Sleep Breath DOI 10.1007/s11325-014-1046-1
ORIGINAL ARTICLE
Trigeminal induced arousals during human sleep Clemens Heiser & Jan Baja & Franziska Lenz & J. Ulrich Sommer & Karl Hörmann & Raphael M. Herr & Boris A. Stuck
Received: 13 April 2014 / Revised: 27 July 2014 / Accepted: 30 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Arousals caused by external stimuli during human sleep have been studied for most of the sensorial systems. It could be shown that a pure nasal trigeminal stimulus leads to arousals during sleep. The frequency of arousals increases dependent on the stimulus concentration. The aim of the study was to evaluate the influence of different stimulus durations on arousal frequency during different sleep stages. Methods Ten young healthy volunteers with 20 nights of polysomnography were included in the study. Pure trigeminal stimulation with both different concentrations of CO2 (0, 10, 20, 40 %v/v) and different stimulus durations (1, 3, 5, and 10 s) were applied during different sleep stages to the volunteers using an olfactometer. The application was performed during different sleep stages (light sleep, deep sleep, REM sleep). Results The number of arousals increased with rising stimulus duration and stimulus concentration during each sleep stage. Conclusion Trigeminal stimuli during sleep led to arousals in dose- and time-dependent manner.
C. Heiser : J. Baja : F. Lenz : J. U. Sommer : K. Hörmann : B. A. Stuck Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany C. Heiser (*) Department of Otorhinolaryngology, Head and Neck Surgery, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany e-mail: [email protected] URL: http://www.schlaf-hno.de R. M. Herr Mannheim Institute of Public Health, Social and Preventive Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
Keywords Sleep . Trigeminal arousals . Arousal latency . Chemosensation . Olfaction
Introduction Sensory activation during sleep has been studied for auditory, visual, somatosensory, and also for olfactory stimuli. It has been shown that olfactory stimuli are processed during sleep with the help of event-related potentials [1]. Olfactory stimuli such as phenylethyl alcohol (PEA, smell of roses) or hydrogen sulfide (smell of rotten eggs) significantly affect dream emotions depending on their hedonicity [2]. In contrast, olfactory stimulation does not induce arousals during sleep as long as the substance used for stimulation has no or only little trigeminal component [3, 4]. These results demonstrate that olfactory information is differently processed than other sensory information, especially during sleep [5]. Most of the odorants have an olfactory as well as a trigeminal compound. Trigeminal stimuli are transferred to the brain by the trigeminal nerve, which supplies the nasal mucosa sensitively. Most of the olfactory stimuli trigger a nasal trigeminal response and also provoke an olfactory sensation [6]. An exception is carbon dioxide (CO2), which is a pure trigeminal stimulus. Skramlik described in 1925, that subjects cannot identify which side of the nose is receiving a pure olfactory stimulus but can do so readily when the stimulus triggers irritation [7]. In the following years, other authors confirmed these early findings [8]. In contrast, humans can distinguish whether the trigeminal stimulus has been applied to the left or right nostril which can be examined by a lateralization test [9]. Wysocki et al. described a technique to determine olfactory detection thresholds and nasal lateralization thresholds for any volatile compound [9, 10]. Furthermore, the
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trigeminal chemoreceptive system shows an age-related functional decline analogous to that of the olfactory system [1]. In previous studies, we were able to show that olfactory stimuli during sleep do not lead to arousals in human sleep [11]. In contrast, trigeminal stimuli above the threshold lead to an increased number of arousals [11]. An increase in the concentration of trigeminal stimuli leads to a linear increase of arousals. For the purpose of these studies, carbon dioxide (CO2) was used as a pure trigeminal stimulus. To avoid adaptation of the olfactory and trigeminal system, the stimulus duration was set to 1 s. Stuck et al. reported in 2007 that short trigeminal stimuli just below the threshold (such as 1 s stimulus time) do not lead to an increase in arousal frequency [11]. In summary for trigeminal stimulation, both the concentration as well as the duration of the stimulus play an important role. This is in contrast to the olfactory system during sleep. The first aim of the present study was to assess the influence of trigeminal stimulus duration on the number of arousals. The second aim of the study was to determine whether different concentrations of the trigeminal stimulus increase arousal frequency.
