2.1 Experimental animals

The experiments were performed in adult, male Hannover–Wistar (HW) rats (weight: 150–200 g; Sino-British SIPPR/BK Lab. Animal Ltd., Co., Shanghai, China, except for animals in motor performance tests, which were from Harlan, Horst, Netherlands). All experiments were approved by the institutional ethics committee and all experimental procedures are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data.

2.2 Techniques for intrathecal drug delivery

For intrathecal (i.t.) drug injections a catheter (PE-10) was administered into the lumbar level of the spinal cord under pentobarbital anesthesia (50 mg/kg i.p.) as described in detail elsewhere [17]. Following recovery from anesthesia, the correct placing of the catheter was verified by administering lidocaine (4%, 7–10 μl followed by a 15 μl of saline for flushing) with a 50 μl Hamilton syringe. Only those rats that had no motor impairment before lidocaine injection but had a bilateral paralysis of hind limbs following i.t. administration of lidocaine were studied further. For i.t. administration, the drugs were microinjected with a 50 μl Hamilton microsyringe in a volume of 5 μl followed by a saline flush in a volume of 15 μl.

2.3 REM sleep deprivation procedure

The pedestal-over-water or flower pot technique of REM sleep deprivation was modified from the method described earlier [18]. Briefly, rat was placed on top of platform surrounded by water. The base of the cage was submerged in 4 cm of water. The platform was 7.5cm in diameter and 7.5cm high. REM sleep deprivation was performed for 48 h. The rat was allowed to recover from sleep deprivation for at least one week before next testing, which has proved sufficient for a complete recovery of pain behavior to baseline levels [19].

Under control conditions, the animals were living in similar cages (one animal/cage) as during sleep-deprivation, except that there was no flower pot or water in the cage.

2.4 Behavioral testing

To assess mechanical hypersensitivity, the frequency of the withdrawal response to the application of monofilaments (von Frey hairs) to the hind paw was examined. Monofilaments with forces varying between 1 g and 60 g, or between 0.16 g and 15 g when assessing t-BOOH-induced effects (Stoelting, Wood Dale, IL, U.S.A.), were applied five times at a frequency of approximately of 0.5 Hz. Monofilaments were tested in ascending order of force. A visible lifting of the stimulated hind limb was considered a withdrawal response. The focus was on mechanical sensitivity, since our earlier study indicated that REM sleep deprivation has a more pronounced effect on mechanical than heat sensitivity [11]. Moreover, central mechanisms that were studied in our experiments play an important role in hypersensitivity to mechanical stimulation [20,21].

2.5 Motor performance test

To exclude the possibility that the drug-induced effects on pain behavior were due to motor rather than sensory action, the potential motor impairment by the studied compounds was assessed in a Rotarod test. In the test, the animals were placed on a revolving drum (a constant speed of 26 rounds/min) of a Rotarod device (Ugo Basile, Varese, Italy). The latency until the animal dropped from the drum was determined with a stop watch. Before any drug testing, the rats were habituated to the Rotarod test during two previous days. The maximum observation period was 1min after which the animal that was still on the drum was removed. The Rotarod test was repeated three times at 1 min intervals and the longest latency for each rat in each condition was used in further calculations. Our previous results indicate that Rotarod test using the same testing parameters as in the present study is sensitive to detect motor impairment induced by a low, sedative dose of sodium pentobarbitone [22].

2.6 Drugs

Phenyl-N-tert-butylnitrone (PBN; an antioxidant), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL; an antioxidant), and tert-butyl-hydroperoxide (t-BOOH; a ROS donor) were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Drugs were dissolved in saline. Physiological saline was used as control.

2.7 Course of the study

In a preliminary test, in which the hypersensitivity effect induced by REMSD per se was assessed, pain behavior was assessed 24 h and 48 h following REM sleep deprivation. Drug effects on pain-related behavior were assessed in two experimental conditions: (1) 48 h after REM sleep deprivation (testing started immediately after the end of the sleep deprivation) and (2) control conditions without REM sleep deprivation. The monofilament test was performed prior to, 5, 15, 30 and 60 min after intrathecal administration of each drug dose or vehicle control. With attempts to prevent the hypersensitivity effect induced by i.t. administration of t-BOOH with i.t. pretreatment with TEMPOL, TEMPOL was administered at the dose of 200 μg 20 min before administration of 1.1 μg of t-BOOH. While the experiments were not formally blinded, it should be pointed out that the experimenter assessing pain behavior was not aware of the nature, known pharmacological effects of the studied compounds, nor on the working hypothesis or expected results. Moreover, it should be emphasized that in the studied rat strain (unlike in most mouse strains), the limb withdrawal response evoked by monofilaments is brisk and easily detectable leaving little, if any possibility for interpretations, while the REMSD- and drug-induced effects in this study were quite large (up to 300–400% change in the response frequency).

