rats using experimental model of lindane-induced refractory seizures. Sleep disruption in male Wistar rats with implanted EEG electrodes was achieved by treadmill method (belt speed set on 0.02 m/s for working and 0.00 m/s for stop mode, respectively). Animals were assigned to experimental conditions lasting 6 h: 1) sleep disruption (sleep interrupted, SI; 30 s working and 90 s stop mode every 2 min; 180 cycles in total); 2) activity control (AC, 10 min working and 30 min stop mode, 9 cycles in total); 3) treadmill chamber control (TC, only stop mode). Afterwards, the animals were intraperitoneally treated with lindane (L, 4 mg/kg, SI + L, AC + L and TC + L groups) or dimethylsulfoxide (DMSO, SIc, ACc and TCc groups). Convulsive behavior was assessed by seizure incidence, latency time to first seizure, and its severity during 30 min after drug administration. Number and duration of ictal periods were determined in recorded EEGs. Incidence and severity of lindane-induced seizures were significantly increased, latency time significantly decreased in animals undergoing sleep disruption (SI + L group) compared with the animals from TC + L. Seizure latency was also significantly decreased in SI + L compared to AC + L groups. Number of ictal periods were increased and duration of it presented tendency to increase in SI + L comparing to AC + L. No convulsive signs were observed in TCc, ACc and SIc groups, as well as no ictal periods in EEG. These results indicate sleep disruption facilitates induction of epileptic activity in rodent model of lindaneepilepsy enabling translational research of this phenomenon. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
⁎ Corresponding author at: Institute of Medical Physiology “Richard Burian”, Belgrade University Faculty of Medicine, Višegradska 26/II, 11000 Belgrade, Serbia. E-mail address: [email protected] (O. Stanojlović).
http://dx.doi.org/10.1016/j.physbeh.2015.12.016 0031-9384/© 2015 Elsevier Inc. All rights reserved.
Sleep is a vital, natural, periodic and reversible physiological state of decreased vigilance. Therefore, changes in sleep duration and architecture are associated with different diseases, including cardiovascular diseases, neurological disorders, psychosis, bipolar disorder, as well as epilepsy [1]. Sleep deprivation has been identified as one of the leading
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
precipitant factors in patients with epilepsy [2]. It is believed that the most important role of sleep is its restorative function which includes recovery of neuronal integrity and maturation of synapses [3,4]. In order to preserve these positive effects, the sleep must remain undisturbed for a specified period of time; otherwise it loses its beneficial effects [5,6]. Hence, beside sleep deprivation, sleep disruption also might have deleterious effects. Sleep disruption occurs in patients with different psychiatric and respiratory diseases, in particular in patients with sleep apnea [1,7]. Sleep apnea is a sleep disorder characterized by abnormal breathing interruptions lasting from 10 s to 1 min [8]. These interruptions in patients with severe form of sleep apnea appear in cycles of 30 or more times per hour [9]. There is evidence on pathogenic link between sleep apnea and seizures: neuronal excitability and possible synchronized discharge of neurons are increased [10]. Obstructive sleep apnea is particularly common in patients with refractory epilepsy as well as in patients with epilepsy which appears later in life, so therapy of sleep apnea can be crucial to the prevention and treatment of epilepsy [11,12]. In fact, about one-third of patients with refractory epilepsy are suffering from symptoms of sleep apnea, with a somewhat higher incidence in males [12]. Recently, Pornsriniyom et al. [13] demonstrated beneficial effects of continuous positive airway pressure (CPAP) therapy for refractory epilepsy patients with comorbid obstructive sleep apnea. There are numerous concerns regarding sleep apnea and the mechanisms responsible for augmentation of CNS excitability. It is believed that its main cause is hypoxemia [14], but interruptions of sleep caused by sleep apnea is also believed to be a powerful contributing factor [15]. However, we must bear in mind that the full effects of sleep disruption and problems associated with it are still unclear, especially the relationship between sleep disruption and epilepsy in experimental models. Many experimental models of epilepsy enable investigation of mechanisms of epileptogenesis and its modulation. Lindane (γchexachlorocyclochexan) is an organochloride pesticide and scabicide, widely used in agriculture, but also in human and veterinary medicine, especially in developing countries [16]. Symptoms of lindane intoxication start from headache and vertigo, leading to expressed seizures and death [17]. The model of lindane-induced seizures in rats is an experimental model of generalized epilepsy with characteristic behavioral and electroencephalographic (EEG) manifestations [18]. Lindaneinduced seizures are refractory to numerous classical antiepileptic drugs such as carbamazepine, phenytoin, felbamate and lamotrigine, gabapetnin and vigabatrin [19]. Consequently, this model is believed to be suitable for exploration of refractory epilepsy. Since obstructive sleep apnea is particularly common in patients with refractory epilepsy and causing factors underlying this are far from being resolved, experimental and translational studies regarding this issue is important. In line with that, the objective of this study was to investigate the effects of sleep disruption on seizure susceptibility using experimental model of lindane-induced seizures and high frequency sleep disruption model of apnea. 2. Materials and methods 2.1. Animals The experiments were conducted on adult male Wistar albino rats (two months old) obtained from the Military Medical Academy breeding laboratory (Belgrade, Serbia). The animals were housed individually in plexiglas cages (55 × 35 × 30 cm) with free access to food and water during the entire experiment. They were kept under controlled ambient conditions (22–24 °C, 50 ± 5% relative humidity, 12/12 h light/dark cycle with light switched on from 8:00 h to 20:00 h). Acclimation period to the general laboratory conditions, from the time animals arrived to the laboratory to any further manipulation or specific adaptation, lasted for one full week. All animals were used only once during the experiment.
189
All experimental procedures were in full compliance with Directive of the European Parliament and of the Council (2010/63/EU) and approved by The Ethical Committee of the University of Belgrade (Permission No. 298/5-2). 2.2. Surgery and EEG electrode implantation Upon being anesthetized (pentobarbital sodium 50 mg/kg, i.p.), rats were placed in a stereotaxic apparatus and three gold-plated recording electrodes were implanted over frontal (2 mm rostrally to bregma and 2 mm from the median line), parietal (2 mm rostrally to lambda and 2 mm laterally to median line) and occipital (2 mm caudally to lambda) cortices for further EEG recordings. All three EEG electrodes were necessary to estimate generalized ictal activity, while EMG electrode wasn't implanted. The electrodes were fixed to the skull with dental acrylic cement and 7-day recovery period was allowed prior to further experiments. Also, 24-hour-habituation to the EEG recording situation with attached cable was allowed to all animals. 2.3. Experimental design and method of sleep disruption Sleep disruption was achieved by the method of McKenna et al. [20] with the use of treadmill apparatus for small animals, during the first 6 h of light phase of the light/dark cycle. The treadmill apparatus for small experimental animals (NeuroSciLaBG-Treadmill, Elunit, Serbia) used in this study consists of conveyor belt, whose motion was programmed in advance with an appropriate control program. The treadmill activity was defined by ON mode (working mode, belt moving forward) at the speed of 0.02 m/s and OFF mode (stop mode) at the speed of 0.00 m/s. All animals were adapted to the treadmill apparatus two days prior to the experiments, in daily sessions of 1 h during which ON and OFF mode alternated in 5 min cycles (5 min ON: 5 min OFF). Every hour encompass 30 consecutive sections, corresponding to fragments of 2 min each with alternate rotations of both ON and OFF treadmill activity. Therefore, the treadmill was programmed to alternately work 30 s ON and 90 s OFF every 2 min during entire period of 6 h for sleep disruption (sleep interrupted, group SI + L, 180 consecutive cycles in total, n = 6). In order to avoid confounding effects of movements itself, the correspondent activity control group was also included in the study. In this group total quantity of motion, i.e. a total movement of rats was equal to SI + L group, but without interruption of their sleep during longer time. Therefore, treadmill program was set to working mode of 10 min ON and 30 min OFF (activity group, AC + L, n = 6). Treadmill control consisted of rats staying in the treadmill apparatus at moving speed of 0 m/s and conditions equivalent to those in cages (treadmill control group: TC + L, 9 consecutive cycles in total over 6 h, n = 6). After 6 h rats from SI + L, AC + L and TC + L groups were transferred to plexiglas cages where lindane was administered intraperitoneally. Subconvulsive dose of lindane (4 mg/kg, Sigma Aldrich Co., USA) were used according to our previous studies [18,24]. Lindane was dissolved in dimethylsulfoxide (DMSO) immediately prior to use. Rats which were underwent the same experimental protocol as those in the SI + L, AC + L, TC + L groups, but received DMSO as a vehicle instead of lindane, encompassed the SIc, ACc and TCc group, respectively (n = 6 per group). 2.4. Convulsive behavior assessment During 30 min upon lindane administration, the following parameters of convulsive behavior were monitored: a) incidence (number of convulsing rats in group, expressed as percentage); b) latency period (time between lindane administration and first convulsion) and c) seizure severity (modified descriptive scale with grades from 0 to 4). The grades were defined as follows: 0 — no seizure; grade 1 — head nodding and lower jaw twitching; grade 2 — myoclonic body jerks (hot
190
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
Texas, USA). Ambient noise was removed using a 50 Hz notch filter and the cutoff frequencies for were set at 0.3 Hz and 100 Hz for the high-pass and low-pass filters, respectively. Data acquisition and signal processing were performed with LabVIEW platform software developed in the Laboratory (NeuroSciLaBG) [21,22]. All EEG recordings were visually monitored and screened for ictal activity and stored on disk for subsequent analysis. The rats was removed from the recording chamber and returned to its home cage upon completion of the 30 min recording sessions. Ictal periods in EEG were defined as follows: 1) spontaneous and generalized spiking activity; 2) lasting N1 s and 3) amplitude of at least twice the background EEG activity [22]. The number and duration of ictal periods were calculated during 30 min period after lindane administration using left frontal–right parietal cortex lead, since recorded electrographic seizures were generalized with simultaneous and comparable ictal activity in all leads. All ictal periods were detected visually. 2.6. Data analysis Fig. 1. The effect of sleep disruption on incidence of lindane-induced seizures in rats. Rats were adapted to appropriate experimental conditions (ON and OFF mode of the treadmill with belt speed set on 0.02 m/s and 0.00 m/s) and assigned to the following groups: 1) treadmill control (TC, only OFF mode), 2) motion, activity control (AC, 10 min ON and 30 min OFF mode) and 3) sleep disruption (sleep interrupted, SI, 30 s ON and 90 s OFF mode). Six hours later, animals were intraperitoneally treated with lindane (L, 4 mg/kg, TC + L, AC + L and SI + L groups) or dimethylsulfoxide (DMSO, TCc, ACc and SIc groups, these showed no signs of convulsions). Seizure incidence was expressed as percentage (%) of convulsive rats in the group. The statistical significance of the differences between the groups was estimated by Fisher's exact probability test (*p b 0.05 vs. TC + L).