Materials and methods The study was conducted at the Sleep Disorders Centre at the Department of Otorhinolaryngology, Head and Neck Surgery Mannheim, Germany. The study protocol was in accordance with the declaration of Helsinki and approved by the local ethics board of the Medical Faculty Mannheim of the University of Heidelberg. Written informed consent was obtained from all participants. Ten (three female, seven male) young healthy volunteers were included in the trial (mean age 25±2 years, range between 22–27 years). Every subject had two nights for sleep recording; hence, in total, 20 nights of testing were performed. Subjects with smell or taste disorders, use of medications known to affect chemosensory function [12], or a history of sleep disorders were excluded from the study. We performed an ENT examination in each subject. This included a nasal/ retronasal endoscopy, an inspection of mouth/oral cavity, and a larynx endoscopy. Pathologies, for instance mucosal inflammation, nasal polyposis, and a significant septal deviation, were exclusion parameters. All recordings were performed during the night and spontaneous sleep. Furthermore, all subjects underwent a “Sniffin’ Stick Test” and “Taste Strips” to rule out participants with a disorder in smell or taste [13, 14]. The overnight sleep protocol and application of CO2 during the night was performed as described in previous studies [4, 11, 15]. The sleep was recorded and scored according to Rechtschaffen and Kales [16]. An overnight
polysomnography with two electroencephalographic channels (C3-A2, C4-A1), two electrooculograms (left, right), two submental, and two leg electromyograms (left, right) were used. Arousals were defined according to the American Sleep Disorders Association as an abrupt shift in EEG frequency, which may include an abrupt shift of EEG frequency including alpha, theta, and/or frequencies greater than 16 Hz (but no spindles) of at least 3 s, with at least 10 s of stable sleep preceding the change. Scoring of arousals during REM required an additional concurrent increase in submental EMG of at least 1 s [17]. For the chemosensory stimulation, an olfactometer (OM6b, Burghart instruments, Wedel, Germany) was used. A constant airstream of 8 l/min over a tube which was placed in the patient’s nostril was applied. Kobal showed in 1981 that this application does not alter the mechanical or thermal conditions at the nasal mucosa. Carbon dioxide (CO2) was used as a pure trigeminal compound in three different concentrations: 10, 20, and 40 %v/v. For every concentration, 4 different stimulus durations were used: 1, 3, 5, and 10 s. Odorless stimuli were presented as control (used in this article with 0 s of CO2). The stimuli were presented in a randomized manner according to a computer-randomized protocol. The interstimulus interval was at least 60 s. Every subject received earplugs in order to dampen external sounds. Two observers (JB, FL) constantly monitored the recordings during the night and administered the stimuli. CO2 was applied during light sleep (LS), during slow wave sleep (SWS) and during rapid eye movement sleep (REM). If a subject woke up during the stimulus, the stimuli were stopped and started again after the participant had fallen asleep again. The two observers set marks in the hypnogramms, and two sleep specialists (BAS, CH) verified sleep stages and arousals. The number of arousals was counted and divided by the number of stimuli, providing the arousal frequency (in %). Additionally, all stimuli of each concentration were added together resulting in a cumulative arousal frequency for the entire patient group. The concentration and duration of exposure were randomized. Furthermore, the arousal latency was listed for every stimulus if an arousal occurred within 30 s after stimulus application. For each group of the same stimulus concentration and stimulus duration, the average latency and standard deviation of arousals were calculated. Arousal frequency was analyzed by repeated measures analysis of variance (rm-ANOVA) with Bonferroni post hoc tests. Therefore, the average value for each subject and for each study condition was submitted into a 4 (stimulus duration of CO2: 1, 3, 5, 10 s) x 4 (stimulus concentration: 0 %, 10 %, 20 %, 40 %) x 3 (sleep stage: LS, SWS, REM) rm-ANOVA. Unfortunately, it was not possible to apply rm-ANOVAs for arousal latency, because arousals did not occur in all settings. We tested four different stimulus durations and four different
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stimulus concentrations in three different sleep stages. This leads to a total of 48 different settings in every subject. At least, we tested every setting 10 times for each subject. Arousals could not be detected in every subject during low stimulus concentrations (0 %, 10 %) and low stimulus durations (1 and 3 s). Due to the missing data, two single factor variance analyses (one way-ANOVAs) were estimated instead. One ANOVA was conducted to compare the effect of stimulus duration (1, 3, 5, 10 s) on arousal latency for 12 combinations of sleep stage and stimulus concentration (4 stimulus concentration: 0 %, 10 %, 20 %, 40 % x 3 sleep stage: LS, SWS, REM). Another ANOVA was used to compare the effect of stimulus concentration (0 %, 10 %, 20 %, 40 %) on arousal latency for the 12 combinations of sleep stage and stimulus duration (4 stimulus duration: 1, 3, 5, 10 s x 3 sleep stage: LS, SWS, REM). All analyses were performed using PASW Statistics 18.0 (SPSS Inc., Chicago; IL, USA).
