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RESEARCH REPORT |
Department of Psychobiology–Universidade Federal de São Paulo, Escola Paulista de Medicina (UNIFESP/EPM), Rua Napoleão de Barros, 925, Vila Clementino-SP-04024-002, São Paulo, SP, Brazil
* corresponding author, tschutz{at}psicobio.epm.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: temporomandibular joint • sleep • cyclo-oxygenase-2 • rat
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Studies have demonstrated that cyclo-oxygenase-2 inhibitors offer substantial benefits in relation to non-selective cyclo-oxygenase inhibitors, because of their favorable gastro-intestinal profiles and innocuous effect on platelet function (Chang et al., 2002; Khan et al., 2002; Cicconetti et al., 2004; Martina et al., 2005).
Prostaglandin-D2 is a major prostanoid in the mammalian brain, and is the endogenous sleep-promoting substance in mice, rats, and monkeys, and probably in humans. The prostaglandin-D2-sensitive sleep-promoting zone was demarcated in the rat within the ventral surface of the rostral basal forebrain, where prostaglandin-D2 applied to the subarachnoid space of the zone increased non-rapid eye movement sleep (nREM) up to the daytime level (Matsumura et al., 1994). The cerebrospinal fluid level of prostaglandin-D2 in rats increased during sleep deprivation and tended to become higher with increasing propensity toward and deepening of sleep under a non-sleep-deprived normal condition (Ram et al., 1997).
When prostaglandin synthase, the enzyme responsible for the byosynthesis of prostaglandin-D2 in the brain, was inhibited in vivo by its selective inhibitors, tetravalent selenium compounds, both nREM and REM sleep were reduced almost completely but reversibly, indicating that prostaglandin synthase is key in sleep regulation (Hayaishi, 1999). In addition, cyclo-oxygenase-2 mediated hyperproduction of prostaglandins, including D2-sensitivity, being critically involved in the enhancement of nREM sleep, but not in the suppression of REM sleep (Terao et al., 1998). Recently, inhibition of cyclo-oxygenase-2 was shown to attenuate spontaneous sleep when NS-398, a highly selective cyclo-oxygenase-2 inhibitor, was injected intracerebroventricularly (Yoshida et al., 2003).
Chronically painful conditions are associated with sleep disturbances, i.e., sleep continuity changes and sleep architecture (Lautenbacher et al., 2006). Sleep disturbances have been reported in acute inflammation of TMJ, described as an orofacial pain model (Schütz et al., 2003, 2004). TMJ conditions represent a clinical entity, and different mechanisms pertaining to the function of the TMJ have been endorsed, as has the efficacy of pharmacological approaches used to treat them (Schütz et al., 2004).
Some compounds (i.e., etoricoxib) act as selective inhibitors of cyclo-oxygenase-2 enzyme, but there is no information of whether cyclo-oxygenase-2 enzyme and prostaglandins may be directly involved in the modulation of the sleep-wake cycle. Little is known about the physiology and pathology of cyclo-oxygenase in acute inflammation of the TMJ. Because of the demonstration that sleep disturbances in animals occur in TMJ acute inflammatory scenarios, we sought to examine the role of cyclo-oxygenase-2 in that painful condition, while trying to establish the extent to which this enzyme influences the sleep patterns of animals, both when subjected to this experimental model and when merely manipulated.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Electrocorticogram Recordings
The recordings were performed on a Nihon Koden Co. (Tokyo, Japan) model QP-223A apparatus, with 3 channels for each animal: 2 for the electrocorticogram, and 1 for head-muscle activity (electromyogram). The electrocorticogram signals were amplified and filtered with the low pass at 0.1 sec (1.6 Hz), and electromyogram activity was filtered with the low pass at 0.03 sec (5.3 Hz). The sleep recording was carried out immediately after the injection of Freund’s adjuvant or saline over 2 light and 2 dark periods of 12 hrs each. The electrocorticogram traces were visually and manually scored blindly for 30-second periods. All recordings were scored by only one researcher, thus ensuring reliability of the data. The sleep stages were identified and scored according to Timo-Iaria et al.(1970).
The following sleep parameters were assessed: sleep efficiency (total sleep time percentage during the recording time); sleep latency (time lag between the onset of recording and the first sleep period); REM sleep latency (time lag between the first sleep period and the first episode of REM); nREM sleep (percentage of all periods of deep sleep throughout the recording) and REM sleep (percentage of all periods of REM sleep throughout the recording); and arousal bouts (events at least 15 sec long with abrupt modification of baseline frequency of electrocorticogram accompanied by high-amplitude electromyogram activity followed by nREM sleep).