plate reaction) and bilateral forelimb clonus with full rearing (Kangaroo position); grade 3 — progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tale; grade 4 — status epilepticus [21]. 2.5. EEG registration and analysis An eight-channel EEG apparatus (RIZ, Zagreb, Croatia) with sampling frequency of 512 Hz/channel and 16-bit A/D conversion was used for EEG recording in freely moving rats. The signals were digitized using a SCB-68 data acquisition card (National Instruments Co, Austin,
The statistically significant difference in the incidence was determined by using Fisher's exact probability test. Since the normal data distribution of the data regarding latency period, number of convulsive episodes per rat and their severity, as well as number and duration of ictal periods in EEG, has not been determined by Kolmogorov– Smirnov test, the non-parametric Kruskal–Wallis ANOVA and Mann Whitney U tests were applied in further data analyses for the assessment of statistically significant difference between groups (*p b 0.05, **p b 0.01). The results were expressed as medians with 25th and 75th percentiles. 3. Results 3.1. Convulsive behavior analysis Seizures have been recorded in all animal groups treated with lindane. The incidence of seizures in group TC + L was 16.67%, and 33.33% in group AC + L. The greatest number of animals with seizures was in the group subjected to sleep disruption (SI + L), where the seizure incidence was 83.33%. The incidence of seizures was significantly higher in group SI + L compared to group TC + L (p b 0.05, Fig. 1).
Fig. 2. The effect of sleep disruption on latency period of lindane-induced seizures in rats (A) and their severity (B). Latency period = time between lindane administration and the first convulsive sign. Severity of seizure episodes was determined by a descriptive — rating scale with following grades: 1 — head nodding, lower jaw twitching; 2 — myoclonic body jerks (hot plate reaction), bilateral forelimb clonus with full rearing (Kangaroo position); 3 — progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tail; 4 — prolonged severe tonic–clonic convulsions lasting over 20 s (status epilepticus) or frequent repeated episodes of clonic convulsions for an extended period of time (over 5 min). The significance of the differences between the groups was estimated by Kruskal–Wallis ANOVA and Mann–Whitney U test (*p b 0.05 vs. TC + L, #p b 0.05 vs. AC + L). For details see caption to Fig. 1.
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
191
Fig. 3. Representative EEG tracings recorded in (A) control groups (TCc, ACc and SIc) showing baseline activity without signs of epileptiform discharges and in (B) TC + L, (C) AC + L and (D) SI + L groups of rats upon lindane administration showing progressively more intensive ictal patterns. In C and D are examples of ictal period quantified according to definition stated in Section 2.5. Lead: left frontal–right parietal cortex. For details see caption to Fig. 1.
192
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
Fig. 4. The number (A) and duration (B) of ictal periods during 30 min EEG recordings of experimental rats upon lindane administration. The significance of the differences between the groups was estimated by Kruskal–Wallis ANOVA and Mann–Whitney U test (*p b 0.05 vs. TC + L, #p b 0.05 vs. AC + L). For details see caption to Figs.1 and 3.
Although the incidence of seizures was higher in group SI + L compared to group AC + L, the recorded difference (83.33% vs. 33.33%) did not reach statistical significance (p N 0.05, Fig. 1). There was no difference in the incidence of seizures between TC + L and AC + L groups (p N 0.05, Fig. 1). When latency period to first convulsive sign was analyzed, it has been determined that the shortest latency period was observed in group SI + L. The latency period was significantly shorter in SI + L comparing to AC + L and TC + L and groups (p b 0.05, Fig. 2A). There were no statistically significant differences in the latency period between AC + L and TC + L groups (p N 0.05, Fig. 2A). The severity of convulsions was significantly higher in SI + L in comparison to TC + L group (p b 0.05, Fig. 2B). There was no statistically significant difference between AC + L and TC + L groups regarding the severity of the convulsions (p N 0.05, Fig. 2B). Rats subjected to the same experimental protocol as those in SI + L, AC + L and TC + L groups, but received DMSO instead of lindane (groups SIc, ACc and TCc, respectively), showed no signs of convulsive behavior. 3.2. EEG analysis EEG recordings in rats from control groups (TCc, ACc and SIc) revealed no signs of ictal activity upon DMSO administration. Rats from these groups showed baseline EEG activity correspondent with grooming behavior (Fig. 3A). Sporadic appearance of isolated spikes was characteristic of EEG recordings in TC + L group upon lindane administration (Fig. 3B). Ictal activity was also recording in AC + L group during the period upon lindane (Fig. 3C). In EEG recording from SI + L group series of highvoltage ictal bursts were commonly registered (Fig 3D). Quantitative analysis of ictal periods, i.e. its number and durations revealed the differences between these groups. Number of ictal periods per rat was significantly higher in SI + L group in comparison to TC + L, (p b 0.05), as well as in comparison to AC + L group (p b 0.05, Fig. 4A). There were no statistically significant difference between AC + L and TC + L groups regarding number of ictal periods per rat in EEG (p N 0.05, Fig 4A). Duration of ictal periods showed tendency to be higher in SI + L group comparing to TC + L and AC + L, but these differences didn't attain statistical significance (p N 0.05, Fig 4A). As for number of ictal periods, there were no statistically significant difference between AC + L and TC + L groups regarding duration of ictal periods per rat in EEG (p N 0.05, Fig. 4B).