Results All subjects were normosmic (mean TDI score 36.4±3.7; range 31.5–39.5). During overnight sleep recordings, no abnormalities could be detected. Ten subjects performed a total of twenty nights of testing. The total number of stimuli for all subjects for every stimulus concentration and stimulus duration is shown in Table 1. The difference of number of stimuli reflects the higher number of arousals and awakenings at higher concentrations and during longer stimuli. The subjects showed arousals during stimulation or even woke up at higher concentrations and longer stimuli, which shortened at least the investigation night. Table 1 Total number of stimuli of CO2 for the different sleep stages, different CO2 concentrations, and different stimulus duration
Light sleep 1s 3s 5s 10 s Slow wave sleep 1s 3s 5s 10 s REM sleep 1s 3s 5s 10 s
0%
10 %
20 %
40 %
95 95 95 95
95 95 95 88
95 94 93 88
93 95 92 79
95
95
95
82
97 55 54
95 93 88
93 90 67
84 74 30
87 88 84 64
90 95 83 62
86 85 69 42
88 74 52 22
Arousal frequency In all sleep stages, the increases in CO2 concentration as well as the increases in stimulus duration (see Figs. 1, 2, and 3 and Table 2) were accompanied by a rise in arousal frequency. During light sleep, the increase of stimulus concentration (CO2 v/v) led to an increase in arousal frequency. The same was observed when increasing the stimulus duration (see Fig. 1). These results in light sleep did not differ from the results during slow wave sleep and REM sleep (see Figs. 2 and 3). Increasing stimulus concentration or stimulus duration always led to an increase in arousal frequency. The assumption of sphericity for the rm-ANOVA was violated for stimulus duration (Mauchly’s test: χ2 =19.13, p=.002). Therefore, the Greenhouse-Geisser correction was used to adjust the degrees of freedom (epsilon=0.61). The results show a significant main effect of the stimulus concentration (F(3,24) = 20.77; p < 0.001), the stimulus duration (F(1.84,14.72) =6.31; p=0.012), and the sleep stage (F(2,16) = 6.25; p=0.010), as well as a significant interaction between stimulus concentration and sleep stage (F(6,48) =3.86; p=0.003). The post-hoc tests revealed a significant difference between the concentration of 40 and 0 % ( p=0.001), 10 ( p=0.007), and 20% ( p=0.014). Moreover, there was also a significant difference between 0 and 20 % (p=0.041). Regarding stimulus duration of CO2, 1 s differed significantly from 3 s ( p=0.008), and 5 s ( p=0.048). In addition, sleep stage LS differed from SWS ( p=0.016), which differed from REM ( p=0.045). Arousal latency Regarding the arousal latency, different effects was observed. For most stimulus concentrations and durations, the increase had no effect on a shortened latency or even longer latency. Just for some different stimulus durations (5 and 10 s) in slow wave sleep and REM sleep, we could see a significant effect on arousal latency when increasing the stimulus concentration. This led to a shortened latency. During slow wave sleep with a duration of 5 s (F(3,17) =8.53; p=0.001), during REM sleep with a duration with 5 s (F(3,17) =4.52; p=0.017), and during light sleep with a duration of 10 s (F(3,18) =13.32; p0.05).
Discussion Our results demonstrate that an increasing concentration of trigeminal stimulus (CO2) led to an increase in the number of
Sleep Breath Fig. 1 Arousal frequency (%) for the different CO2 concentrations and stimulus durations in light sleep (means)
arousals. An increase in stimulus duration of the same stimulus concentration significantly increased the arousal frequency. We could visually not detect any other changes in EEG patterns except of arousals. No automated analysis of EEG frequencies was employed. Even a moderate stimulus of the trigeminal nerve— which is not perceived as painful during wakefulness— already showed an increase in the arousal frequency. A concentration close to the threshold to sense of the stimulus (10 %v/v CO2) led to an increase in the number of arousals.
Fig. 2 Arousal frequency (%) for the different CO2 concentrations and stimulus durations in slow wave sleep (means)
As already known from further studies during healthy sleep, most of humans have a basal frequency of 3–4 % on arousals, which can be detected in all three sleep stages without stimulation [11]. Previous studies from our group described that nociceptive stimuli as well as trigeminal stimuli lead to statistical significant increases in arousal frequency both for concentrations around the threshold as well as concentrations above the threshold [11, 18, 19]. The processing of strong nociceptive stimuli—that represent a significant alarm mechanism—is attenuated in all sleep
Sleep Breath Fig. 3 Arousal frequency (%) for the different CO2 concentrations and stimulus durations in REM sleep (means)
stages [19]. The data from our study and Lavigne et al. clearly showed that pain sensation (high concentrations of CO2 are uncomfortable—at least known during alertness) represents a significant alarm mechanism. Lavigne et al. used different thermal nociceptive stimulations (24, 37, and 47 °C) during sleep with one stimulus duration (either 6 s or 12 s of stimulus duration). The group demonstrated that an increase in temperature next to the pain level (47 °C) led to an increase in arousals. Compared to our results with an increasing stimulus concentration next to the discomfort threshold (20 %v/v CO2)
Table 2 Arousal frequency (%) for the different CO2 concentrations and stimulus durations during different sleep stages (mean±SD) 0% Light sleep 1s 6.7±7.1 3s 3.3±7.1 5s 3.3±5.0 10 s 2.2±4.4 Slow wave sleep 1s 1.1±3.3 3s 5s 10 s REM sleep 1s 3s 5s 10 s
10 %
20 %
40 %
5.6±5.3 10.6±13.3 8.9±10.5 20.1±32.0
7.8±12.0 10.6±12.9 16.1±13.6 23.3±23.5
29.7±21.1 35.0±25.7 59.2±29.6 51.1±32.7
2.2±4.4
0.0±0.0
10.4±18.5
2.7±5.5 2.2±4.4 0.0±0.0
3.3±7.1 12.2±26.4 14.9±32.4
4.4±7.3 20.3±31.5 16.3±26.1
38.0±28.3 31.2±29.8 25.6±42.8
2.2±4.4 0.0±0.0 3.5±7.2 7.8±8.3
4.4±7.3 5.6±13.3 11.9±21.7 20.7±19.2
2.2±6.7 25.9±18.9 37.1±39.2 32.6±38.4
14.2±15.4 34.1±25.5 40.8±20.0 44.4±46.7