TMJ Injection Procedure
The animals were lightly anesthetized by inhalation of halothane. A 0.1-mL quantity of Freund’s adjuvant (complete fraction of denatured Mycobacterium butyricum suspended in mineral oil; Sigma Chemical Co., St. Louis, MO, USA) was injected into the TMJ at 0700 a.m. The SHAM group received 0.1 mL of saline. The injections were performed with a 30-G needle introduced into the TMJ capsule on both sides. Electrocorticogram recording started immediately upon the animals’ return to their cages. In the treated groups, the sleep recording also took place after the etoricoxib administration by mouth, along with Freund’s adjuvant or saline.
Groups
Animals were randomly distributed among the following 4 groups (N = 8/group): (a) saline (SHAM group) and (b) Freund’s adjuvant (FA group) injected into the temporomandibular joint; (c) saline (SHAM/COX2i) and (d) Freund’s adjuvant (FA/COX2i) injected into the TMJ treated with etoricoxib (COX-2i, 90 mg/kg, by mouth).
The protocol was chosen as in our previous study (Schütz et al., 2003). Etoricoxib administration was done only on the first recording day, so that the selective cyclo-oxygenase-2 inhibitor’s residual effect could be verified on the second day of recording.
Statistical Analysis
Data were analyzed statistically by two-way analysis of variance (ANOVA), with group (4 groups) and time (periods of recording) as main factors, followed Tukey’s test. Values are expressed as the mean ± standard deviation. The level of significance was set at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Still, when Freund’s adjuvant animals were compared with those in the SHAM group, both treated with cyclo-oxygenase-2 inhibitor, we verified that, on the first day of recording, the cyclo-oxygenase-2 inhibitor improved sleep efficiency in the Freund’s adjuvant group. However, this improvement was not enough to return parameters to levels similar to those found in the SHAM/COX2i group (day 1).
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). Cyclo-oxygenase-2 inhibitor administered to the Freund’s adjuvant group diminished the total awake time significantly on 2 days of recording (p = 0.004 and p = 0.001) when compared with Freund’s adjuvant animals not receiving such treatment, but the onset of the effects of this medication took some time, because the value of this parameter was statistically different from the value found in the SHAM/COX2i animals (p = 0.04). The values became statistically similar on the second day of recording.
nREM Sleep
The Freund’s adjuvant-treated rats showed diurnal variation in sleep percentage time, with significantly less nREM [F(3,27) = 23.74; p = 0.00001]. Reduced nREM was observed on both days compared with the SHAM group (p = 0.01 and p = 0.001). The former group treated with cyclo-oxygenase-2 inhibitor had more nREM on both days, but on the first day the compound did not prompt nREM sleep values similar (p = 0.03) to those found in SHAM animals. On the second day, the cyclo-oxygenase-2 inhibitor returned nREM to the SHAM values, differing statistically from that of untreated Freund’s adjuvant-treated animals (p = 0.0061). To determine the isolated effect of this drug, we evaluated SHAM animals treated with cyclo-oxygenase-2 inhibitor and verified that this group did not statistically differ in sleep pattern from the untreated SHAM rats (Fig. 1B).
REM Sleep
The percentage of REM sleep decreased [F(3,27) = 29.83; p = 0.00001] in the Freund’s adjuvant-treated group compared with the SHAM group on both days (p = 0.01 and p = 0.04, respectively). The per-oral administration of cyclo-oxygenase-2 inhibitor to the Freund’s adjuvant-treated animals was followed by a significant increase (p = 0.0001 and p = 0.001) in REM sleep compared with Freund’s adjuvant rats not receiving such treatment (Fig. 1B). When the isolated effect of this compound was evaluated in the REM sleep of SHAM animals, we verified that, on the first day, the percentage of REM increased significantly (p = 0.001) when compared with that in SHAM rats. On the second day, a trend toward increase in this parameter was observed (p = 0.07).
Sleep Latency
The amounts of sleep latency were markedly above those of the SHAM group [F(3,27) = 20.31; p = 0.00001] in animals subjected to Freund’s adjuvant treatment. In the FA/COX2i group, on the first day of recording, sleep latency differed from that in the SHAM group (p = 0.005, Fig. 2A) and from that in the SHAM/COX2i group (p = 0.01). However, on the second day, sleep latency in FA/COX2i-treated animals decreased significantly when compared with that in the SHAM group and with that on the first recording day in the FA/COX2i group (p = 0.03 and p = 0.0001, respectively). Evaluating the values of the SHAM/COX2i treatment, we verified that, on the second day, there was a significant reduction of this parameter when compared with the values found in the SHAM and SHAM/COX2i groups on the first day (p = 0.02 and p = 0.01, respectively).