4. Discussion Sleep disruption in this study was achieved by treadmill which working regime (30 interruptions per 1 h) was programmed to simulate sleep interruption frequency in patients with severe sleep apnea. The findings of this study implicated that sleep disruption potentiated both behavioral and EEG epileptic activity in rat. Namely, sleep disruption significantly increased the seizure incidence and severity, whereas it shortened its latency period induced by subconvulsive dose of lindane. Furthermore, described sleep disruption significantly increased the number of ictal periods in EEG of these rats. Numerous studies investigated the effects of total sleep deprivation, as well as REM sleep deprivation on psychological disorders and behavior [5], also its relationship with epilepsy [23]. However, notably smaller number of studies dealt with sleep disruption and its effects on different body functions. The focus of these few studies [5,24,25] was to discriminate sleep disruption from sleep deprivation and specifically to determine the minimum period of uninterrupted sleep in order not to lose its beneficial restorative role in humans. Based on these clinical studies, we can draw an assumption that sleep disruption refers to the sleep interruption with frequency in the range from 6 to 60 per hour, and everything above or below this limit should not be considered as sleep disruption. In our current study, sleep of rats in sleep disruption groups (SI + L and SIc) was interrupted with frequency of 30 per hour, while this frequency in activity control groups (AC + L and ACc) was 1.5 per hour. The shorter sleep cycle of both REM and NREM sleep in experimental rodents, i.e. faster sleep cycles in rodents (few min) comparing to humans (90–110 min) together with differences in sleep patterns (monophasic vs. polyphasic) [26–28] have to be taken into account when translatability of sleep studies from rats to humans is in question. However, brain mechanisms controlling sleep and wakefulness, both homeostatic and circadian, are highly conserved through evolution and among species [29]. As an experimental model of epilepsy, we used animal model of generalized seizures induced by lindane and ictal activity obtained in our current study corroborated with previous findings in this model [18]. Sleep disruption alone did not evoke any signs of ictal activity in control group of rats (SIc). Also, it should be noted that both rat behavior and EEG activity were not altered in TCc and ACc groups. On the other hand, sleep disruption significantly augmented both behavioral and EEG parameters of lindane -induced seizures. It has been demonstrated that there is increased number of patients suffering from epilepsy among those with sleep apnea [30]. Epilepsy and obstructive sleep apnea are very common diseases. Their
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
concomitant occurrence might be accidental, but when presented together, they reciprocally interact, facilitating and emphasizing comorbidity [31]. Moreover, CPAP as a therapy of sleep apnea beneficially affects refractory epilepsy in these patients [13]. Also, daily somnolence and lack of attention usually appear due to sleep disruption [32,33]. Intermittent hypoxia with consecutive hypoxemia is well-established hypothesis for the augmentation of CNS excitability associated with sleep apnea [14]. Namely, it is proved that hypoxia acts proconvulsively [34, 35] by different mechanisms including facilitation of glutamatergic neurotransmission and the role of microglia [36,37]. Intermittent hypoxia and sleep disruption might have additive or possibly synergistic effects, but they occur simultaneously in apneic patients and therefore it is very difficult to study them separately in clinical settings. We applied here experimental design developed by McKenna et al. [20] which enable us to show that sleep disruption independently (without the concomitant hypoxia) increases seizure susceptibility. However, the importance of experimental sleep disruption relative to intermittent hypoxia is still questionable and need to be elucidated. Namely, unequal durations of these phenomena are necessary to produce notable effects on different neuronal functions like cognition [38]. The deleterious effects of high frequency sleep disruption contributing to increased seizure susceptibility are potentially the consequence of either sleep architecture interruption and fragmentation [39,40] or loss of total sleep time [41]. McKenna et al. [20] reported that 6 h — protocol of high frequency sleep interruption, applied in our study, resulted in substantial active wake increase and significantly shortened REM and NREM phases, as well as in reduction in total sleep time. This procedure was also accompanied with increased homeostatic sleep drive, as evident by: decreased latency to sleep onset in rodents multiple sleep latency test, increased delta power in NREM and its duration during recovery period. Therefore, this partial sleep deprivation should be taken into account for the explanation of the results of our study. Also, shortening of certain sleep phases could help to understand the mechanisms of modulatory effects of sleep-apnea-accompanying sleep disruption on the onset of seizures, showed in this study. Namely, it has been demonstrated that REM sleep is crucial for the occurrence of epilepsy, i.e. sufficient amount of REM sleep decreased excitability [42], while REM sleep deprivation potentiated epileptic activity [23]. It should be pointed out that this manipulation mimics the high frequency sleep disruption associated with apnea. However, the effects of manipulation when total sleep levels are held constant on seizure susceptibility remain to be clarified. Recently, Park et al. [43] identified that synapsin II expression is significantly reduced in rat hippocampus by sleep disruption. It is known that mice lacking synapsin IIa or IIb isoforms display an epileptic phenotype [44]. These molecular changes in level of this phosphoprotein regulating neurotransmitter release could further support the results of this study showing increased susceptibility for epileptic activity of rats experiencing sleep disruption. Although numerous studies support the hypothesis that sleep disturbances affect excitability to a significant extent, there are some that oppose it. Namely, clinical study performed by Marlow et al. [45] involving 84 patients showed that sleep disruption did not significantly affect the occurrence and severity of seizures, even though there were differences and specific characteristics in the reaction to disruption in certain patients. Also, it has been demonstrated that sleep deprivation did not change the frequency of focal seizures in patients of same age and sex [45]. Apart from this, it should be mentioned that many experiments prove that the treatment of sleep apnea significantly decreased the intensity and frequency of convulsions [13,46]. Sleep disorder is probably one of the factors which potentiate seizure in patients whose basic disease is not epilepsy. The clinical study, conducted on 400 patients with epilepsy suggested that apart from sleep disruption, stress or fatigue also have significant effect in facilitating seizures, so one epileptic attack per se occurs with the accumulation of all these effects [47]. It should be pointed out that the high frequency sleep disruption (SI, 30 s treadmill
193
on, 90 s off every 2 min for 6 h) applied in our study may represent a stressful condition comparing to activity control condition (AC, 10 min on, 30 min off), what might also contribute to potentiation of seizure susceptibility [48]. However, SI and AC groups involved equal locomotors activity. Recently, Ferlisi et al. [2] identified a list of precipitant factors in patients with epilepsy. Our results indicate that sleep disruption is also one of them. In summary, presented results obtained in experimental model of lindane-induced epilepsy suggest that high frequency disruptions of sleep increases sensitivity to seizure susceptibility in rats. Conflict of interest None of the authors has to declare any conflict of interest. Acknowledgments This study was supported by the Ministry of Education, Science and Technological Development of Serbia, Grant No. 175032. References [1] R. Day, R. Gerhardstein, A. Lumley, T. Roth, L. Rosenthal, The behavioral morbidity of obstructive sleep apnea, Prog. Cardiovasc. Dis. 41 (1999) 341–354. [2] M. Ferlisi, S. Shorvon, Seizure precipitants (triggering factors) in patient with epilepsy, Epilepsy Behav. 33 (2014) 101–105. [3] A.A. Borbély, A two process model of sleep regulation, Hum. Neurobiol. 1 (3) (1982) 195–204. [4] I. Feinberg, Changes in sleep cycle patterns with age, J. Psychiatr. Res. 10 (3–4) (1974) 283–306. [5] M.H. Bonnet, Performance and sleepiness as a function of frequency and placement of sleep disruption, Psychophysiology 23 (3) (1986) 263–271. [6] P. Franken, D. Chollet, M. Tafti, The homeostatic regulation of sleep need is under genetic control, J. Neurosci. 21 (8) (2001) 2610–2621. [7] T. Roth, T.A. Roehrs, Etiologies and sequelae of excessive daytime sleepiness, Clin. Ther. 18 (4) (1996) 562–576. [8] American Academy of Sleep Medicine, International Classification of Sleep Disorders, third ed. American Academy of Sleep Medicine, Darien, 2014. [9] American Academy of Sleep Medicine Task Force, Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research, Sleep 22 (5) (1999) 667–689. [10] O. Devinsky, B. Ehrenberg, G.M. Barthlen, H.S. Abramson, D. Luciano, Epilepsy and sleep apnea syndrome, Neurology 44 (11) (1994) 2060–2064. [11] A.M. Chihorek, B. Abou-Khalil, B.A. Malow, Obstructive sleep apnea is associated with seizure occurrence in older adults with epilepsy, Neurology 69 (19) (2007) 1823–1827. [12] B.A. Malow, K. Levy, K. Maturen, R. Bowes, Obstructive sleep apnea is common in medically refractory epilepsy patients, Neurology 55 (7) (2000) 1002–1007. [13] D. Pornsriniyom, K. Shinlapawittayatorn, J. Fong, N.D. Andrews, N. FoldvarySchaefer, Continuous positive airway pressure therapy for obstructive sleep apnea reduces interictal epileptiform discharges in adults with epilepsy, Epilepsy Behav. 37 (2014) 171–174. [14] M. Seyal, L.M. Bateman, T.E. Albertson, T.C. Lin, C.S. Li, Respiratory changes with seizures in localization-related epilepsy: analysis of periictal hypercapnia and airflow patterns, Epilepsia 51 (8) (2010) 1359–1364. [15] N. Foldvary-Schaefer, N.D. Andrews, D. Pornsriniyom, D.E. Moul, Z. Sun, J. Bena, Sleep apnea and epilepsy: who's at risk? Epilepsy Behav. 25 (3) (2012) 363–367. [16] Y.F. Li, Global technical hexachlorocyclohexane usage and its contamination consequences in the environment from: 1948–1997, Sci. Total Environ. 232 (1999) 121–158. [17] Centers for Disease Control and Prevention (CDC), Unintentional topical lindane ingestions—United States, 1998–2003, MMWR Morb Mortal Wkly. Rep. 54 (21) (2005) 533–535. [18] D. Vučević, D. Hrnčić, T. Radosavljević, D. Mladenović, A. Rašić-Marković, H. LončarStevanović, et al., Correlation between electrocorticographic and motor phenomena in lindane-induced experimental epilepsy in rats, Can. J. Physiol. Pharmacol. 86 (4) (2008) 173–179. [19] A.M. Tochman, R. Kamiński, W.A. Turski, S.J. Czuczwar, Protection by conventional and new antiepileptic drugs against lindane-induced seizures and lethal effects in mice, Neurotox. Res. 2 (1) (2000) 63–70. [20] J.T. McKenna, J.L. Tartar, C.P. Ward, M.M. Thakkar, J.W. Cordeira, R.W. McCarley, et al., Sleep fragmentation elevates behavioral, electrographic and neurochemical measures of sleepiness, Neuroscience 146 (4) (2007) 1462–1473. [21] O. Stanojlović, A. Rasić-Marković, D. Hrncić, V. Susić, D. Macut, T. Radosavljević, et al., Two types of seizures in homocysteine thiolactone-treated adult rats, behavioral and electroencephalographic study, Cell. Mol. Neurobiol. 29 (3) (2009) 329–339. [22] D. Hrnčić, A. Rašić-Marković, D. Djuric, V. Sušić, O. Stanojlović, The role of nitric oxide in convulsions induced by lindane in rats, Food Chem. Toxicol. 49 (4) (2011) 947–954.