the same observations for trigeminal stimulus were detected. Higher concentrations of CO2 during alertness are perceived as strong stinging painful sensations at the nasal mucosa. During sleep on the one hand, the stimulus concentration was important and on the other hand, the stimulus duration played a crucial role in arousal frequency. Trigeminal stimuli during awake can be experienced by the patient as painful and unpleasant. It seems logical that pain can disturb sleep. In different diseases, which are associated with pain (such as oesophageal reflux, low back pain, angina pectoris), disturbances in sleep structure during polysomnography could be detected [20, 21]. That makes sense in the view of human evolution. Drewes et al. figured out that pain from different body structures may give different responses in sleep microstructures [18]. Muscle pain stimulus as well as joint pain stimulus provoked changes in EEG frequency. In contrast, cutaneous pain did not caused any changes in the EEG activity. No derivations seemed to be sensitive to detect the evoked changes in the EEG. Drewes et al. tested stimulus concentrations and stimulus durations above the pain threshold. Tuladhar et al. examined nasal trigeminal stimuli during sleep in infants. They detected a decrease of the heart rate dependent on sleep stage and body sleep position after trigeminal stimulation [22]. In 2003, Nordin et al. showed that both a circadian rhythm and desensitization are involved in triggering trigeminal-induced event-related potentials (ERP) [23]. During different times (4:00, 08:00, 12:00, 16:00, 20:00, and 24:00 h), event-related potentials with an olfactory stimulus (H2S) and a trigeminal stimulus (CO2) were recorded in young, awake men. The authors suggested that trigeminal ERP amplitudes follow a circadian rhythm that is similar to
Sleep Breath Fig. 4 Arousal latency (s) for the different CO2 concentrations and stimulus durations during light sleep (mean±SD)
oral temperature and opposite to sleepiness. The strongest effect was at 16:00 and 20:00 and smallest amplitude at 04:00. The latencies of ERPs did not follow a circadian rhythm. In our clinical trial, we also could not see any differences in arousal frequencies between the beginning and at the end of the night. In a former study, we demonstrated that ERP
Fig. 5 Arousal latency (s) for the different CO2 concentrations and stimulus durations in slow wave sleep (mean±SD). In the 1 s group of 20 % CO2, no arousals could be detected; hence, no bar graph was plotted
can be measured during sleep [15]. Latencies of ERPs are longer, and amplitudes are larger during light sleep and slow wave sleep compared to wakefulness. These results are congruent with previous research which showed that cognitive processing of chemosensory information is dependent on the individual condition [24]. Regarding the arousals, we could
Sleep Breath Fig. 6 Arousal latency (s) for the different CO2 concentrations and stimulus durations in REM sleep (mean±SD). In the 3 s group of odorless stimuli (0 % CO2) no arousals could be detected; hence, no bar graph was plotted
not see any changes in duration, amplitude, or latency for most of the different conditions (concentration, stimulus duration, sleep stage). But with our current results, we demonstrate that certain processes regarding the trigeminal sensory system remain operative during sleep. In the previous studies, we did not record ERPs. During wakefulness, arousals cannot be recorded because the “WakeEEG” superimposes arousals and the EEG- frequencies between both do not differ. Table 3 Arousal latency (in seconds) for the different CO2 concentrations and stimulus durations in different sleep stages (mean±SD) 0% Light sleep 1s 12.3±6.9 3s 11.3±7.8 5s 9.5±6.4 10 s 22.5±2.1 Slow wave sleep 1s 4.0±0.0 3s 14.3±6.5 5s 18.8±1.9 10 s 11.0±0.0 REM sleep 1s 7.5±7.8 3s 0.0±0.0 5s 13.0±2.8 10 s 12.1±6.6
10 %
20 %
40 %
11.2±11.7 8.6±4.3 5.9±5.1 6.8±0.6
5.7±3.7 7.4±1.4 7.8±1.2 7.6±2.3
7.6±3.5 5.8±2.2 6.7±1.4 8.2±4.2
16.0±10.4 12.3±4.5 6.4±2.6 9.0±5.0
0.0±0.0 9.3±2.3 9.0±2.6 12.0±11.4
6.7±1.2 6.8±2.6 8.4±4.6 5.7±2.7
5.2±0.8 6.7±4.2 10.9±4.1 7.3±4.7
7.0±0.0 9.8±5.2 6.9±2.9 8.2±4.6
6.6±3.1 5.7±2.2 6.5±2.0 4.6±0.8
A pure olfactory stimulus with highly supra-threshold concentration does not increase arousal frequency [11]. Both systems—the olfactory and trigeminal system which coordinates the olfaction and the olfactory impression—seem to be differently processed on the cortical level during sleep [15]. This phenomenon makes sense with regard to the human evolution. During sleep, olfaction is normally not needed. The trigeminal system is an alarm system, which can be important. It seems that during sleep, the central processing changes for olfactory stimuli, while trigeminal stimuli are processed in the same manner as awake. During alertness, the thalamus processes olfactory and trigeminal stimuli [25]. The thalamic gating is highly significant for the processing of most sensory input [26]. It seems that gating of the olfactory stimuli is governed by the ascending reticular activating system; meanwhile, the trigeminal system seems to be unaffected [27]. This might be the reason that odor alarms (such as smoke) are not working for humans. In a further study, we investigated artificial smoke during sleep, which did not elicit arousals during human sleep [3]. The trigeminal system appears to work during sleep as fast as during wakefulness. It probably belongs to the pain alarm system as a sensory part of the body. We demonstrated that the trigeminal system plays a unique role in the human sensory system. Trigeminal stimuli seemed to be processed similarly during sleep and wakefulness. The results demonstrated that trigeminal stimuli during sleep led to arousals in a dose- and time-dependent manner.
Sleep Breath Acknowledgments The study was supported by a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft STU 488/2-1).
14.
Conflict of interest The authors declare that they have no conflict of interest. 15.
