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Influence of Temporomandibular Joint Pain on Sleep Patterns: Role of Nitric OxideDepartment 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; Correspondence: * corresponding author, tschutz{at}psicobio.epm.br
Since nitric oxide is related to nociception and the sleep-wake cycle, this study sought to determine its involvement in the altered sleep pattern in a temporomandibular joint pain model by investigating the effect of the inhibitor of nitric oxide synthase (L-NAME) and that of its precursor (L-arginine). The temporomandibular joints of test animals were injected with Freund’s adjuvant or saline, and their sleep was recorded. The procedure was repeated after the administration of L-NAME and L-arginine. L-NAME increased rapid eye movement (REM) sleep in the control group. The orofacial pain group showed a reduction in total sleep time and an increase in sleep latency compared with the SHAM group. L-NAME increased sleep time, non-rapid eye movement (NREM), and REM sleep and reduced sleep latency in the orofacial pain group. L-arginine did not alter sleep parameters. Thus, L-NAME improved sleep efficiency, whereas L-arginine did not modify it, suggesting the involvement of nitric oxide in painful temporomandibular joint conditions.
Key Words: orofacial pain • sleep • nitric oxide • L-NAME • L-arginine • rat
Nitric oxide is an unconventional neurotransmitter. Its synthesis by nitric oxide synthase occurs through the oxidation of amino acid L-arginine and results in an equimolar production of nitric oxide and L-citrulline (Burlet and Cespuglio, 1997; Monti et al., 1999). Nitric oxide, a free radical gas, is now widely suggested as being an important biological messenger in either the peripheral or the central nervous system (Burlet and Cespuglio, 1997). Nitric oxide has various functions in the central nervous system, which may partly be related to its facilitatory influence on the release of neurotransmitters (Aoki et al., 1995). It is believed that nitric oxide is involved in mediating nociception, food intake, drinking, sexual behavior, neuroendocrine function, circadian rhythms, and modulation of the sleep-wake cycle (Leonard et al., 1995; Dawson and Dawson, 1996; Datta et al., 1997; Burlet et al., 1999; Cudeiro et al., 2000). Several authors have studied the association between nitric oxide and sleep. Kapás and Krueger (1996) found that intracerebroventricular injection of nitric oxide donors (L-arginine) in rats promoted NREM sleep. In contrast, micro-injection of NG-nitro-L-arginine methyl ester (L-NAME, by nitric oxide synthase inhibitor) into the pontine reticular formation suppressed Rapid Eye Movement (REM) and/or NREM sleep in rats. Dugovic et al.(1995) showed that intraperitoneal injection of L-NAME produced no change in the amount of waking, reduced NREM, and concomitantly increased REM in rats. When 7-NI, another nitric oxide synthase inhibitor, was given intraperitoneally to rats, as described by Dzoljic et al.(1996), there was a decrease in NREM and in REM duration, whereas the latencies for NREM and REM occurrence increased. Waking decreased when another nitric oxide synthase inhibitor (L-NMMA) was injection through the same route, whereas NREM and REM were enhanced (Dzoljic and De Vries, 1994). The fraction of nitric oxide present in the cortex is higher during waking than during NREM and REM (Burlet and Cespuglio, 1997). Furthermore, Datta et al.(1997) reported that nitric oxide synthase activity was highest during the behaviorally active period and lowest in the middle of the rest period. It has been reported that inhibition of nitric oxide synthesis either produced a suppression of sleep (Kapás et al., 1994) or, conversely, promoted sleep (Dzoljic and De Vries, 1994) in rats. It is not possible to establish whether nitric oxide is associated with the maintenance of sleep or with the initiation of wakefulness. Nitric oxide may have an important function in pain perception in rats (Dawson and Dawson, 1996). According to Santos et al.(2001), painful temporomandibular joint conditions may be associated with higher levels of nitric oxide in the synovial fluid, but its involvement in these cases has not been fully described. Temporomandibular joint pain conditions represent an important clinical entity. A study performed in our laboratory investigated the mechanisms underlying orofacial pain conditions and the efficacy of pharmacological approaches that may be used to treat them (Schütz et al., 2003). Various substances have been used to elucidate the role of nitric oxide in the brain and in painful conditions. Some compounds act as competitive inhibitors of enzyme nitric oxide synthase (i.e., L-NAME); others act as nitric oxide donors (i.e., L-arginine). This study aimed at elucidating temporomandibular joint issues in relation to its influence on sleep patterns, focusing on nitric oxide mechanisms.
Subjects Male Wistar rats were bred in the animal facility of the Department of Psychobiology, Universidade Federal de São Paulo. The entire study was conducted under conditions of a controlled 12-hour light-dark cycle (lights on at 0700 hrs) and environmental temperature (23 ± 1°C). The animals were maintained in cages with food and water provided ad libitum. The study was conducted in accordance with the ethical guidelines for investigations of experimental pain in conscious animals (Zimmermann, 1983; Andersen et al., 2004), and the number of animals was kept to a minimum. All experimental procedures were approved by the Ethics Committee of the Universidade Federal de São Paulo (CEP No. 703/02).
Groups
Surgical Preparation
Electrocorticogram Recording The following sleep parameters were considered: total sleep time (percentage of total sleep time during the recording time); sleep latency (time lag between the onset of recording and the first sleep period); sleep onset (considered to occur when the animal presented cortical synchronization and muscle hypotonia during 5 consecutive scoring epochs); 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 throughout the recording).