194
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
[23] D. Hrncić, A. Rasić-Marković, J. Bjekić-Macut, V. Susić, D. Djuric, O. Stanojlović, Paradoxical sleep deprivation potentiates epilepsy induced by homocysteine thiolactone in adult rats, Exp. Biol. Med. (Maywood) 238 (1) (2013) 77–83. [24] B. Levine, T. Roehrs, E. Stepanski, F. Zorick, T. Roth, Fragmenting sleep diminishes its recuperative value, Sleep 10 (1987) 590–599. [25] R. Downey, M.H. Bonnet, Performance during frequent sleep disruption, Sleep 10 (1987) 354–363. [26] S.S. Campbell, I. Tobler, Animal sleep: a review of sleep duration across phylogeny, Neurosci. Biobehav. Rev. 8 (1984) 269–300. [27] L. Trachel, I. Tobler, P. Achermann, A.A. Borbely, Sleep continuity and the REM– nonREM cycle in the rat under baseline conditions and after sleep deprivation, Physiol. Behav. 49 (1991) 575–580. [28] C.G. Diniz Behn, V. Booth, Modeling the temporal architecture of rat sleep–wake behavior, Conf. Proc. IEEE Eng. Med. Biol. Soc. 4713–4716 (2011). [29] R.E. Brown, R. Basheer, J.T. McKenna, R.E. Strecker, R.W. McCarley, Control of sleep and wakefulness, Physiol. Rev. 92 (3) (2012) 1087–1187. [30] K. Sonka, M. Juklícková, M. Pretl, S. Dostálová, D. Horínek, S. Nevsímalová, Seizures in sleep apnea patients: occurrence and time distribution, Sb. Lek. 101 (3) (2000) 229–232. [31] R. Manni, M. Terzaghi, C. Arbasino, I. Sartori, C.A. Galimberti, A. Tartara, Obstructive sleep apnea in a clinical series of adult epilepsy patients: frequency and features of the comorbidity, Epilepsia 44 (6) (2003) 836–840. [32] B.D. Hoyt, Sleep in patients with neurologic and psychiatric disorders, Prim. Care. 32 (2) (2005) 535–548. [33] C.R. Soldatos, T.J. Paparrigopoulos, Sleep physiology and pathology: pertinence to psychiatry, Int. Rev. Psychiatry 17 (2005) 213–228. [34] F.E. Jensen, C.D. Applegate, D. Holtzman, T.R. Belin, J.L. Burchfiel, Epileptogenic effect of hypoxia in the immature rodent brain, Ann. Neurol. 29 (6) (1991) 629–637. [35] F.E. Jensen, G.L. Holmes, C.T. Lombroso, H.K. Blume, I.R. Firkusny, Age dependent changes in long-term seizure susceptibility and behavior after hypoxia in rats, Epilepsia 33 (1992) 971–980. [36] A. Rubaj, W. Zgodziński, M. Sieklucka-Dziuba, The epileptogenic effect of seizures induced by hypoxia: the role of NMDA and AMPA/KA antagonists, Pharmacol. Biochem. Behav. 74 (2) (2003) 303–311.
[37] Q. Yang, Y. Wang, J. Feng, J. Cao, B. Chen, Intermittent hypoxia from obstructive sleep apnea may cause neuronal impairment and dysfunction in central nervous system: the potential roles played by microglia, Neuropsychiatr. Dis. Treat. 9 (2013) 1077–1086. [38] C.P. Ward, J.G. McCoy, J.T. McKenna, N.P. Connolly, R.W. McCarley, R.E. Strecker, Spatial learning and memory deficits following exposure to 24 h of sleep fragmentation or intermittent hypoxia in a rat model of obstructive sleep apnea, Brain Res. 1294 (2009) 128–137. [39] E.J. Stepanski, The effect of sleep fragmentation on daytime function, Sleep 25 (3) (2002) 268–276. [40] M.H. Bonnet, Sleep restoration as a function of periodic awakening, movement, or electroencephalographic change, Sleep 10 (1987) 364–373. [41] S.V. Jain, P.S. Horn, N. Simakajornboon, T.A. Glauser, Obstructive sleep apnea and primary snoring in children with epilepsy, J. Child Neurol. 28 (1) (2013) 77–82. [42] P. Kumar, T.R. Raju, Seizure susceptibility decreases with enhancement of rapid eye movement sleep, Brain Res. 922 (2) (2001) 299–304. [43] D.S. Park, D.W. Yoon, W.B. Yoo, S.K. Lee, C.H. Yun, S.J. Kim, et al., Sleep fragmentation induces reduction of synapsin II in rat hippocampus, Sleep Biologic Rhythms 12 (2014) 135–144. [44] A. Fassio, A. Raimondi, G. Lignani, F. Benfenati, P. Baldelli, Synapsins: from synapse to network hyperexcitability and epilepsy, Semin. Cell Dev. Biol. 22 (2011) 408–415. [45] B.A. Malow, E. Passaro, C. Milling, D.N. Minecan, K. Levy, Sleep deprivation does not affect seizure frequency during inpatient video-EEG monitoring, Neurology 59 (2002) 1371–1374. [46] B.A. Malow, K.J. Weatherwax, R.D. Chervin, T.F. Hoban, M.L. Marzec, C. Martin, et al., Identification and treatment of obstructive sleep apnea in adults and children with epilepsy: a prospective pilot study, Sleep Med. 4 (2003) 509–515. [47] M.M. Frucht, M. Quigg, C. Schwaner, N.B. Fountain, Distribution of seizure precipitants among epilepsy syndromes, Epilepsia 41 (2000) 1534–1539. [48] Y.H. Ho, Y.T. Lin, C.W. Wu, Y.M. Chao, A.Y. Chang, J.Y. Chan, Peripheral inflammation increases seizure susceptibility via the induction of neuroinflammation and oxidative stress in the hippocampus, J. Biomed. Sci. 22 (1) (2015) 46.
