PBB-72193; No of Pages 9 Pharmacology, Biochemistry and Behavior xxx (2015) xxx–xxx

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Article history: Received 10 February 2015 Received in revised form 28 April 2015 Accepted 4 May 2015 Available online xxxx

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Keywords: Anterior cingulate cortex c-Fos Neuropathic pain Pregabalin Sleep disturbance

Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai, China Department of Pharmacology, Wannan Medical College, Wuhu, China Department of Pharmacology, College of Pharmaceutical Science, Fudan University, Shanghai, China d Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China e Baker-Norton Pharmaceutical Co., Ltd, Kunming, China f State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China

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To evaluate the antinociceptive and hypnotic effects of pregabalin, a neuropathic pain-like model was established in mice using partial sciatic nerve ligation (PSNL), and examined thermal hyperalgesia, mechanical allodynia, electroencephalography, rota-rod testing, and c-Fos expression in the anterior cingulate cortex. Gabapentin was used as a reference drug in the study. Pregabalin administered i.g. at 12.5 and 25 mg/kg prolonged the duration of thermal latencies by 1.4- and 1.6-fold and increased the mechanical threshold by 2.2- and 3.1-fold 3 h after administration, respectively, but did not affect motor coordination in PSNL mice, compared with vehicle control. Pregabalin (12.5 and 25 mg/kg) given at 6:30 increased the amount of non-rapid eye movement sleep in a 4-h period by 1.3- and 1.4-fold, respectively, in PSNL mice. However, pregabalin (25 mg/kg) given at 20:30 did not alter the sleep pattern in normal mice. Immunohistochemical study showed that PSNL increased c-Fos expression in the neurons of anterior cingulate cortex by 2.1-fold, which could be reversed by pregabalin. These results indicate that pregabalin is an effective treatment for both neuropathic pain and sleep disturbance in PSNL mice. © 2015 Published by Elsevier Inc.

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Neuropathic pain, which is caused by lesions or diseases of the sensory transmission pathways in the peripheral or central nervous system (Treede et al., 2008), seriously affects human quality of life. The efficacy of therapeutic drugs is only 50% (Gilron and Dickenson, 2014), making it the most difficult type of pain to manage in clinical settings. In addition to pain, sensory abnormalities in specific parts of the body, and loss of normal sensory innervation (Jensen and Finnerup, 2009), more than 70% of sufferers of neuropathic pain and other chronic pain conditions complain of significant sleep disturbances, such as increased sleep

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1. Introduction

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Tian-Xiao Wang a,b, Dou Yin c, Wei Guo a, Yuan-Yuan Liu a, Ya-Dong Li b, Wei-Min Qu a,d, Wu-Jian Han e, Zong-Yuan Hong b,⁎, Zhi-Li Huang a,d,f,⁎⁎

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Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice

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Abbreviations: ACC, anterior cingulate cortex; EEG, electroencephalogram; EMG, electromyogram; NREM, non-rapid eye movement; PSNL, partial sciatic nerve ligation; REM, rapid eye movement. ⁎ Correspondence to: Z.-Y. Hong, Laboratory of Quantitative Pharmacology, Department of Pharmacology, Wannan Medical College, Wuhu, 241002, P. R. China. Tel.: +86 553 3932003; fax: +86 553 3932671. ⁎⁎ Correspondence to: Z.-L. Huang, Department of Pharmacology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, P. R. China. Tel.: + 86 21 54237043; fax: +86 21 54237103. E-mail addresses: [email protected] (Z.-Y. Hong), [email protected] (Z.-L. Huang).

latency, reduced slow-wave sleep, and shifts in sleep stage (Morin et al., 1998; Smith and Haythornthwaite, 2004). Evidence mounts that restricting sleep can cause a range of neurobehavioral deficits, such as lapses of attention, slowed working memory, reduced cognitive throughput (Walker, 2009). However, clinical drugs currently used to treat neuropathic pain and sleep disorders are relatively inadequate. Therefore, developing new effective sleep-promoting and analgesic agents remains a scientific and medical challenge. Pregabalin, (S)-3-(aminomethyl)-5-methylhexanoic acid, rac-CI1008, S(+)-3-isobutyl GABA, has been shown to be such a candidate (Stahl et al., 2013). Previous studies have shown pregabalin to be effective for treatment of neuropathic pain associated with post-herpetic neuralgia, diabetic peripheral neuropathy, fibromyalgia in double-blind clinical studies(Dworkin et al., 2003; Gore et al., 2007). Pregabalin has been shown to positively affect subjective ratings of pain-related sleep interference in clinical trials. For example, pregabalin (600 mg/d) increased total duration and efficiency of sleep efficiency over placebo values in patients with diabetic peripheral neuropathic pain (Boyle et al., 2012). Improvement of slow-wave sleep has also been observed in patients with fibromyalgia (Russell et al., 2009). An ideal agent should effectively improve both chronic pain and sleep but fails to affect the patient's daily life. However, the mechanism by which and degree to which

http://dx.doi.org/10.1016/j.pbb.2015.05.007 0091-3057/© 2015 Published by Elsevier Inc.