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Arousals
All the groups that were examined presented a higher statistical significance [F(3,27) = 19.46; p = 0.00001] in the number of arousals when compared with the SHAM group. When evaluating rats with Freund’s adjuvant treated with cyclo-oxygenase-2 inhibitor (35.75 ± 8.94) in relation to untreated animals (44.42 ± 4.19), we verified a statistically significant reduction in the number of arousals (p = 0.02). Still, these values did not reach those of the SHAM (20.20 ± 5.22) group (p = 0.001). On the second day, the significance remained similar to those of day 1 (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The Role of the Cyclo-oxygenase-2 Enzyme in the Sleep-Wake Cycle
The experiments showed that the administration of a cyclo-oxygenase-2 inhibitor was not per se capable of altering sleep efficiency or REM sleep latency in the SHAM group. However, when sleep latency was evaluated, there was a decrease in this parameter on day 2, indicating that SHAM animals treated with cyclo-oxygenase-2 inhibitor that day took less time to fall asleep, but no alteration was verified in the percentage of nREM. Such information contradicts Hayaishi’s (1999) report, which stated that when the biosynthesis of prostaglandin-D2 was inhibited, both nREM and REM sleep reduced almost completely, but reversibly. Notwithstanding, the cyclo-oxygenase-2 inhibitor injected intracerebroventricularly decreased nREM sleep (Yoshida et al., 2003). However, Terao et al.(1998) found that cyclo-oxygenase-2-mediated hyperproduction of prostaglandins, including D2-sensitive, is critically involved in nREM sleep enhancement, but not in REM suppression. Thus, it seems that cyclo-oxygenase plays a role in physiological sleep regulation (Yoshida et al., 2003).
According to our data, REM sleep increased on the first day of recording, and there was a trend for increase on the second day. Therefore, it seems that inhibition of cyclo-oxygenase-2 modulates REM sleep exclusively. The increased REM sleep following cyclo-oxygenase-2 inhibitor might be due to the route of administration, which was oral, and this differed from the methods of administrations adopted in the studies described previously.
With respect to arousals, we observed that when both groups were treated with cyclo-oxygenase-2 inhibitor, there was a statistically significant reduction in awake time and an increased number of arousals, in comparison with the respective controls. Such findings do not necessarily reflect that animals were experiencing a worsening of sleep quality, because such arousals were brief (Fig. 4).
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We may hypothesize that the chemical mediator is the cyclo-oxygenase-2 enzyme-synthesized prostaglandin-D2 within this model. This would be in accordance with several reports in which activation of this enzyme occurs in response to inflammatory processes (O’Banion, 1999; Samad et al., 2001; Simon et al., 2002).
The therapeutic effect of analgesia from such drugs was reported and confirmed in this investigation, in view of the improvements in animals seen in the experimental model of acute inflammation of the TMJ (Cicconetti et al., 2004; Martina et al., 2005).
In all the sleep parameters that were studied, we verified that there was a progressive improvement on the 2 days of recording in Freund’s adjuvant-injected rats, but it was only on the second day that values reached both SHAM groups’ values, suggesting that the drug requires a certain amount of time to reach its maximum effect. The inflammatory condition increased the sleep latency onset, and the treatment of these rats produced values similar to those encountered in the SHAM group. Interestingly, there was a significant reduction of this parameter on day 2 when compared with the values found on day 1 in the SHAM and SHAM/COX2i groups. REM sleep latency returned to values similar to those in SHAM animals. The significant differences between days 1 and 2 reflect the importance of making prolonged recordings in animals that are under an inflammatory protocol and in those still under the influence of agents that modify physiological parameters and that have different effect times. The evaluation of arousals of the groups revealed that the values obtained from all groups were higher than those encountered in the SHAM group, and the treatment of Freund’s adjuvant rats decreased the number of arousals in relation to those in untreated rats. Finally, analysis of our data suggests that acute inflammation of the TMJ may be associated with increased prostaglandin synthesized by the cyclo-oxygenase-2 enzyme, and that its inhibition affects the nociceptive condition.
It seems that prostaglandin-D2 and cyclo-oxygenase-2 enzyme are involved specifically in REM sleep. The results from rats subjected to the acute inflammatory model showed that the decrease in the prostaglandin-D2 concentration through cyclo-oxygenase-2 inhibitor administration led to a sleep pattern restoration for the different parameters studied.
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ACKNOWLEDGMENTS |
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Received September 6, 2005; Last revision December 20, 2006; Accepted January 15, 2007
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REFERENCES |
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| Journal of Dental Research ® | Critical Reviews (1990-2004) |
Differ from first day of SHAM/COX2i group (p < 0.01). N = 8/group.
Differ from first day of the FA/COX2i group (p < 0.05). N = 8/group.