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/phb
Sleep disruption increases seizure susceptibility: Behavioral and EEG evaluation of an experimental model of sleep apnea Dragan Hrnčić a, Željko Grubač a, Aleksandra Rašić-Marković a, Nikola Šutulović a, Veselinka Šušić b, Jelica Bjekić-Macut c, Olivera Stanojlović a,⁎ a b c
Laboratory of Neurophysiology, Institute of Medical Physiology “Richard Burian”, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia Serbian Academy of Sciences and Arts, 11000 Belgrade, Serbia CHC Bežanijska Kosa, Faculty of Medicine, University of Belgrade, 11000 Belgrade, Serbia
H I G H L I G H T S • • • • •
We investigated the effects of sleep disruption on seizure susceptibility of rats. We used experimental model of lindane-induced refractory seizures. Sleep disruption frequency resembled that in patients with severe sleep apnea. Sleep disruption potentiated both behavioral and EEG epileptic activity in rat. This could explain the occurrence of epileptic symptoms in sleep apnea patients.
a r t i c l e
i n f o
Article history: Received 11 June 2015 Received in revised form 14 December 2015 Accepted 15 December 2015 Available online 17 December 2015 Keywords: Sleep disruption Epilepsy Convulsive behavior EEG Rats
a b s t r a c t Sleep disruption accompanies sleep apnea as one of its major symptoms. Obstructive sleep apnea is particularly common in patients with refractory epilepsy, but causing factors underlying this are far from being resolved. Therefore, translational studies regarding this issue are important. Our aim was to investigate the effects of sleep disruption on seizure susceptibility of rats using experimental model of lindane-induced refractory seizures. Sleep disruption in male Wistar rats with implanted EEG electrodes was achieved by treadmill method (belt speed set on 0.02 m/s for working and 0.00 m/s for stop mode, respectively). Animals were assigned to experimental conditions lasting 6 h: 1) sleep disruption (sleep interrupted, SI; 30 s working and 90 s stop mode every 2 min; 180 cycles in total); 2) activity control (AC, 10 min working and 30 min stop mode, 9 cycles in total); 3) treadmill chamber control (TC, only stop mode). Afterwards, the animals were intraperitoneally treated with lindane (L, 4 mg/kg, SI + L, AC + L and TC + L groups) or dimethylsulfoxide (DMSO, SIc, ACc and TCc groups). Convulsive behavior was assessed by seizure incidence, latency time to first seizure, and its severity during 30 min after drug administration. Number and duration of ictal periods were determined in recorded EEGs. Incidence and severity of lindane-induced seizures were significantly increased, latency time significantly decreased in animals undergoing sleep disruption (SI + L group) compared with the animals from TC + L. Seizure latency was also significantly decreased in SI + L compared to AC + L groups. Number of ictal periods were increased and duration of it presented tendency to increase in SI + L comparing to AC + L. No convulsive signs were observed in TCc, ACc and SIc groups, as well as no ictal periods in EEG. These results indicate sleep disruption facilitates induction of epileptic activity in rodent model of lindaneepilepsy enabling translational research of this phenomenon. © 2015 Elsevier Inc. All rights reserved.
1. Introduction
⁎ Corresponding author at: Institute of Medical Physiology “Richard Burian”, Belgrade University Faculty of Medicine, Višegradska 26/II, 11000 Belgrade, Serbia. E-mail address: [email protected] (O. Stanojlović).
http://dx.doi.org/10.1016/j.physbeh.2015.12.016 0031-9384/© 2015 Elsevier Inc. All rights reserved.
Sleep is a vital, natural, periodic and reversible physiological state of decreased vigilance. Therefore, changes in sleep duration and architecture are associated with different diseases, including cardiovascular diseases, neurological disorders, psychosis, bipolar disorder, as well as epilepsy [1]. Sleep deprivation has been identified as one of the leading
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
precipitant factors in patients with epilepsy [2]. It is believed that the most important role of sleep is its restorative function which includes recovery of neuronal integrity and maturation of synapses [3,4]. In order to preserve these positive effects, the sleep must remain undisturbed for a specified period of time; otherwise it loses its beneficial effects [5,6]. Hence, beside sleep deprivation, sleep disruption also might have deleterious effects. Sleep disruption occurs in patients with different psychiatric and respiratory diseases, in particular in patients with sleep apnea [1,7]. Sleep apnea is a sleep disorder characterized by abnormal breathing interruptions lasting from 10 s to 1 min [8]. These interruptions in patients with severe form of sleep apnea appear in cycles of 30 or more times per hour [9]. There is evidence on pathogenic link between sleep apnea and seizures: neuronal excitability and possible synchronized discharge of neurons are increased [10]. Obstructive sleep apnea is particularly common in patients with refractory epilepsy as well as in patients with epilepsy which appears later in life, so therapy of sleep apnea can be crucial to the prevention and treatment of epilepsy [11,12]. In fact, about one-third of patients with refractory epilepsy are suffering from symptoms of sleep apnea, with a somewhat higher incidence in males [12]. Recently, Pornsriniyom et al. [13] demonstrated beneficial effects of continuous positive airway pressure (CPAP) therapy for refractory epilepsy patients with comorbid obstructive sleep apnea. There are numerous concerns regarding sleep apnea and the mechanisms responsible for augmentation of CNS excitability. It is believed that its main cause is hypoxemia [14], but interruptions of sleep caused by sleep apnea is also believed to be a powerful contributing factor [15]. However, we must bear in mind that the full effects of sleep disruption and problems associated with it are still unclear, especially the relationship between sleep disruption and epilepsy in experimental models. Many experimental models of epilepsy enable investigation of mechanisms of epileptogenesis and its modulation. Lindane (γchexachlorocyclochexan) is an organochloride pesticide and scabicide, widely used in agriculture, but also in human and veterinary medicine, especially in developing countries [16]. Symptoms of lindane intoxication start from headache and vertigo, leading to expressed seizures and death [17]. The model of lindane-induced seizures in rats is an experimental model of generalized epilepsy with characteristic behavioral and electroencephalographic (EEG) manifestations [18]. Lindaneinduced seizures are refractory to numerous classical antiepileptic drugs such as carbamazepine, phenytoin, felbamate and lamotrigine, gabapetnin and vigabatrin [19]. Consequently, this model is believed to be suitable for exploration of refractory epilepsy. Since obstructive sleep apnea is particularly common in patients with refractory epilepsy and causing factors underlying this are far from being resolved, experimental and translational studies regarding this issue is important. In line with that, the objective of this study was to investigate the effects of sleep disruption on seizure susceptibility using experimental model of lindane-induced seizures and high frequency sleep disruption model of apnea. 2. Materials and methods 2.1. Animals The experiments were conducted on adult male Wistar albino rats (two months old) obtained from the Military Medical Academy breeding laboratory (Belgrade, Serbia). The animals were housed individually in plexiglas cages (55 × 35 × 30 cm) with free access to food and water during the entire experiment. They were kept under controlled ambient conditions (22–24 °C, 50 ± 5% relative humidity, 12/12 h light/dark cycle with light switched on from 8:00 h to 20:00 h). Acclimation period to the general laboratory conditions, from the time animals arrived to the laboratory to any further manipulation or specific adaptation, lasted for one full week. All animals were used only once during the experiment.
