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Am J Physiol Regul Integr Comp Physiol 285: R99-R109, 2003. First published March 6, 2003; doi:10.1152/ajpregu.00609.2002
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COMPLEX FUNCTIONS OF THE CENTRAL NERVOUS SYSTEM, SLEEP AND LOCOMOTION

A cyclooxygenase-2 inhibitor attenuates spontaneous and TNF-{alpha}-induced non-rapid eye movement sleep in rabbits

Hitoshi Yoshida, Takeshi Kubota, and James M. Krueger

Department of Veterinary, Comparative Anatomy, Pharmacology and Physiology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164-6520

Submitted 3 October 2002 ; accepted in final form 5 March 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sleep is regulated in part by the brain cytokine network, including tumor necrosis factor-{alpha} (TNF-{alpha}). TNF-{alpha} activates the transcription factor nuclear factor-{kappa}B, which in turn promotes transcription of many genes, including cyclooxygenase-2 (COX-2). COX-2 is in the brain and is an enzyme responsible for production of prostaglandin D2. The hypothesis that central COX-2 plays a role in the regulation of spontaneous and TNF-{alpha}-induced sleep was investigated. Three doses (0.5, 5, and 50 µg) of NS-398, a highly selective COX-2 inhibitor, were injected intracerebroventricularly. The highest dose decreased non-rapid eye movement sleep. The intermediate and highest doses decreased electroencephalographic slow-wave activity; the greatest reduction occurred after 50 µg of NS-398 during the first 3-h postinjection period. Rapid eye movement sleep and brain temperature were not altered by any dose of NS-398. Pretreatment of rabbits with 5 or 50 µg of NS-398 blocked the TNF-{alpha}-induced increases in non-rapid eye movement sleep, electroencephalographic slow-wave activity, and brain temperature. These data suggest that COX-2 is involved in the regulation of spontaneous and TNF-{alpha}-induced sleep.

electroencephalogram; cytokine; slow-wave activity

SLEEP IS REGULATED IN PART by the brain cytokine network. For instance, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin (IL)-1{beta} play important roles in the regulation of sleep. Thus administration of exogenous TNF-{alpha} enhances non-rapid eye movement sleep (NREMS) in various species (8, 12, 21, 31, 46, 48, 49, 54, 55). TNF-{alpha} and TNF-{alpha} mRNA are present in many regions in the brain, and their levels vary with the sleep-wake cycle (3, 13). Moreover, inhibition of TNF-{alpha} with an anti-TNF-{alpha} antibody (47), the soluble TNF-binding protein I (52), or a fragment of a soluble TNF receptor (48, 50) reduces spontaneous NREMS. This inhibition also reduces the NREMS enhanced by sleep deprivation (48, 50), increased ambient temperature (51), or microbial challenge (49). Moreover, TNF-{alpha} activates the transcription factor nuclear factor-{kappa}B (NF-{kappa}B), which in turn enhances TNF-{alpha} production (19, 38). Inhibition of NF-{kappa}B decreases sleep (30), and activation of NF-{kappa}B is enhanced in the cerebral cortex by sleep deprivation (6). Furthermore, NF-{kappa}B induces production of many sleep-regulatory substances, including IL-1 (38), IL-2 (17), the adenosine A1 receptor (37), and nitric oxide (NO) synthase (56, 59).

Cyclooxygenase (COX) is a rate-limiting enzyme that catalyzes the biosynthesis of prostanoids, including prostaglandin (PG) D2 (7, 16, 58). PGD2 is posited to be one of the substances involved in sleep regulation (14, 15, 57). COX exists in two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues. In contrast, COX-2 is present in only a few tissues, including the brain, and it is induced by proinflammatory cytokines such as IL-1{beta} (4) and TNF-{alpha} (5). Furthermore, NF-{kappa}B promotes transcription of the COX-2 gene (18, 36). Therefore, COX-2 could be expected to be a constituent of the biochemical network regulating sleep. Previously, it was reported that systemic administration of a COX-2 inhibitor attenuated TNF-{alpha}- and IL-1{beta}-induced sleep in rats (54, 55), although at the dose used, the COX-2 inhibitor failed to alter spontaneous sleep. It is hypothesized, however, that central COX-2 plays a role in the regulation of spontaneous and TNF-{alpha}-induced sleep. In this report, we extend the previous work demonstrating the involvement of COX-2 in TNF-{alpha}-induced sleep to another species, rabbits, and thereby provide evidence for its broader biological relevance. In addition, selective COX-2 inhibitors have been recently used as drugs with an improved risk-to-benefit ratio for treatment of diseases including arthritis, cancer, and Alzheimer's disease in humans. Others have emphasized that further investigations of the physiological effects of selective COX-2 inhibitors are needed (16), and sleep directly affects the quality of life. We now show for the first time that spontaneous sleep is inhibited by the highly selective COX-2 inhibitor NS-398.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Agents