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ORIGINAL ARTICLE
Trigeminal induced arousals during human sleep Clemens Heiser & Jan Baja & Franziska Lenz & J. Ulrich Sommer & Karl Hörmann & Raphael M. Herr & Boris A. Stuck
Received: 13 April 2014 / Revised: 27 July 2014 / Accepted: 30 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Background Arousals caused by external stimuli during human sleep have been studied for most of the sensorial systems. It could be shown that a pure nasal trigeminal stimulus leads to arousals during sleep. The frequency of arousals increases dependent on the stimulus concentration. The aim of the study was to evaluate the influence of different stimulus durations on arousal frequency during different sleep stages. Methods Ten young healthy volunteers with 20 nights of polysomnography were included in the study. Pure trigeminal stimulation with both different concentrations of CO2 (0, 10, 20, 40 %v/v) and different stimulus durations (1, 3, 5, and 10 s) were applied during different sleep stages to the volunteers using an olfactometer. The application was performed during different sleep stages (light sleep, deep sleep, REM sleep). Results The number of arousals increased with rising stimulus duration and stimulus concentration during each sleep stage. Conclusion Trigeminal stimuli during sleep led to arousals in dose- and time-dependent manner.
C. Heiser : J. Baja : F. Lenz : J. U. Sommer : K. Hörmann : B. A. Stuck Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany C. Heiser (*) Department of Otorhinolaryngology, Head and Neck Surgery, Technische Universität München, Ismaninger Str. 22, 81675 Munich, Germany e-mail: [email protected] URL: http://www.schlaf-hno.de R. M. Herr Mannheim Institute of Public Health, Social and Preventive Medicine, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
Keywords Sleep . Trigeminal arousals . Arousal latency . Chemosensation . Olfaction
Introduction Sensory activation during sleep has been studied for auditory, visual, somatosensory, and also for olfactory stimuli. It has been shown that olfactory stimuli are processed during sleep with the help of event-related potentials [1]. Olfactory stimuli such as phenylethyl alcohol (PEA, smell of roses) or hydrogen sulfide (smell of rotten eggs) significantly affect dream emotions depending on their hedonicity [2]. In contrast, olfactory stimulation does not induce arousals during sleep as long as the substance used for stimulation has no or only little trigeminal component [3, 4]. These results demonstrate that olfactory information is differently processed than other sensory information, especially during sleep [5]. Most of the odorants have an olfactory as well as a trigeminal compound. Trigeminal stimuli are transferred to the brain by the trigeminal nerve, which supplies the nasal mucosa sensitively. Most of the olfactory stimuli trigger a nasal trigeminal response and also provoke an olfactory sensation [6]. An exception is carbon dioxide (CO2), which is a pure trigeminal stimulus. Skramlik described in 1925, that subjects cannot identify which side of the nose is receiving a pure olfactory stimulus but can do so readily when the stimulus triggers irritation [7]. In the following years, other authors confirmed these early findings [8]. In contrast, humans can distinguish whether the trigeminal stimulus has been applied to the left or right nostril which can be examined by a lateralization test [9]. Wysocki et al. described a technique to determine olfactory detection thresholds and nasal lateralization thresholds for any volatile compound [9, 10]. Furthermore, the
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trigeminal chemoreceptive system shows an age-related functional decline analogous to that of the olfactory system [1]. In previous studies, we were able to show that olfactory stimuli during sleep do not lead to arousals in human sleep [11]. In contrast, trigeminal stimuli above the threshold lead to an increased number of arousals [11]. An increase in the concentration of trigeminal stimuli leads to a linear increase of arousals. For the purpose of these studies, carbon dioxide (CO2) was used as a pure trigeminal stimulus. To avoid adaptation of the olfactory and trigeminal system, the stimulus duration was set to 1 s. Stuck et al. reported in 2007 that short trigeminal stimuli just below the threshold (such as 1 s stimulus time) do not lead to an increase in arousal frequency [11]. In summary for trigeminal stimulation, both the concentration as well as the duration of the stimulus play an important role. This is in contrast to the olfactory system during sleep. The first aim of the present study was to assess the influence of trigeminal stimulus duration on the number of arousals. The second aim of the study was to determine whether different concentrations of the trigeminal stimulus increase arousal frequency.