189
All experimental procedures were in full compliance with Directive of the European Parliament and of the Council (2010/63/EU) and approved by The Ethical Committee of the University of Belgrade (Permission No. 298/5-2). 2.2. Surgery and EEG electrode implantation Upon being anesthetized (pentobarbital sodium 50 mg/kg, i.p.), rats were placed in a stereotaxic apparatus and three gold-plated recording electrodes were implanted over frontal (2 mm rostrally to bregma and 2 mm from the median line), parietal (2 mm rostrally to lambda and 2 mm laterally to median line) and occipital (2 mm caudally to lambda) cortices for further EEG recordings. All three EEG electrodes were necessary to estimate generalized ictal activity, while EMG electrode wasn't implanted. The electrodes were fixed to the skull with dental acrylic cement and 7-day recovery period was allowed prior to further experiments. Also, 24-hour-habituation to the EEG recording situation with attached cable was allowed to all animals. 2.3. Experimental design and method of sleep disruption Sleep disruption was achieved by the method of McKenna et al. [20] with the use of treadmill apparatus for small animals, during the first 6 h of light phase of the light/dark cycle. The treadmill apparatus for small experimental animals (NeuroSciLaBG-Treadmill, Elunit, Serbia) used in this study consists of conveyor belt, whose motion was programmed in advance with an appropriate control program. The treadmill activity was defined by ON mode (working mode, belt moving forward) at the speed of 0.02 m/s and OFF mode (stop mode) at the speed of 0.00 m/s. All animals were adapted to the treadmill apparatus two days prior to the experiments, in daily sessions of 1 h during which ON and OFF mode alternated in 5 min cycles (5 min ON: 5 min OFF). Every hour encompass 30 consecutive sections, corresponding to fragments of 2 min each with alternate rotations of both ON and OFF treadmill activity. Therefore, the treadmill was programmed to alternately work 30 s ON and 90 s OFF every 2 min during entire period of 6 h for sleep disruption (sleep interrupted, group SI + L, 180 consecutive cycles in total, n = 6). In order to avoid confounding effects of movements itself, the correspondent activity control group was also included in the study. In this group total quantity of motion, i.e. a total movement of rats was equal to SI + L group, but without interruption of their sleep during longer time. Therefore, treadmill program was set to working mode of 10 min ON and 30 min OFF (activity group, AC + L, n = 6). Treadmill control consisted of rats staying in the treadmill apparatus at moving speed of 0 m/s and conditions equivalent to those in cages (treadmill control group: TC + L, 9 consecutive cycles in total over 6 h, n = 6). After 6 h rats from SI + L, AC + L and TC + L groups were transferred to plexiglas cages where lindane was administered intraperitoneally. Subconvulsive dose of lindane (4 mg/kg, Sigma Aldrich Co., USA) were used according to our previous studies [18,24]. Lindane was dissolved in dimethylsulfoxide (DMSO) immediately prior to use. Rats which were underwent the same experimental protocol as those in the SI + L, AC + L, TC + L groups, but received DMSO as a vehicle instead of lindane, encompassed the SIc, ACc and TCc group, respectively (n = 6 per group). 2.4. Convulsive behavior assessment During 30 min upon lindane administration, the following parameters of convulsive behavior were monitored: a) incidence (number of convulsing rats in group, expressed as percentage); b) latency period (time between lindane administration and first convulsion) and c) seizure severity (modified descriptive scale with grades from 0 to 4). The grades were defined as follows: 0 — no seizure; grade 1 — head nodding and lower jaw twitching; grade 2 — myoclonic body jerks (hot
190
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
Texas, USA). Ambient noise was removed using a 50 Hz notch filter and the cutoff frequencies for were set at 0.3 Hz and 100 Hz for the high-pass and low-pass filters, respectively. Data acquisition and signal processing were performed with LabVIEW platform software developed in the Laboratory (NeuroSciLaBG) [21,22]. All EEG recordings were visually monitored and screened for ictal activity and stored on disk for subsequent analysis. The rats was removed from the recording chamber and returned to its home cage upon completion of the 30 min recording sessions. Ictal periods in EEG were defined as follows: 1) spontaneous and generalized spiking activity; 2) lasting N1 s and 3) amplitude of at least twice the background EEG activity [22]. The number and duration of ictal periods were calculated during 30 min period after lindane administration using left frontal–right parietal cortex lead, since recorded electrographic seizures were generalized with simultaneous and comparable ictal activity in all leads. All ictal periods were detected visually. 2.6. Data analysis Fig. 1. The effect of sleep disruption on incidence of lindane-induced seizures in rats. Rats were adapted to appropriate experimental conditions (ON and OFF mode of the treadmill with belt speed set on 0.02 m/s and 0.00 m/s) and assigned to the following groups: 1) treadmill control (TC, only OFF mode), 2) motion, activity control (AC, 10 min ON and 30 min OFF mode) and 3) sleep disruption (sleep interrupted, SI, 30 s ON and 90 s OFF mode). Six hours later, animals were intraperitoneally treated with lindane (L, 4 mg/kg, TC + L, AC + L and SI + L groups) or dimethylsulfoxide (DMSO, TCc, ACc and SIc groups, these showed no signs of convulsions). Seizure incidence was expressed as percentage (%) of convulsive rats in the group. The statistical significance of the differences between the groups was estimated by Fisher's exact probability test (*p b 0.05 vs. TC + L).
plate reaction) and bilateral forelimb clonus with full rearing (Kangaroo position); grade 3 — progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tale; grade 4 — status epilepticus [21]. 2.5. EEG registration and analysis An eight-channel EEG apparatus (RIZ, Zagreb, Croatia) with sampling frequency of 512 Hz/channel and 16-bit A/D conversion was used for EEG recording in freely moving rats. The signals were digitized using a SCB-68 data acquisition card (National Instruments Co, Austin,
The statistically significant difference in the incidence was determined by using Fisher's exact probability test. Since the normal data distribution of the data regarding latency period, number of convulsive episodes per rat and their severity, as well as number and duration of ictal periods in EEG, has not been determined by Kolmogorov– Smirnov test, the non-parametric Kruskal–Wallis ANOVA and Mann Whitney U tests were applied in further data analyses for the assessment of statistically significant difference between groups (*p b 0.05, **p b 0.01). The results were expressed as medians with 25th and 75th percentiles. 3. Results 3.1. Convulsive behavior analysis Seizures have been recorded in all animal groups treated with lindane. The incidence of seizures in group TC + L was 16.67%, and 33.33% in group AC + L. The greatest number of animals with seizures was in the group subjected to sleep disruption (SI + L), where the seizure incidence was 83.33%. The incidence of seizures was significantly higher in group SI + L compared to group TC + L (p b 0.05, Fig. 1).
Fig. 2. The effect of sleep disruption on latency period of lindane-induced seizures in rats (A) and their severity (B). Latency period = time between lindane administration and the first convulsive sign. Severity of seizure episodes was determined by a descriptive — rating scale with following grades: 1 — head nodding, lower jaw twitching; 2 — myoclonic body jerks (hot plate reaction), bilateral forelimb clonus with full rearing (Kangaroo position); 3 — progression to generalized clonic convulsions followed by tonic extension of fore and hind limbs and tail; 4 — prolonged severe tonic–clonic convulsions lasting over 20 s (status epilepticus) or frequent repeated episodes of clonic convulsions for an extended period of time (over 5 min). The significance of the differences between the groups was estimated by Kruskal–Wallis ANOVA and Mann–Whitney U test (*p b 0.05 vs. TC + L, #p b 0.05 vs. AC + L). For details see caption to Fig. 1.
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
191
Fig. 3. Representative EEG tracings recorded in (A) control groups (TCc, ACc and SIc) showing baseline activity without signs of epileptiform discharges and in (B) TC + L, (C) AC + L and (D) SI + L groups of rats upon lindane administration showing progressively more intensive ictal patterns. In C and D are examples of ictal period quantified according to definition stated in Section 2.5. Lead: left frontal–right parietal cortex. For details see caption to Fig. 1.
192
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
Fig. 4. The number (A) and duration (B) of ictal periods during 30 min EEG recordings of experimental rats upon lindane administration. The significance of the differences between the groups was estimated by Kruskal–Wallis ANOVA and Mann–Whitney U test (*p b 0.05 vs. TC + L, #p b 0.05 vs. AC + L). For details see caption to Figs.1 and 3.
Although the incidence of seizures was higher in group SI + L compared to group AC + L, the recorded difference (83.33% vs. 33.33%) did not reach statistical significance (p N 0.05, Fig. 1). There was no difference in the incidence of seizures between TC + L and AC + L groups (p N 0.05, Fig. 1). When latency period to first convulsive sign was analyzed, it has been determined that the shortest latency period was observed in group SI + L. The latency period was significantly shorter in SI + L comparing to AC + L and TC + L and groups (p b 0.05, Fig. 2A). There were no statistically significant differences in the latency period between AC + L and TC + L groups (p N 0.05, Fig. 2A). The severity of convulsions was significantly higher in SI + L in comparison to TC + L group (p b 0.05, Fig. 2B). There was no statistically significant difference between AC + L and TC + L groups regarding the severity of the convulsions (p N 0.05, Fig. 2B). Rats subjected to the same experimental protocol as those in SI + L, AC + L and TC + L groups, but received DMSO instead of lindane (groups SIc, ACc and TCc, respectively), showed no signs of convulsive behavior. 3.2. EEG analysis EEG recordings in rats from control groups (TCc, ACc and SIc) revealed no signs of ictal activity upon DMSO administration. Rats from these groups showed baseline EEG activity correspondent with grooming behavior (Fig. 3A). Sporadic appearance of isolated spikes was characteristic of EEG recordings in TC + L group upon lindane administration (Fig. 3B). Ictal activity was also recording in AC + L group during the period upon lindane (Fig. 3C). In EEG recording from SI + L group series of highvoltage ictal bursts were commonly registered (Fig 3D). Quantitative analysis of ictal periods, i.e. its number and durations revealed the differences between these groups. Number of ictal periods per rat was significantly higher in SI + L group in comparison to TC + L, (p b 0.05), as well as in comparison to AC + L group (p b 0.05, Fig. 4A). There were no statistically significant difference between AC + L and TC + L groups regarding number of ictal periods per rat in EEG (p N 0.05, Fig 4A). Duration of ictal periods showed tendency to be higher in SI + L group comparing to TC + L and AC + L, but these differences didn't attain statistical significance (p N 0.05, Fig 4A). As for number of ictal periods, there were no statistically significant difference between AC + L and TC + L groups regarding duration of ictal periods per rat in EEG (p N 0.05, Fig. 4B).