Recombinant human TNF-{alpha} (Peprotech, Rocky Hill, NJ) was dissolved in pyrogen-free isotonic saline (PFS; Abbot Laboratories, North Chicago, IL) at a concentration of 125 ng in a volume of 25 µl and stored at -80°C until used in the experiments. NS-398 (ICN Biomedicals, Aurora, OH) was dissolved in pyrogen-free dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO) and stored at -20°C. It was diluted with PFS and DMSO at concentrations of 0.5, 5, and 50 µg in a volume of 25 µl before the experiments; the vehicle for NS-398 was 40% DMSO.

Animals

The study was approved by the Washington State University Institutional Animal Care and Use Committee. Male New Zealand White Pasteurella-free rabbits weighing 3.5–4.5 kg (Western Oregon Rabbit, Philomath, OR) were implanted with a lateral ventricular guide cannula, stainless steel electroencephalogram (EEG) electrodes, and a brain thermistor during ketamine and xylazine (35 and 5 mg/kg, respectively) anesthesia, as previously described (28, 30). Briefly, the guide cannula was placed above the left lateral ventricle 2.7 mm lateral of the bregma for intracerebroventricular injections. The EEG electrodes were placed over the frontal and parietal cortexes. A calibrated 30-k{Omega} thermistor (model 44008, Omega Engineering, Stamford, CT) was implanted on the dura mater over the parietal cortex to measure brain temperature (Tbr). The leads from the EEG electrodes and the thermistor were routed to a Teflon pedestal (Plastic One, Roanoke, VA). The pedestal, guide cannula, and leads were attached to the skull with resin (P-10 Resin Bonded Ceramic, 3M Dental Products, St. Paul, MN) and cement (Cranioplastic Powder, Plastic One). After ≥2 wk of recovery, the rabbits were placed in experimental chambers (model 352600, Hot Pack, Philadelphia, PA). They were kept on a 12:12-h light-dark cycle (light on at 0600) at 21 ± 1°C ambient temperature. Water and food were available ad libitum throughout the experiment.

Recording and Analysis

A flexible tether connecting the EEG electrodes and the thermistor was led to an electronic swivel (model SL6C, Plastic One). Body movements were detected by ultrasonic detectors (Biomedical Instrumentation, University of Tennessee). The leads from the swivel and the movement detectors were routed to polygraphs (model 7D, Grass Instrument, Quincy, MA) in an adjacent room. The EEG was filtered below 0.1 Hz and above 35 Hz. The amplified signals were digitized at a frequency of 128 Hz for the EEG and 2 Hz for Tbr and motor activity. Tbr data were saved on a computer in 10-s intervals. Because of technical problems, the number of animals from which Tbr data could be obtained was less than the number used for sleep. Tbr results from two rabbits in the group receiving 5 µg of NS-398 + TNF-{alpha} and one from the group receiving 50 µg of NS-398 + TNF-{alpha} were lost because of technical problems. The vigilance states of wakefulness, NREMS, and rapid eye movement sleep (REMS) were visually determined off-line in 10-s epochs by using criteria previously reported (28, 30). Briefly, wakefulness was characterized by fast low-amplitude EEG waves, gradually increasing Tbr, and a high incidence of gross body movements. NREMS was associated with slow high-amplitude EEG waves, slowly decreasing Tbr, and lack of body movements. In contrast, REMS was characterized by fast low-amplitude EEG waves, appearance of theta activity in the EEG, rapidly increasing Tbr at REMS onset, and a lack of body movement. On-line Fourier analysis of the EEG was performed. The average of EEG power density in the delta frequency band (0.5–4.0 Hz) during NREMS, also called EEG slow-wave activity (SWA), was calculated. The average power of EEG SWA throughout the entire 23-h control-recording period was normalized to 100% for each animal. Then all EEG SWA data were expressed as a percentage of that control value. Furthermore, EEG power spectrum analyses during NREMS were performed for the 0.5- and 25-Hz frequency range. The average power in each 1-Hz frequency bandwidth during NREMS of control recordings was normalized to 100%, and then all EEG power data during the treatment-recording period were converted to a percentage of these values. The average amount of time spent in each vigilance state, EEG SWA, and Tbr were calculated for 3-h intervals. In addition, the number of NREMS and REMS episodes and the mean episode lengths were determined by a computer program with the criterion that each episode lasted ≥30 s.