Materials and methods The study was conducted at the Sleep Disorders Centre at the Department of Otorhinolaryngology, Head and Neck Surgery Mannheim, Germany. The study protocol was in accordance with the declaration of Helsinki and approved by the local ethics board of the Medical Faculty Mannheim of the University of Heidelberg. Written informed consent was obtained from all participants. Ten (three female, seven male) young healthy volunteers were included in the trial (mean age 25±2 years, range between 22–27 years). Every subject had two nights for sleep recording; hence, in total, 20 nights of testing were performed. Subjects with smell or taste disorders, use of medications known to affect chemosensory function [12], or a history of sleep disorders were excluded from the study. We performed an ENT examination in each subject. This included a nasal/ retronasal endoscopy, an inspection of mouth/oral cavity, and a larynx endoscopy. Pathologies, for instance mucosal inflammation, nasal polyposis, and a significant septal deviation, were exclusion parameters. All recordings were performed during the night and spontaneous sleep. Furthermore, all subjects underwent a “Sniffin’ Stick Test” and “Taste Strips” to rule out participants with a disorder in smell or taste [13, 14]. The overnight sleep protocol and application of CO2 during the night was performed as described in previous studies [4, 11, 15]. The sleep was recorded and scored according to Rechtschaffen and Kales [16]. An overnight
polysomnography with two electroencephalographic channels (C3-A2, C4-A1), two electrooculograms (left, right), two submental, and two leg electromyograms (left, right) were used. Arousals were defined according to the American Sleep Disorders Association as an abrupt shift in EEG frequency, which may include an abrupt shift of EEG frequency including alpha, theta, and/or frequencies greater than 16 Hz (but no spindles) of at least 3 s, with at least 10 s of stable sleep preceding the change. Scoring of arousals during REM required an additional concurrent increase in submental EMG of at least 1 s [17]. For the chemosensory stimulation, an olfactometer (OM6b, Burghart instruments, Wedel, Germany) was used. A constant airstream of 8 l/min over a tube which was placed in the patient’s nostril was applied. Kobal showed in 1981 that this application does not alter the mechanical or thermal conditions at the nasal mucosa. Carbon dioxide (CO2) was used as a pure trigeminal compound in three different concentrations: 10, 20, and 40 %v/v. For every concentration, 4 different stimulus durations were used: 1, 3, 5, and 10 s. Odorless stimuli were presented as control (used in this article with 0 s of CO2). The stimuli were presented in a randomized manner according to a computer-randomized protocol. The interstimulus interval was at least 60 s. Every subject received earplugs in order to dampen external sounds. Two observers (JB, FL) constantly monitored the recordings during the night and administered the stimuli. CO2 was applied during light sleep (LS), during slow wave sleep (SWS) and during rapid eye movement sleep (REM). If a subject woke up during the stimulus, the stimuli were stopped and started again after the participant had fallen asleep again. The two observers set marks in the hypnogramms, and two sleep specialists (BAS, CH) verified sleep stages and arousals. The number of arousals was counted and divided by the number of stimuli, providing the arousal frequency (in %). Additionally, all stimuli of each concentration were added together resulting in a cumulative arousal frequency for the entire patient group. The concentration and duration of exposure were randomized. Furthermore, the arousal latency was listed for every stimulus if an arousal occurred within 30 s after stimulus application. For each group of the same stimulus concentration and stimulus duration, the average latency and standard deviation of arousals were calculated. Arousal frequency was analyzed by repeated measures analysis of variance (rm-ANOVA) with Bonferroni post hoc tests. Therefore, the average value for each subject and for each study condition was submitted into a 4 (stimulus duration of CO2: 1, 3, 5, 10 s) x 4 (stimulus concentration: 0 %, 10 %, 20 %, 40 %) x 3 (sleep stage: LS, SWS, REM) rm-ANOVA. Unfortunately, it was not possible to apply rm-ANOVAs for arousal latency, because arousals did not occur in all settings. We tested four different stimulus durations and four different
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stimulus concentrations in three different sleep stages. This leads to a total of 48 different settings in every subject. At least, we tested every setting 10 times for each subject. Arousals could not be detected in every subject during low stimulus concentrations (0 %, 10 %) and low stimulus durations (1 and 3 s). Due to the missing data, two single factor variance analyses (one way-ANOVAs) were estimated instead. One ANOVA was conducted to compare the effect of stimulus duration (1, 3, 5, 10 s) on arousal latency for 12 combinations of sleep stage and stimulus concentration (4 stimulus concentration: 0 %, 10 %, 20 %, 40 % x 3 sleep stage: LS, SWS, REM). Another ANOVA was used to compare the effect of stimulus concentration (0 %, 10 %, 20 %, 40 %) on arousal latency for the 12 combinations of sleep stage and stimulus duration (4 stimulus duration: 1, 3, 5, 10 s x 3 sleep stage: LS, SWS, REM). All analyses were performed using PASW Statistics 18.0 (SPSS Inc., Chicago; IL, USA).