4. Discussion Sleep disruption in this study was achieved by treadmill which working regime (30 interruptions per 1 h) was programmed to simulate sleep interruption frequency in patients with severe sleep apnea. The findings of this study implicated that sleep disruption potentiated both behavioral and EEG epileptic activity in rat. Namely, sleep disruption significantly increased the seizure incidence and severity, whereas it shortened its latency period induced by subconvulsive dose of lindane. Furthermore, described sleep disruption significantly increased the number of ictal periods in EEG of these rats. Numerous studies investigated the effects of total sleep deprivation, as well as REM sleep deprivation on psychological disorders and behavior [5], also its relationship with epilepsy [23]. However, notably smaller number of studies dealt with sleep disruption and its effects on different body functions. The focus of these few studies [5,24,25] was to discriminate sleep disruption from sleep deprivation and specifically to determine the minimum period of uninterrupted sleep in order not to lose its beneficial restorative role in humans. Based on these clinical studies, we can draw an assumption that sleep disruption refers to the sleep interruption with frequency in the range from 6 to 60 per hour, and everything above or below this limit should not be considered as sleep disruption. In our current study, sleep of rats in sleep disruption groups (SI + L and SIc) was interrupted with frequency of 30 per hour, while this frequency in activity control groups (AC + L and ACc) was 1.5 per hour. The shorter sleep cycle of both REM and NREM sleep in experimental rodents, i.e. faster sleep cycles in rodents (few min) comparing to humans (90–110 min) together with differences in sleep patterns (monophasic vs. polyphasic) [26–28] have to be taken into account when translatability of sleep studies from rats to humans is in question. However, brain mechanisms controlling sleep and wakefulness, both homeostatic and circadian, are highly conserved through evolution and among species [29]. As an experimental model of epilepsy, we used animal model of generalized seizures induced by lindane and ictal activity obtained in our current study corroborated with previous findings in this model [18]. Sleep disruption alone did not evoke any signs of ictal activity in control group of rats (SIc). Also, it should be noted that both rat behavior and EEG activity were not altered in TCc and ACc groups. On the other hand, sleep disruption significantly augmented both behavioral and EEG parameters of lindane -induced seizures. It has been demonstrated that there is increased number of patients suffering from epilepsy among those with sleep apnea [30]. Epilepsy and obstructive sleep apnea are very common diseases. Their
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
concomitant occurrence might be accidental, but when presented together, they reciprocally interact, facilitating and emphasizing comorbidity [31]. Moreover, CPAP as a therapy of sleep apnea beneficially affects refractory epilepsy in these patients [13]. Also, daily somnolence and lack of attention usually appear due to sleep disruption [32,33]. Intermittent hypoxia with consecutive hypoxemia is well-established hypothesis for the augmentation of CNS excitability associated with sleep apnea [14]. Namely, it is proved that hypoxia acts proconvulsively [34, 35] by different mechanisms including facilitation of glutamatergic neurotransmission and the role of microglia [36,37]. Intermittent hypoxia and sleep disruption might have additive or possibly synergistic effects, but they occur simultaneously in apneic patients and therefore it is very difficult to study them separately in clinical settings. We applied here experimental design developed by McKenna et al. [20] which enable us to show that sleep disruption independently (without the concomitant hypoxia) increases seizure susceptibility. However, the importance of experimental sleep disruption relative to intermittent hypoxia is still questionable and need to be elucidated. Namely, unequal durations of these phenomena are necessary to produce notable effects on different neuronal functions like cognition [38]. The deleterious effects of high frequency sleep disruption contributing to increased seizure susceptibility are potentially the consequence of either sleep architecture interruption and fragmentation [39,40] or loss of total sleep time [41]. McKenna et al. [20] reported that 6 h — protocol of high frequency sleep interruption, applied in our study, resulted in substantial active wake increase and significantly shortened REM and NREM phases, as well as in reduction in total sleep time. This procedure was also accompanied with increased homeostatic sleep drive, as evident by: decreased latency to sleep onset in rodents multiple sleep latency test, increased delta power in NREM and its duration during recovery period. Therefore, this partial sleep deprivation should be taken into account for the explanation of the results of our study. Also, shortening of certain sleep phases could help to understand the mechanisms of modulatory effects of sleep-apnea-accompanying sleep disruption on the onset of seizures, showed in this study. Namely, it has been demonstrated that REM sleep is crucial for the occurrence of epilepsy, i.e. sufficient amount of REM sleep decreased excitability [42], while REM sleep deprivation potentiated epileptic activity [23]. It should be pointed out that this manipulation mimics the high frequency sleep disruption associated with apnea. However, the effects of manipulation when total sleep levels are held constant on seizure susceptibility remain to be clarified. Recently, Park et al. [43] identified that synapsin II expression is significantly reduced in rat hippocampus by sleep disruption. It is known that mice lacking synapsin IIa or IIb isoforms display an epileptic phenotype [44]. These molecular changes in level of this phosphoprotein regulating neurotransmitter release could further support the results of this study showing increased susceptibility for epileptic activity of rats experiencing sleep disruption. Although numerous studies support the hypothesis that sleep disturbances affect excitability to a significant extent, there are some that oppose it. Namely, clinical study performed by Marlow et al. [45] involving 84 patients showed that sleep disruption did not significantly affect the occurrence and severity of seizures, even though there were differences and specific characteristics in the reaction to disruption in certain patients. Also, it has been demonstrated that sleep deprivation did not change the frequency of focal seizures in patients of same age and sex [45]. Apart from this, it should be mentioned that many experiments prove that the treatment of sleep apnea significantly decreased the intensity and frequency of convulsions [13,46]. Sleep disorder is probably one of the factors which potentiate seizure in patients whose basic disease is not epilepsy. The clinical study, conducted on 400 patients with epilepsy suggested that apart from sleep disruption, stress or fatigue also have significant effect in facilitating seizures, so one epileptic attack per se occurs with the accumulation of all these effects [47]. It should be pointed out that the high frequency sleep disruption (SI, 30 s treadmill
193