Experimental Protocol

In a preliminary experiment, intracerebroventricular injection of 25 µl of 40% DMSO did not alter sleep or Tbr in rabbits (Fig. 1).



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  		Fig. 1. Effects of intracerebroventricular injection of 40% DMSO on spontaneous non-rapid eye movement sleep (NREMS), rapid eye movement sleep (REMS), electroencephalogram (EEG) slow-wave activity (SWA), and brain temperature (Tbr). {circ}, Pyrogen-free saline (PFS); {bullet}, 40% DMSO (n = 6 each). Horizontal hatched bar, dark phase of the day. Average power of EEG SWA throughout entire 23-h control recording period was normalized to 100% for each animal, and then all EEG SWA data were expressed as percentage of control value.

 

Experiment I: effects of intracerebroventricular administration of NS-398 on spontaneous sleep. Twenty-three rabbits were injected intracerebroventricularly with the vehicle (40% DMSO) on a control day. On the next day, the rabbits received 0.5 (n = 8), 5 (n = 8), or 50 µg (n = 7) of NS-398 during the light period. These injections took place between 0845 and 0915. We chose daylight hours for the injections, because rabbits sleep more during the day than at night, and it seemed likely that it would be easier to observe an inhibition of sleep if the baseline were higher. Furthermore, TNF-{alpha} promotes sleep in rabbits if injected during the daylight hours (46). After injections, EEG, Tbr, and motor activity were recorded for the next 23 h.

Experiment II: effects of intracerebroventricular administration of NS-398 on TNF-{alpha}-induced sleep in rabbits. Of the 16 rabbits used for this experiment, 8 received injections of the vehicles used for NS-398 and TNF-{alpha} on the control day; 40% DMSO (25 µl) was followed 30 min later by 25 µl of PFS. On the next day, four of the rabbits were injected with 25 µl of 40% DMSO and the other four with 5 µg of NS-398 in a volume of 25 µl of 40% DMSO; 30 min later all eight rabbits received 125 ng of TNF-{alpha} in 25 µl of PFS. Seven days later, the rabbits received 40% DMSO or 5 µg of NS-398 (each rabbit received a substance different from that administered previously) followed 30 min later by 125 ng of TNF-{alpha}. The first and second injection times were 0815–0845 and 0845–0915, respectively. After the second injection, EEG, Tbr, and motor activity were recorded for the next 23 h. The same series of injections was repeated with another group of eight rabbits with administration of 50 µg of NS-398.

Statistical Analysis

Two-way ANOVA for repeated measures followed by the Student-Newman-Keuls (SNK) test was used to analyze data concerning time spent in each vigilance state, EEG SWA, and Tbr; 3-h time blocks were used for these analyses. For the sleep-episode data, one-way ANOVA for repeated measures followed by the SNK test was used for experiment II. For power spectrum analysis data, the EEG power density values were summed in four frequency bands [delta (0.5–4.0 Hz), theta (4.5–8.0 Hz), alpha (8.5–12.0 Hz), and beta (12.5–25.0 Hz)], and then one-way ANOVA, which was followed by the SNK test for experiment II, was performed for between-group comparisons. P < 0.05 was considered significantly different.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment I: Effects of Intracerebroventricular Administration of NS-398 on Spontaneous Sleep

The lower two doses of NS-398 failed to affect NREMS. The highest dose of NS-398, however, significantly decreased NREMS (Fig. 2, Tables 1 and 2). This effect was due to a decrease in the time spent in NREMS during the light period (Table 3). Changes in the number or duration of NREMS episodes did not reach significance after the highest dose of NS-398 (Table 4). REMS was not changed for the entire 23-h period (Fig. 2), although after the lowest dose of NS-398, the time spent in REMS during the light period was decreased (Tables 1 and 3). The number or duration of REMS episodes was not significantly altered by any dose of NS-398 (Table 5). EEG SWA was decreased after the higher two doses of NS-398 (Fig. 2, Table 2). The largest decreases in EEG SWA occurred during the initial 3-h period (Fig. 2), and these effects are clearly seen in the EEG power spectrum analysis performed for the first 3 h after injection (Fig. 3). After 50 µg of NS-398, EEG delta activity was significantly decreased (Table 6). All doses of NS-398 failed to affect spontaneous Tbr (Fig. 2).