Results All subjects were normosmic (mean TDI score 36.4±3.7; range 31.5–39.5). During overnight sleep recordings, no abnormalities could be detected. Ten subjects performed a total of twenty nights of testing. The total number of stimuli for all subjects for every stimulus concentration and stimulus duration is shown in Table 1. The difference of number of stimuli reflects the higher number of arousals and awakenings at higher concentrations and during longer stimuli. The subjects showed arousals during stimulation or even woke up at higher concentrations and longer stimuli, which shortened at least the investigation night. Table 1 Total number of stimuli of CO2 for the different sleep stages, different CO2 concentrations, and different stimulus duration
Light sleep 1s 3s 5s 10 s Slow wave sleep 1s 3s 5s 10 s REM sleep 1s 3s 5s 10 s
0%
10 %
20 %
40 %
95 95 95 95
95 95 95 88
95 94 93 88
93 95 92 79
95
95
95
82
97 55 54
95 93 88
93 90 67
84 74 30
87 88 84 64
90 95 83 62
86 85 69 42
88 74 52 22
Arousal frequency In all sleep stages, the increases in CO2 concentration as well as the increases in stimulus duration (see Figs. 1, 2, and 3 and Table 2) were accompanied by a rise in arousal frequency. During light sleep, the increase of stimulus concentration (CO2 v/v) led to an increase in arousal frequency. The same was observed when increasing the stimulus duration (see Fig. 1). These results in light sleep did not differ from the results during slow wave sleep and REM sleep (see Figs. 2 and 3). Increasing stimulus concentration or stimulus duration always led to an increase in arousal frequency. The assumption of sphericity for the rm-ANOVA was violated for stimulus duration (Mauchly’s test: χ2 =19.13, p=.002). Therefore, the Greenhouse-Geisser correction was used to adjust the degrees of freedom (epsilon=0.61). The results show a significant main effect of the stimulus concentration (F(3,24) = 20.77; p < 0.001), the stimulus duration (F(1.84,14.72) =6.31; p=0.012), and the sleep stage (F(2,16) = 6.25; p=0.010), as well as a significant interaction between stimulus concentration and sleep stage (F(6,48) =3.86; p=0.003). The post-hoc tests revealed a significant difference between the concentration of 40 and 0 % ( p=0.001), 10 ( p=0.007), and 20% ( p=0.014). Moreover, there was also a significant difference between 0 and 20 % (p=0.041). Regarding stimulus duration of CO2, 1 s differed significantly from 3 s ( p=0.008), and 5 s ( p=0.048). In addition, sleep stage LS differed from SWS ( p=0.016), which differed from REM ( p=0.045). Arousal latency Regarding the arousal latency, different effects was observed. For most stimulus concentrations and durations, the increase had no effect on a shortened latency or even longer latency. Just for some different stimulus durations (5 and 10 s) in slow wave sleep and REM sleep, we could see a significant effect on arousal latency when increasing the stimulus concentration. This led to a shortened latency. During slow wave sleep with a duration of 5 s (F(3,17) =8.53; p=0.001), during REM sleep with a duration with 5 s (F(3,17) =4.52; p=0.017), and during light sleep with a duration of 10 s (F(3,18) =13.32; p0.05).
Discussion Our results demonstrate that an increasing concentration of trigeminal stimulus (CO2) led to an increase in the number of
Sleep Breath Fig. 1 Arousal frequency (%) for the different CO2 concentrations and stimulus durations in light sleep (means)
arousals. An increase in stimulus duration of the same stimulus concentration significantly increased the arousal frequency. We could visually not detect any other changes in EEG patterns except of arousals. No automated analysis of EEG frequencies was employed. Even a moderate stimulus of the trigeminal nerve— which is not perceived as painful during wakefulness— already showed an increase in the arousal frequency. A concentration close to the threshold to sense of the stimulus (10 %v/v CO2) led to an increase in the number of arousals.
Fig. 2 Arousal frequency (%) for the different CO2 concentrations and stimulus durations in slow wave sleep (means)
As already known from further studies during healthy sleep, most of humans have a basal frequency of 3–4 % on arousals, which can be detected in all three sleep stages without stimulation [11]. Previous studies from our group described that nociceptive stimuli as well as trigeminal stimuli lead to statistical significant increases in arousal frequency both for concentrations around the threshold as well as concentrations above the threshold [11, 18, 19]. The processing of strong nociceptive stimuli—that represent a significant alarm mechanism—is attenuated in all sleep
Sleep Breath Fig. 3 Arousal frequency (%) for the different CO2 concentrations and stimulus durations in REM sleep (means)
stages [19]. The data from our study and Lavigne et al. clearly showed that pain sensation (high concentrations of CO2 are uncomfortable—at least known during alertness) represents a significant alarm mechanism. Lavigne et al. used different thermal nociceptive stimulations (24, 37, and 47 °C) during sleep with one stimulus duration (either 6 s or 12 s of stimulus duration). The group demonstrated that an increase in temperature next to the pain level (47 °C) led to an increase in arousals. Compared to our results with an increasing stimulus concentration next to the discomfort threshold (20 %v/v CO2)
Table 2 Arousal frequency (%) for the different CO2 concentrations and stimulus durations during different sleep stages (mean±SD) 0% Light sleep 1s 6.7±7.1 3s 3.3±7.1 5s 3.3±5.0 10 s 2.2±4.4 Slow wave sleep 1s 1.1±3.3 3s 5s 10 s REM sleep 1s 3s 5s 10 s
10 %
20 %
40 %
5.6±5.3 10.6±13.3 8.9±10.5 20.1±32.0
7.8±12.0 10.6±12.9 16.1±13.6 23.3±23.5
29.7±21.1 35.0±25.7 59.2±29.6 51.1±32.7
2.2±4.4
0.0±0.0
10.4±18.5
2.7±5.5 2.2±4.4 0.0±0.0
3.3±7.1 12.2±26.4 14.9±32.4
4.4±7.3 20.3±31.5 16.3±26.1
38.0±28.3 31.2±29.8 25.6±42.8
2.2±4.4 0.0±0.0 3.5±7.2 7.8±8.3
4.4±7.3 5.6±13.3 11.9±21.7 20.7±19.2
2.2±6.7 25.9±18.9 37.1±39.2 32.6±38.4
14.2±15.4 34.1±25.5 40.8±20.0 44.4±46.7