Temporomandibular Joint Injection Procedure
Pharmacological Approaches
Statistical Analysis
Total Sleep Time Total sleep decreased in the orofacial pain group compared with the SHAM group (p < 0.001). When L-NAME was administered to the orofacial pain group, the animals presented higher sleep time on the 2 days of experimental recording than did the untreated animals (p < 0.001). L-arginine administered to the SHAM or orofacial pain group did not modify sleep time (p > 0.05) (Fig. 1  ).
Sleep Latency Animals subjected to orofacial pain took longer to initiate sleep than did SHAM animals (p < 0.001) on the 2 recording days. Latency was reduced in the orofacial pain group treated with L-NAME when compared with the respective untreated rats (p < 0.001). The administration of L-arginine to the SHAM group showed that it did not modify latency parameters. Its administration to the orofacial pain group significantly reduced latency compared with the untreated group (p < 0.001); however, levels did not return to the values found in the SHAM group (p < 0.001, Fig. 2A ).
REM Sleep Latency Orofacial pain rats presented longer latency to REM compared with SHAM animals (p < 0.001). When this experimental pain group was treated with L-NAME, we found a reduction in latency compared with orofacial pain rats (p < 0.001). The latency in the orofacial pain group that received L-arginine was lower compared with that in untreated orofacial pain rats (p < 0.001, Fig. 2B  ).
NREM Sleep
REM Sleep REM was lower in the orofacial pain group compared with the SHAM group (p < 0.001); however, the orofacial pain animals treated with L-NAME presented more REM on the 2 days of sleep recording (p < 0.001) than did the untreated orofacial pain rats (Figs. 3B , 4). The SHAM group treated with L-NAME showed a significant increase in REM compared with the untreated SHAM group (p < 0.001). However, the L-arginine administered to the SHAM group did not differ from SHAM, although a tendency was observed (p < 0.07). Finally, in the orofacial pain/L-arginine group, REM values remained similar to those found in the orofacial pain group (Fig. 3B).
These findings show that orofacial pain conditions led to reduced sleep time, suggesting that this pain model altered sleep patterns in rats, as described previously (Schütz et al., 2003). Nitric oxide seems to be directly linked to REM percentage, and administering L-NAME substantially increased REM in both SHAM and orofacial pain groups.
The Role of Nitric Oxide in Orofacial Pain Animals Upon administering L-arginine, we found no alteration in sleep time and latency, although these parameters tended toward normal, and alterations were not significant compared with the SHAM group. Likewise, L-arginine did not restore NREM and REM. These parameters remained similar to those found in rats belonging to the orofacial pain group. Latency to REM was the only parameter studied in which there was a return to a level similar to that of the SHAM group. However, Kapás and Krueger (1996) reported that NREM increased upon administration of L-arginine. Perhaps the divergent results are due to the means of administration, since the above authors injected the amino acid intracerebroventricularly, while our study used oral administration to mimic the way in which the medication is most often used by people experiencing painful conditions.
The Role of Nitric Oxide in SHAM Animals L-arginine was unable to modify NREM and REM significantly, although there was a non-significant tendency for lower REM in the group studied, which indicates that there is a saturation effect for the nitric oxide synthase enzymes, causing oxidation of L-arginine amino acid and production of nitric oxide and L-citrulline. Our finding of a higher REM following the administration of L-NAME agrees with the results of previous research by Dzoljic and De Vries (1994) and Dugovic et al.(1995), although the nitric oxide synthase inhibitor in the latter studies was administered intraperitoneally. The same authors published further, divergent, results, reporting that intraperitoneal administration of nitric oxide synthase inhibitor reduced NREM and REM sleep (Dzoljic et al., 1996). According to Burlet and Cespuglio (1997) and Datta et al.(1997), nitric oxide synthase enzyme activity was greater in the rats’ active period (the dark period), when nitric oxide release was higher, whereas the light periods showed less nitric oxide release and longer sleep time. When nitric oxide synthase inhibitor is administered, there may be a reduction in normal rat activity, thus facilitating longer REM time. As mentioned earlier, Kapás and Krueger (1996) found that direct administration of L-arginine to the central nervous system led to increased NREM, and that micro-injections of the inhibitor in the pontine reticular formation suppressed NREM and REM. Interestingly, it seems that nitric oxide is involved specifically in the REM of rats injected with saline in the temporomandibular joint. Analysis of the results from rats in our orofacial pain model showed that the decrease in the concentration of free nitric oxide through the administration of L-NAME led to a restoration of sleep patterns in the different parameters studied, whereas the administration of nitric oxide precursor did not modify them, seemingly a saturation effect for this neurotransmitter.
The authors express their sincere thanks to Waldemarks Leite, Tomé dos Anjos, and Ricardo Machado. This work was supported by grants from Associação Fundo de Incentivo à Psicofarmacologia (AFIP) and FAPESP (CEPID #98/14303-3 to S.T. and 01/04329-0 to M.L.A.). Received for publication November 14, 2003. Revision received April 22, 2004. Accepted for publication July 2, 2004.
Journal of Dental Research, Vol. 83, No. 9,
693-697 (2004) This article has been cited by other articles:
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