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  		Fig. 2. Effects of intracerebroventricular injections of NS-398 on spontaneous NREMS, REMS, EEG SWA, and Tbr. {circ}, Control (40% DMSO); {bullet}, NS-398 (0.5, 5, or 50 µg). Treatment effects of SWA after 5 µg of NS-398 and NREMS after 50 µg of NS-398 were significantly different. *P < 0.05, control vs. treatment.

 

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  		Table 1. Effects of a COX-2 inhibitor, NS-398, on spontaneous sleep

 

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  		Table 2. Statistical results of sleep, SWA, and Tbr for 23-h recording period

 

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  		Table 3. Statistical results of time in and episode duration of NREMS or REMS

 

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  		Table 4. Effects of a COX-2 inhibitor, NS-398, on NREMS episodes

 

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  		Table 5. Effects of a COX-2 inhibitor, NS-398, on REMS episodes

 


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  		Fig. 3. EEG power spectrum analysis during NREMS after NS-398 for the initial 3-h postinjection period. Average power values for each animal and for each frequency band during control recordings were normalized to 100%, and then all frequency band densities in the NS-398 treatment groups were converted to relative power data.

 

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  		Table 6. Statistical results of EEG power spectrum analysis during NREMS

 


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  		Table 7. A COX-2 inhibitor, NS-398, attenuates TNF-{alpha}-induced sleep

 

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  		Table 8. Effects of a COX-2 inhibitor, NS-398, on TNF-{alpha}-induced NREMS episodes

 

Experiment II: Effects of Intracerebroventricular Administration of NS-398 on TNF-{alpha}-Induced Sleep in Rabbits

TNF-{alpha} at 125 ng enhanced NREMS (Fig. 4, Table 2). The NREMS-enhancing actions occurred predominantly during the initial light period (Table 7). These TNF-{alpha}-induced NREMS enhancements were significantly inhibited by each dose of NS-398 (Tables 3 and 7). The TNF-{alpha}-induced increases in NREMS during the initial light period were due to the increases in the duration of NREMS episodes (Table 8). Both doses of NS-398 also inhibited the TNF-{alpha}-induced changes in the duration of NREMS episodes (Tables 3 and 8). Although, TNF-{alpha} tended to decrease REMS, REMS was not significantly changed for the entire 23-h period (Fig. 4, Table 7). Furthermore, TNF-{alpha} or TNF-{alpha} + NS-398 failed to affect REMS episode duration or number (Table 9). The only parameter of REMS that was significantly affected by TNF-{alpha} was the time spent in REMS during the dark period in the group of rabbits that also received 5 µg of NS-398 on another day. This TNF-{alpha}-induced decrease in REMS was also inhibited by 5 µg of NS-398 (Tables 3 and 7). EEG SWA had a biphasic pattern after TNF-{alpha} treatment (Fig. 4). It was increased during the initial 9 h after injection and then decreased during the rest of the recording period. In addition, this TNF-{alpha}-induced effect was also significantly inhibited by pretreatment with NS-398 (Table 2). The largest increases in EEG SWA occurred during the initial 3-h period (Fig. 4). EEG power spectrum analysis during NREMS in the initial 3-h postinjection period showed that TNF-{alpha} enhanced EEG power in the low-frequency bands, and this TNF-{alpha}-induced effect was inhibited by NS-398 (Fig. 5, A and B, Table 6). In contrast, TNF-{alpha} decreased EEG SWA after the onset of the dark period (Fig. 4). These TNF-{alpha}-induced effects were also evident in the EEG power spectrum analysis 12–15 h after injection (Fig. 5, C and D). NS-398 at 50 µg tended to inhibit these TNF-{alpha}-induced effects, although these changes did not reach significance [delta: F(2,7) = 2.705, P = 0.102 by ANOVA]. Tbr significantly increased after TNF-{alpha} treatment, and these effects were also inhibited by pretreatment of rabbits with NS-398 (Fig. 4, Table 2).