Drug doses were selected based on literature and our preliminary experiments. The i.t. doses of PBN were 54.4 μg and 108.8 μg, the i.t. doses of TEMPOL were 100μg and 200 μg, and the i.t. dose of t-BOOH was 1.1 μg [15]. In the vehicle session, physiological saline was administered intrathecally at a volume of 5 μl.

Each animal participated in 3–5 testing sessions on pain behavior. If animals were not deprived of sleep, the minimum interval between two drug testing sessions in one animal was 3 days. The minimum interval between two REM sleep deprivation sessions in one animal was 1 week. When testing same animals in different experimental conditions, the order of testing different drugs, drug doses and experimental conditions was counterbalanced to avoid serial effects. Moreover, it was verified that the pre-drug response of the animal was not different from the corresponding pre-drug response in the preceding test session. Since none of the animals was tested in all experimental conditions and the numbers of animals tested in multiple conditions varied between the conditions, results for each animal in each condition were considered to represent independent observations.

Rotarod test for assessment of motor performance was performed in a separate group of control animals. Each animal participated in all Rotarod testing sessions that were performed at 3 day intervals. To avoid serial effects, the order of testing different compounds was varied between the animals.

2.8 Statistical analysis of data

Results are presented as mean ± S.E.M. Statistical analysis was performed using two-way non-repeated analysis of variance (2- w-ANOVA) followed by a t-test with a Bonferroni correction for multiple comparisons. Results of the Rotarod test were evaluated using Kruskall–Wallis test followed by Dunn’s test. For comparisons of the efficacy of drug treatments in control versus sleep-deprived animals, the mean drug-induced change in the cumulative response rate at the time point of the maximum drug effect was calculated in each condition (cumulative response rate at the maximum time point of the drug effect – cumulative response rate prior to administration of the studied drug). The differences in the druginduced changes of response rates between sleep-deprived and control animals were compared with t-test. P < 0.05 was considered to represent a significant difference.

3.1 Pain hypersensitivity induced by REMSD

REMSD induced marked pain hypersensitivity as indicated by increased limb withdrawal rates to mechanical stimulation with monofilaments (Fig. 1). Pain hypersensitivity was increased with duration of REMSD from 24 h to 48 h (F_8,171 = 80.8, P < 0.0001; Fig. 1).

Fig. 1

Mechanical sensitivity before (baseline) and 24 h or 48 h after rapid eye movement sleep deprivation (REMSD). The graph shows the limb withdrawal response rate to repeated monofilament stimulation at different forces. Increase in the response rate indicates hypersensitivity. Error bars represent S.E.M. (n = 8). The horizontal line with three asterisks above it indicates the test force values at which the significance of difference between the response rate in the baseline condition and the corresponding response rate obtained after 48 h of REMSD was P < 0.005 (t-test with a Bonferroni correction for multiple comparisons). For sake of clarity, significance levels of differences between baseline values and the corresponding values after 24 h of REMSD are not shown.

3.2 Attenuation of the REMSD-induced pain hypersensitivity by i.t. administration of PBN

Pain hypersensitivity induced by 48 h of REMSD was attenuated by i.t. treatment with PBN (an antioxidant) in a dose-related fashion (54.4 μg and 108.8 μg; F_2,171 = 121.26, P < 0.0001; Fig. 2A). The maximum antihypersensitivity effect induced by PBN was obtained 30 min after its i.t. administration. By 60 min after its administration, the PBN-induced antihypersensitivity effect was not anymore significant (Fig. 2C). PBN attenuated mechanically induced pain behavior also in control animals (F_2,171 = 8.74, P = 0.0002; Fig. 2B).However, the suppression of pain behavior following 108.8 μg of PBN, as revealed by a decrease in the cumulative response rate by the 30 min time point, was significantly stronger in the REMSD (373 ± 29%; mean ± S.E.M.) than the control group (114 ± 23%; t₁₃ = 6.9, P < 0.0001).