on, 90 s off every 2 min for 6 h) applied in our study may represent a stressful condition comparing to activity control condition (AC, 10 min on, 30 min off), what might also contribute to potentiation of seizure susceptibility [48]. However, SI and AC groups involved equal locomotors activity. Recently, Ferlisi et al. [2] identified a list of precipitant factors in patients with epilepsy. Our results indicate that sleep disruption is also one of them. In summary, presented results obtained in experimental model of lindane-induced epilepsy suggest that high frequency disruptions of sleep increases sensitivity to seizure susceptibility in rats. Conflict of interest None of the authors has to declare any conflict of interest. Acknowledgments This study was supported by the Ministry of Education, Science and Technological Development of Serbia, Grant No. 175032. References [1] R. Day, R. Gerhardstein, A. Lumley, T. Roth, L. Rosenthal, The behavioral morbidity of obstructive sleep apnea, Prog. Cardiovasc. Dis. 41 (1999) 341–354. [2] M. Ferlisi, S. Shorvon, Seizure precipitants (triggering factors) in patient with epilepsy, Epilepsy Behav. 33 (2014) 101–105. [3] A.A. Borbély, A two process model of sleep regulation, Hum. Neurobiol. 1 (3) (1982) 195–204. [4] I. Feinberg, Changes in sleep cycle patterns with age, J. Psychiatr. Res. 10 (3–4) (1974) 283–306. [5] M.H. Bonnet, Performance and sleepiness as a function of frequency and placement of sleep disruption, Psychophysiology 23 (3) (1986) 263–271. [6] P. Franken, D. Chollet, M. Tafti, The homeostatic regulation of sleep need is under genetic control, J. Neurosci. 21 (8) (2001) 2610–2621. [7] T. Roth, T.A. Roehrs, Etiologies and sequelae of excessive daytime sleepiness, Clin. Ther. 18 (4) (1996) 562–576. [8] American Academy of Sleep Medicine, International Classification of Sleep Disorders, third ed. American Academy of Sleep Medicine, Darien, 2014. [9] American Academy of Sleep Medicine Task Force, Sleep-related breathing disorders in adults: recommendations for syndrome definition and measurement techniques in clinical research, Sleep 22 (5) (1999) 667–689. [10] O. Devinsky, B. Ehrenberg, G.M. Barthlen, H.S. Abramson, D. Luciano, Epilepsy and sleep apnea syndrome, Neurology 44 (11) (1994) 2060–2064. [11] A.M. Chihorek, B. Abou-Khalil, B.A. Malow, Obstructive sleep apnea is associated with seizure occurrence in older adults with epilepsy, Neurology 69 (19) (2007) 1823–1827. [12] B.A. Malow, K. Levy, K. Maturen, R. Bowes, Obstructive sleep apnea is common in medically refractory epilepsy patients, Neurology 55 (7) (2000) 1002–1007. [13] D. Pornsriniyom, K. Shinlapawittayatorn, J. Fong, N.D. Andrews, N. FoldvarySchaefer, Continuous positive airway pressure therapy for obstructive sleep apnea reduces interictal epileptiform discharges in adults with epilepsy, Epilepsy Behav. 37 (2014) 171–174. [14] M. Seyal, L.M. Bateman, T.E. Albertson, T.C. Lin, C.S. Li, Respiratory changes with seizures in localization-related epilepsy: analysis of periictal hypercapnia and airflow patterns, Epilepsia 51 (8) (2010) 1359–1364. [15] N. Foldvary-Schaefer, N.D. Andrews, D. Pornsriniyom, D.E. Moul, Z. Sun, J. Bena, Sleep apnea and epilepsy: who's at risk? Epilepsy Behav. 25 (3) (2012) 363–367. [16] Y.F. Li, Global technical hexachlorocyclohexane usage and its contamination consequences in the environment from: 1948–1997, Sci. Total Environ. 232 (1999) 121–158. [17] Centers for Disease Control and Prevention (CDC), Unintentional topical lindane ingestions—United States, 1998–2003, MMWR Morb Mortal Wkly. Rep. 54 (21) (2005) 533–535. [18] D. Vučević, D. Hrnčić, T. Radosavljević, D. Mladenović, A. Rašić-Marković, H. LončarStevanović, et al., Correlation between electrocorticographic and motor phenomena in lindane-induced experimental epilepsy in rats, Can. J. Physiol. Pharmacol. 86 (4) (2008) 173–179. [19] A.M. Tochman, R. Kamiński, W.A. Turski, S.J. Czuczwar, Protection by conventional and new antiepileptic drugs against lindane-induced seizures and lethal effects in mice, Neurotox. Res. 2 (1) (2000) 63–70. [20] J.T. McKenna, J.L. Tartar, C.P. Ward, M.M. Thakkar, J.W. Cordeira, R.W. McCarley, et al., Sleep fragmentation elevates behavioral, electrographic and neurochemical measures of sleepiness, Neuroscience 146 (4) (2007) 1462–1473. [21] O. Stanojlović, A. Rasić-Marković, D. Hrncić, V. Susić, D. Macut, T. Radosavljević, et al., Two types of seizures in homocysteine thiolactone-treated adult rats, behavioral and electroencephalographic study, Cell. Mol. Neurobiol. 29 (3) (2009) 329–339. [22] D. Hrnčić, A. Rašić-Marković, D. Djuric, V. Sušić, O. Stanojlović, The role of nitric oxide in convulsions induced by lindane in rats, Food Chem. Toxicol. 49 (4) (2011) 947–954.
194
D. Hrnčić et al. / Physiology & Behavior 155 (2016) 188–194
[23] D. Hrncić, A. Rasić-Marković, J. Bjekić-Macut, V. Susić, D. Djuric, O. Stanojlović, Paradoxical sleep deprivation potentiates epilepsy induced by homocysteine thiolactone in adult rats, Exp. Biol. Med. (Maywood) 238 (1) (2013) 77–83. [24] B. Levine, T. Roehrs, E. Stepanski, F. Zorick, T. Roth, Fragmenting sleep diminishes its recuperative value, Sleep 10 (1987) 590–599. [25] R. Downey, M.H. Bonnet, Performance during frequent sleep disruption, Sleep 10 (1987) 354–363. [26] S.S. Campbell, I. Tobler, Animal sleep: a review of sleep duration across phylogeny, Neurosci. Biobehav. Rev. 8 (1984) 269–300. [27] L. Trachel, I. Tobler, P. Achermann, A.A. Borbely, Sleep continuity and the REM– nonREM cycle in the rat under baseline conditions and after sleep deprivation, Physiol. Behav. 49 (1991) 575–580. [28] C.G. Diniz Behn, V. Booth, Modeling the temporal architecture of rat sleep–wake behavior, Conf. Proc. IEEE Eng. Med. Biol. Soc. 4713–4716 (2011). [29] R.E. Brown, R. Basheer, J.T. McKenna, R.E. Strecker, R.W. McCarley, Control of sleep and wakefulness, Physiol. Rev. 92 (3) (2012) 1087–1187. [30] K. Sonka, M. Juklícková, M. Pretl, S. Dostálová, D. Horínek, S. Nevsímalová, Seizures in sleep apnea patients: occurrence and time distribution, Sb. Lek. 101 (3) (2000) 229–232. [31] R. Manni, M. Terzaghi, C. Arbasino, I. Sartori, C.A. Galimberti, A. Tartara, Obstructive sleep apnea in a clinical series of adult epilepsy patients: frequency and features of the comorbidity, Epilepsia 44 (6) (2003) 836–840. [32] B.D. Hoyt, Sleep in patients with neurologic and psychiatric disorders, Prim. Care. 32 (2) (2005) 535–548. [33] C.R. Soldatos, T.J. Paparrigopoulos, Sleep physiology and pathology: pertinence to psychiatry, Int. Rev. Psychiatry 17 (2005) 213–228. [34] F.E. Jensen, C.D. Applegate, D. Holtzman, T.R. Belin, J.L. Burchfiel, Epileptogenic effect of hypoxia in the immature rodent brain, Ann. Neurol. 29 (6) (1991) 629–637. [35] F.E. Jensen, G.L. Holmes, C.T. Lombroso, H.K. Blume, I.R. Firkusny, Age dependent changes in long-term seizure susceptibility and behavior after hypoxia in rats, Epilepsia 33 (1992) 971–980. [36] A. Rubaj, W. Zgodziński, M. Sieklucka-Dziuba, The epileptogenic effect of seizures induced by hypoxia: the role of NMDA and AMPA/KA antagonists, Pharmacol. Biochem. Behav. 74 (2) (2003) 303–311.
[37] Q. Yang, Y. Wang, J. Feng, J. Cao, B. Chen, Intermittent hypoxia from obstructive sleep apnea may cause neuronal impairment and dysfunction in central nervous system: the potential roles played by microglia, Neuropsychiatr. Dis. Treat. 9 (2013) 1077–1086. [38] C.P. Ward, J.G. McCoy, J.T. McKenna, N.P. Connolly, R.W. McCarley, R.E. Strecker, Spatial learning and memory deficits following exposure to 24 h of sleep fragmentation or intermittent hypoxia in a rat model of obstructive sleep apnea, Brain Res. 1294 (2009) 128–137. [39] E.J. Stepanski, The effect of sleep fragmentation on daytime function, Sleep 25 (3) (2002) 268–276. [40] M.H. Bonnet, Sleep restoration as a function of periodic awakening, movement, or electroencephalographic change, Sleep 10 (1987) 364–373. [41] S.V. Jain, P.S. Horn, N. Simakajornboon, T.A. Glauser, Obstructive sleep apnea and primary snoring in children with epilepsy, J. Child Neurol. 28 (1) (2013) 77–82. [42] P. Kumar, T.R. Raju, Seizure susceptibility decreases with enhancement of rapid eye movement sleep, Brain Res. 922 (2) (2001) 299–304. [43] D.S. Park, D.W. Yoon, W.B. Yoo, S.K. Lee, C.H. Yun, S.J. Kim, et al., Sleep fragmentation induces reduction of synapsin II in rat hippocampus, Sleep Biologic Rhythms 12 (2014) 135–144. [44] A. Fassio, A. Raimondi, G. Lignani, F. Benfenati, P. Baldelli, Synapsins: from synapse to network hyperexcitability and epilepsy, Semin. Cell Dev. Biol. 22 (2011) 408–415. [45] B.A. Malow, E. Passaro, C. Milling, D.N. Minecan, K. Levy, Sleep deprivation does not affect seizure frequency during inpatient video-EEG monitoring, Neurology 59 (2002) 1371–1374. [46] B.A. Malow, K.J. Weatherwax, R.D. Chervin, T.F. Hoban, M.L. Marzec, C. Martin, et al., Identification and treatment of obstructive sleep apnea in adults and children with epilepsy: a prospective pilot study, Sleep Med. 4 (2003) 509–515. [47] M.M. Frucht, M. Quigg, C. Schwaner, N.B. Fountain, Distribution of seizure precipitants among epilepsy syndromes, Epilepsia 41 (2000) 1534–1539. [48] Y.H. Ho, Y.T. Lin, C.W. Wu, Y.M. Chao, A.Y. Chang, J.Y. Chan, Peripheral inflammation increases seizure susceptibility via the induction of neuroinflammation and oxidative stress in the hippocampus, J. Biomed. Sci. 22 (1) (2015) 46.