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  		Fig. 4. Effects of intracerebroventricular injections of NS-398 on tumor necrosis factor-{alpha} (TNF-{alpha})-induced NREMS, REMS, EEG SWA, and Tbr. {circ}, 40% DMSO + PFS (control); {bullet}, 40% DMSO + TNF-{alpha} (125 ng); {triangleup}, NS-398 (5 or 50 µg) + TNF-{alpha} (125 ng). Treatment effect of Tbr in the 50 µg of NS-398 treatment group was significantly different. *P < 0.05, control vs. TNF-{alpha}. {dagger}P < 0.05, TNF-{alpha} vs. other 2 groups. {ddagger}P < 0.05, TNF-{alpha} vs. NS-398 + TNF-{alpha}.

 

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  		Table 9. Effects of a COX-2 inhibitor, NS-398, on TNF-{alpha}-induced REMS episodes

 


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  		Fig. 5. EEG power spectrum analysis during NREMS after TNF-{alpha} or NS-398 + TNF-{alpha} for initial 3-h postinjection period. {circ}, 40% DMSO + TNF-{alpha} (125 ng); {bullet}, NS-398 (5 or 50 µg) + TNF-{alpha} (125 ng).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A major finding in the present study is that the inhibition of COX-2 attenuated spontaneous sleep. Furthermore, TNF-{alpha}-induced NREMS was blocked by the inhibition of COX-2. This latter action occurred even after administration of 5 µg of NS-398, a dose that by itself does not alter spontaneous sleep. PGD2, which is formed by the catalytic action of COX, is one of the well-characterized sleep-regulatory substances in various mammals, including mice (43), rats (33), and monkeys (39). Two previous studies from our laboratory failed to suggest the involvement of PGD2 in the regulation of sleep in rabbits. In most of the studies that clearly showed PGD2 induction of NREMS, PGD2 was administered continuously over prolonged periods. In contrast, we gave single bolus injections of PGD2 (24). In another study, we used acetaminophen, a nonselective COX inhibitor. It did not alter spontaneous NREMS or NREMS induced by muramyl dipeptide in rabbits (27). Nevertheless, other investigators reported that another nonselective COX inhibitor, diclofenac, blocked TNF-{alpha}- or IL-1{beta}-induced NREMS in rats (54, 55). Acetaminophen is a less effective inhibitor of COX-1 or COX-2 than is diclofenac (2, 34). Indeed, in the present study, the highest dose of NS-398, a highly selective COX-2 inhibitor, was needed to attenuate spontaneous NREMS. Collectively, the results reported here suggest that PGD2 also plays an important role in the regulation of NREMS in rabbits.

Downstream events of TNF-{alpha}-induced NREMS (25, 26) likely involve TNF-{alpha} activation of NF-{kappa}B, NF-{kappa}B induction of COX-2 expression, and COX-2 enhancement of PGD2 production. The present results and those from a previous report using rats (55) are consistent with this hypothesis. The concentration of PGD2 in cerebrospinal fluid (CSF) varies with sleep-wake cycle (40, 41) and increases after sleep deprivation (44). COX is the rate-limiting enzyme that catalyzes the production of prostanoids including PGD2, and COX-2 is present in the brain under normal conditions (60). However, lipocalin-type PGD synthase (L-PGDS) is one of the enzymes that controls the production of PGD2 (57). In L-PGDS transgenic or knockout mice, spontaneous sleep patterns are relatively normal (14, 43). It is possible that these mutant mice developmentally compensated for the loss of PGD2 via use of other somnogenic PGs such as PGE2, which would be COX, but not L-PGDS, dependent.

Hayaishi and colleagues (14, 15) suggested that the region responsible for the NREMS-promoting activity of PGD2 includes the surface of the basal forebrain. Thus PGD2 injected into the subarachnoid space ventral to the basal forebrain increases NREMS (33). PGD2 is produced in the arachnoid membrane and secreted into CSF, and PGD2 receptors are on the surface of the basal forebrain (15). Other PGs, including PGE2 and PGF2{alpha}, if injected in this region, also increase NREMS in rats (44). Finally, TNF-{alpha} infused into this subarachnoid space increases NREMS, and this TNF-{alpha}-induced NREMS is blocked by the inhibition of COX-2 in rats (55). However, it is also possible that TNF-{alpha} and NS-398 injected intracerebroventricularly act in the preoptic area (POA). Our previous study indicates that an effective site of TNF-{alpha}-induced NREMS in the brain is the POA. The microinjection of TNF-{alpha} into the POA increases NREMS in rats, whereas the inhibition of TNF-{alpha} by microinjection of a fragment of a soluble TNF receptor into the POA decreases NREMS in rats (31). PGD2 also excites and inhibits sleep- and wake-active POA neurons, respectively (23). Finally, recent immunohistochemistry studies indicate that COX-2 and L-PGDS exist in the POA (11, 35).