Fig. 2

Influence by i.t. treatment with phenyl-N-tert-butylnitrone (PBN; an antioxidant) on mechanical sensitivity 48 h after REMSD (A and C), or in control conditions (B). Sal represents vehicle control, 54.4 μg and 108.8_g represent PBN doses. Graphs A and B show pain behavior 30 min after i.t. treatment. In C, the response to a monofilament force of 4 g is shown. Error bars represent S.E.M. (n = 7–8). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test with a Bonferroni correction for multiple comparisons; reference: the corresponding value in the Sal group). For the sake of clarity, X-axis is not linear in graphs A and B.

3.3 Attenuation of the REMSD-induced pain hypersensitivity by i.t. administration of TEMPOL

Pain hypersensitivity induced by 48 h of REMSD was attenuated by i.t. treatment with TEMPOL (an antioxidant) in a dose-related fashion (100 μg and 200 μg; F_2,171 = 181.1, P < 0.0001; Fig. 3A). The maximum antihypersensitivity effect induced by TEMPOL was obtained 30 min after its i.t. administration (Fig. 3C). TEMPOL attenuated mechanical pain behavior also in control animals (F_2,171 = 28.0, P < 0.0001; Fig. 3B). However, the suppression of pain behavior following 200 μg of TEMPOL, as revealed by a decrease in the cumulative response rate by the 30 min time point, was significantly stronger in the REMSD (358 ± 36%) than the control group (157 ± 18%; t₁₃ = 4.9, P = 0.0003).

Fig. 3

Influence by i.t. treatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL; an antioxidant) on mechanical sensitivity 48 h after REMSD (A and C), or in control conditions (B). Sal represents vehicle control, 100 μg and 200 μg represent TEMPOL doses. Graphs A and B show pain behavior 30 min after i.t. treatment. In C, the response to amonofilament force of 4 g is shown. Error bars represent S.E.M. (n = 7–8). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test with a Bonferroni correction for multiple comparisons; reference: the corresponding value in the Sal group). For the sake of clarity, X-axis is not linear in graphs A and B.

3.4 Pain hypersensitivity induced by i.t. administration of t-BOOH, and its prevention by TEMPOL in control animals

In control animals, i.t. administration of t-BOOH (a ROS donor) at the dose of 1.1 μmol produced within 5 min strong hypersensitivity to mechanical stimulation (F_1,90 = 513.2, P < 0.0001; Fig. 4). I.t. pretreatment with TEMPOL (200 μg) attenuated pain hypersensitivity induced by t-BOOH (F_1,90 = 607.1, P < 0.0001; Fig. 4). Mechanically induced pain behavior of saline-treated control animals was not significantly different from that in controls that were treated with a combination of TEMPOL and t-BOOH (F_1,90 = 0.26, not shown).

Fig. 4

Mechanical hypersensitivity effect induced by i.t. treatment with tert-butylhydroperoxide (t-BOOH; a ROS donor) and its prevention by i.t. pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL; an antioxidant) in control conditions. t-BOOH was applied at the dose of 1.1 μg. TEMPOL was administered at the dose of 200 μg 20min before t-BOOH. Error bars represent S.E.M. (n = 6). ***P < 0.005 (t-test with a Bonferroni correction for multiple comparisons). For the sake of clarity, X-axis is not linear.

3.5 Lack of motor effects by i.t. administration of PBN or TEMPOL

To exclude the possibility that suppression of pain behavior induced by PBN or TEMPOL was due to motor impairment rather than suppression of sensory signals, a Rotarod test was performed in control animals. Motor behavior was not impaired by i.t. administration of PBN (108.8 μg) or TEMPOL (200 μg) as indicated by the finding that all rats treated with these two compounds as well as saline-treated animals reached the cut-off value in the test (Fig. 5).

Fig. 5

Influence by i.t. treatment with 108.8 μg of phenyl-N-tert-butylnitrone (PBN; an antioxidant) or 200 μg of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL; an antioxidant) on motor performance of control animals in the Rotarod test. Sal indicates saline control. I.t. treatments were given 30 min before the Rotarod test. Each symbol represents one animal. The upper dotted horizontal line (corresponding to a drop latency of 60 s) represents the cut-off value. For comparison and demonstration of the sensitivity of the assay, earlier result in the same assay [22] following intraperitoneal treatment with 20 mg/kg of pentobarbitone (Barb) is shown with open symbols; the horizontal line represents the mean drop latency in the Barb group. ***P < 0.005 (Dunn’s test; reference: the Sal group).