the same observations for trigeminal stimulus were detected. Higher concentrations of CO2 during alertness are perceived as strong stinging painful sensations at the nasal mucosa. During sleep on the one hand, the stimulus concentration was important and on the other hand, the stimulus duration played a crucial role in arousal frequency. Trigeminal stimuli during awake can be experienced by the patient as painful and unpleasant. It seems logical that pain can disturb sleep. In different diseases, which are associated with pain (such as oesophageal reflux, low back pain, angina pectoris), disturbances in sleep structure during polysomnography could be detected [20, 21]. That makes sense in the view of human evolution. Drewes et al. figured out that pain from different body structures may give different responses in sleep microstructures [18]. Muscle pain stimulus as well as joint pain stimulus provoked changes in EEG frequency. In contrast, cutaneous pain did not caused any changes in the EEG activity. No derivations seemed to be sensitive to detect the evoked changes in the EEG. Drewes et al. tested stimulus concentrations and stimulus durations above the pain threshold. Tuladhar et al. examined nasal trigeminal stimuli during sleep in infants. They detected a decrease of the heart rate dependent on sleep stage and body sleep position after trigeminal stimulation [22]. In 2003, Nordin et al. showed that both a circadian rhythm and desensitization are involved in triggering trigeminal-induced event-related potentials (ERP) [23]. During different times (4:00, 08:00, 12:00, 16:00, 20:00, and 24:00 h), event-related potentials with an olfactory stimulus (H2S) and a trigeminal stimulus (CO2) were recorded in young, awake men. The authors suggested that trigeminal ERP amplitudes follow a circadian rhythm that is similar to
Sleep Breath Fig. 4 Arousal latency (s) for the different CO2 concentrations and stimulus durations during light sleep (mean±SD)
oral temperature and opposite to sleepiness. The strongest effect was at 16:00 and 20:00 and smallest amplitude at 04:00. The latencies of ERPs did not follow a circadian rhythm. In our clinical trial, we also could not see any differences in arousal frequencies between the beginning and at the end of the night. In a former study, we demonstrated that ERP
Fig. 5 Arousal latency (s) for the different CO2 concentrations and stimulus durations in slow wave sleep (mean±SD). In the 1 s group of 20 % CO2, no arousals could be detected; hence, no bar graph was plotted
can be measured during sleep [15]. Latencies of ERPs are longer, and amplitudes are larger during light sleep and slow wave sleep compared to wakefulness. These results are congruent with previous research which showed that cognitive processing of chemosensory information is dependent on the individual condition [24]. Regarding the arousals, we could
Sleep Breath Fig. 6 Arousal latency (s) for the different CO2 concentrations and stimulus durations in REM sleep (mean±SD). In the 3 s group of odorless stimuli (0 % CO2) no arousals could be detected; hence, no bar graph was plotted
not see any changes in duration, amplitude, or latency for most of the different conditions (concentration, stimulus duration, sleep stage). But with our current results, we demonstrate that certain processes regarding the trigeminal sensory system remain operative during sleep. In the previous studies, we did not record ERPs. During wakefulness, arousals cannot be recorded because the “WakeEEG” superimposes arousals and the EEG- frequencies between both do not differ. Table 3 Arousal latency (in seconds) for the different CO2 concentrations and stimulus durations in different sleep stages (mean±SD) 0% Light sleep 1s 12.3±6.9 3s 11.3±7.8 5s 9.5±6.4 10 s 22.5±2.1 Slow wave sleep 1s 4.0±0.0 3s 14.3±6.5 5s 18.8±1.9 10 s 11.0±0.0 REM sleep 1s 7.5±7.8 3s 0.0±0.0 5s 13.0±2.8 10 s 12.1±6.6
10 %
20 %
40 %
11.2±11.7 8.6±4.3 5.9±5.1 6.8±0.6
5.7±3.7 7.4±1.4 7.8±1.2 7.6±2.3
7.6±3.5 5.8±2.2 6.7±1.4 8.2±4.2
16.0±10.4 12.3±4.5 6.4±2.6 9.0±5.0
0.0±0.0 9.3±2.3 9.0±2.6 12.0±11.4
6.7±1.2 6.8±2.6 8.4±4.6 5.7±2.7
5.2±0.8 6.7±4.2 10.9±4.1 7.3±4.7
7.0±0.0 9.8±5.2 6.9±2.9 8.2±4.6
6.6±3.1 5.7±2.2 6.5±2.0 4.6±0.8
A pure olfactory stimulus with highly supra-threshold concentration does not increase arousal frequency [11]. Both systems—the olfactory and trigeminal system which coordinates the olfaction and the olfactory impression—seem to be differently processed on the cortical level during sleep [15]. This phenomenon makes sense with regard to the human evolution. During sleep, olfaction is normally not needed. The trigeminal system is an alarm system, which can be important. It seems that during sleep, the central processing changes for olfactory stimuli, while trigeminal stimuli are processed in the same manner as awake. During alertness, the thalamus processes olfactory and trigeminal stimuli [25]. The thalamic gating is highly significant for the processing of most sensory input [26]. It seems that gating of the olfactory stimuli is governed by the ascending reticular activating system; meanwhile, the trigeminal system seems to be unaffected [27]. This might be the reason that odor alarms (such as smoke) are not working for humans. In a further study, we investigated artificial smoke during sleep, which did not elicit arousals during human sleep [3]. The trigeminal system appears to work during sleep as fast as during wakefulness. It probably belongs to the pain alarm system as a sensory part of the body. We demonstrated that the trigeminal system plays a unique role in the human sensory system. Trigeminal stimuli seemed to be processed similarly during sleep and wakefulness. The results demonstrated that trigeminal stimuli during sleep led to arousals in a dose- and time-dependent manner.
Sleep Breath Acknowledgments The study was supported by a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft STU 488/2-1).
14.
Conflict of interest The authors declare that they have no conflict of interest. 15.
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