The COX-2 inhibitor NS-398 used in this study attenuated EEG SWA during spontaneous NREMS. TNF-{alpha} increases EEG SWA, as do other cytokines (28–30), and pretreatment of rabbits with NS-398 blocked the TNF-{alpha}-induced increases in EEG SWA. EEG SWA is thought to reflect the intensity of NREMS. For instance, it markedly increases during the deep sleep after sleep deprivation (42). It is known that the concentration of PGD2 and other PGs in CSF is increased by sleep deprivation (40, 41, 44), suggesting that PGs are also involved in the depth of sleep. After the initial increase in EEG SWA induced by TNF-{alpha}, EEG SWA declined below the control level. This effect was also blocked by NS-398. Interestingly, EEG SWA after sleep deprivation also shows a biphasic change; the initial increase in EEG SWA during NREMS is followed by a decrease (45). Although the mechanism of the biphasic effect of sleep deprivation on EEG SWA remains unknown, it seems likely to be related to changes in brain levels of sleep-regulatory substances such as TNF-{alpha} and PGs. Another substance that affects EEG SWA is NO. EEG SWA is enhanced and suppressed by the intracerebroventricular injection of NO donors (22) and inhibitors (20), respectively. PGs regulate in part the production of NO. Thus nonselective COX inhibitors or selective COX-2 inhibitors decrease the expression of NO synthase in various tissues including the brain (1, 9, 10, 32). PGD2 and PGE2 mediate some of these actions (9, 10). In addition, NO may also be involved in TNF-{alpha}-induced sleep, because TNF-{alpha} enhances NO production (26). Collectively, it seems likely that NO is a downstream event in TNF-{alpha}- and PG-induced changes in EEG SWA.

In contrast to NREMS, REMS is not greatly altered by NS-398. However, it was previously reported that the decrease in the production of PGD2 leads to reduction of REMS. Thus selenium, a reversible L-PGDS inhibitor, if administered intracerebroventricularly, decreases NREMS and REMS in a dose-dependent manner in rats (53). In that study, however, the lowest dose of selenium, which attenuated NREMS, was not accompanied by an REMS reduction. In the present study, the highest dose of NS-398 was needed to decrease spontaneous NREMS. It is thus possible that inhibition of COX-2 with a much higher dose of NS-398 might decrease REMS. However, because of the solubility limitations, higher doses of NS-398 were not used.

The inhibition of COX-2 clearly blocked the increase in Tbr induced by TNF-{alpha}, and this action is consistent with previous reports in which rats were used. Thus TNF-{alpha}-induced increases in body temperature are reversed by the intraperitoneal injection of NS-398 (5, 55). In contrast, all three doses of NS-398 failed to affect the physiological Tbr, suggesting that, unlike NREMS, PGs play little role in normal Tbr regulation.

The clinical use of selective COX-2 inhibitors is attractive because most of the adverse effects of nonselective COX inhibitors are due to the inhibition of COX-1 (16). However, the physiological effects of the selective COX-2 inhibitors on the central nervous system have not been thoroughly clarified. COX-2 inhibitors administered systemically may pass the blood-brain barrier and affect brain PG production (58). In a previous study, the systemic administration of NS-398 did not affect spontaneous NREMS. However, it seems likely that NS-398 passes the blood-brain barrier to some extent and acts in the brain (54, 55). We show here that the high dose of NS-398 administered centrally inhibits spontaneous sleep. Thus the possibility remains that chronic clinical use of high doses of COX-2 inhibitors may affect human sleep.

In conclusion, COX-2 seems to play a role in the regulation of physiological sleep. The present data also support the hypothesis that COX-2 is involved in the brain cytokine network regulating sleep.


   ACKNOWLEDGMENTS
 
We thank Dr. Akira Terao for information on COX-2 inhibitors and Dr. Jidong Fang and Richard A. Brown for assistance.

This work was supported, in part, by National Institute of Neurological Disorders and Stroke Grant NS-31453.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. M. Krueger, Dept. of VCAPP, PO Box 646520, Washington State University, Pullman, WA 99164-6520 (E-mail: krueger{at}vetmed.wsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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