4.1 Antihypersensitivity effect induced by antioxidants after REMSD

In the present study, mechanical hypersensitivity induced by REMSD was attenuated in a dose-related fashion by i.t. administration of two different antioxidant compounds PBN and TEMPOL. Conversely, i.t. administration of a ROS donor, t-BOOH, recapitulated the mechanical hypersensitivity effect in control animals. These findings indicate that oxidative stress in the spinal cord plays a role in the REMSD-induced pain hypersensitivity. Previous studies have suggested that spinal oxidative stress contributes to pain hypersensitivity induced by cutaneous neurogenic inflammation or peripheral nerve injury [13,14,15]. In these peripherally induced central pain hypersensitivity conditions, spinal oxidative stress played a role particularly in secondary hypersensitivity outside of the injured area that is due to central mechanisms [15]. In line with this, the antioxidant-reversible hypersensitivity induced by REMSD is also due to central mechanisms, since REMSD is not associated with peripheral injury, while it has an influence on various sleep-regulating brain structures [2], some of which regulate, through their descending projections, excitability of spinal painrelay neurons [3].

Previous studies indicate that ROS in the spinal cord is increased in animals with peripheral nerve injury [14] or cutaneous neurogenic inflammation [15]. In particular, inactivation of mitochondrial superoxide dismutase 2 and the consequently increased mitochondrial superoxide accumulation in the spinal cord has been associated with central pain hypersensitivity induced by neurogenic inflammation [23]. In analogy, a similar mechanism inducing oxidative stress in the spinal cord may underlie the antioxidantreversible hypersensitivity in the sleep-deprived animals of the present study. In line with this proposal, there is previous evidence indicating that sleep deprivation induces oxidative stress in the central nervous system [16]. While the focus of the present study was on central mechanisms of hypersensitivity, it should be noted that oxidative stress in peripheral tissues plays an important role in a number of pathophysiological conditions involving peripheral injury (e.g., [24,25]).

4.2 REMSD and peripheral neuropathy: common spinal mechanisms of hypersensitivity

Earlier studies suggest that among common spinal mechanisms that contribute to pain hypersensitivity after REMSD and peripheral nerve injury are those involving the NMDA receptor, type I metabotropic glutamate receptor, nitric oxide [11], coupling of glial gap junctions, and Na⁺–K⁺–2Cl⁻ cotransporter 1 [22]. Moreover, the TRPA1 ion channel that is activated by reactive chemicals generated in oxidative stress was recently shown to play a role in mediating spinal facilitation of pain induced by REMSD as well as peripheral nerve injury [26]. The present results add to the evidence indicating that spinal oxidative stress plays a role in hypersensitivity induced by REMSD as shown earlier in peripheral nerve injury or inflammation [13,14,15]. Changes in glutamatergic signaling [11], glial activation [22] and oxidative stress may be co-players in the same pronociceptive cascade as suggested by previous studies [27]. Interestingly, while brainstem–spinal pathways are likely to play a key role in the spinal hypersensitivity effect induced by REMSD, earlier results indicate that descending pathways also contribute to spinal hypersensitivity induced by peripheral nerve injury or inflammation [12]. It remains to be studied whether descending facilitation, through activation of a pronociceptive spinal–brainstem–spinal loop, contributes to spinal oxidative stress following nerve injury or inflammation.

4.3 Vicious circle between sleep loss and pain

Pain and sleep are known to have reciprocal interactions: pain may interrupt sleep and sleep deprivation can facilitate pain perception [4,5,6]. This reciprocal interaction is noteworthy when taking into account present and earlier results indicating that sleep-deprivation as well as some pathophysiological pain conditions, such as nerve injury or inflammation [13,14], induce spinal oxidative stress that contributes to pain hypersensitivity. It may be speculated that chronic pain per se promotes sleep-deprivation that facilitates chronic pain by increasing spinal oxidative stress. Aggravation of chronic pain is expected to increase sleep-deprivation further and thereby promote oxidative stress and pain; this reciprocal interaction may lead to a vicious circle that increases both pain and sleep-deprivation. It may be suggested that a compound reducing spinal oxidative stress could suppress the vicious pronociceptive interaction between chronic pain and sleep-deprivation.