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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2. Material and methods

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2.1. Animals

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Male SPF C57BL/6j mice, weighing 20–24 g (10–12 weeks old), were obtained from the Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). These animals were housed individually under ambient temperature of 22 ± 0.5 °C with a relative humidity of 60 ± 2% and an automatically controlled 12 h light/12 h dark cycle (lights on at 07:00, illumination intensity ≈ 100 lux), with free access to food and water. Experimental protocols were approved by the Medical Experimental Animal Administrative Committee of Fudan University. Every effort was made to minimize the number of animals used and minimize their pain and discomfort.

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Pregabalin and gabapentin were obtained from the Chongqing Sai Wei Pharmaceutical Co. Ltd. (Chongqing, China). Rabbit polyclonal anti-c-Fos antibody was purchased from Abcam (Cambridge, MA, U.S.), Biotinylated donkey anti-rabbit IgG and avidin–biotin–peroxidase from Vector Laboratories (Burlingame, CA, U.S.), and 3, 3-diaminobenzidine-tetra-hydrochloride (DAB) from Sigma-Aldrich (St. Louis, MO, U.S.). Pregabalin was dissolved in sterile saline before use.

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2.3. Neuropathic pain model

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The animals were anesthetized with 5% chloral hydrate (360 mg/kg, i.p.). A partial nerve injury model was produced by tying a tight ligature with 7–0 silk suture around approximately 1/3 to 1/2 the diameter of the sciatic nerve on the right side (ipsilateral side) as described previously (Liu et al., 2014; Narita et al., 2011; Zhao et al., 2012). At least 7 d was allowed for recovery from surgery.

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2.4. Measurement of thermal hyperalgesia and mechanical allodynia

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Thermal hyperalgesia was assessed with a plantar test apparatus (IITC Inc./Life Science Instruments, Woodland Hills, CA, U.S.) as previously reported, by measuring hind paw withdrawal latency in response to radiant heat (Hargreaves et al., 1988). The intensity of the thermal stimulus was adjusted to produce an average baseline paw-withdrawal latency of approximately 9–11 s in naive mice. Before the behavioral responses to the thermal stimulus were tested, mice were habituated for at least 60 min in compartment enclosures on a glass surface. A mobile radiant heat source was focused on the right hind paw and the paw withdrawal latency was defined as the time taken by the mouse to remove its hind paw from the heat source (maximum of 20 s to avoid tissue damage). The paw-withdrawal latency was determined as the average of five measurements of right hind paw, taken at 5 min intervals to prevent thermal sensitization and behavioral disturbances. Mechanical allodynia was quantified by measuring hind paw withdrawal response to von Frey filament stimulation (Chaplan et al., 1994). Briefly, animals were placed in a clear test chamber with a

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The test was performed according to the method described by Kuribara et al. (1977). The rota-rod apparatus (Med Associates, Georgia, VT, U.S.) consisted of a bar with a diameter of 3 cm, subdivided into five compartments. The time taken to fall off the rota-rod was recorded as the latency. Before drug administration, the mice were trained daily for 3 d, and the rod was rotated at a constant speed of 16 revolutions per minute. On the day of the test, only the mice that were able to stay balanced on the rotating rod for 180 s (cut-off time) were selected for testing.

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Under chloral hydrate anesthesia (360 mg/kg, i.p.), PSNL or shamoperated mice were implanted simultaneously with electrodes for polysomnographic recordings using electroencephalography (EEG) and electromyography (EMG). The implant consisted of two stainless-steel screws (1 mm diameter) inserted through the skull of the cortex (anteroposterior, +1.0 mm; left–right, −1.5 mm from bregma or lambda) according to the atlas of Franklin and Paxinos and served as EEG electrodes. Two insulated stainless-steel, Teflon-coated wires were bilaterally placed into both trapezius muscles. These served as EMG electrodes. All electrodes were attached to a microconnector and fixed to the skull with dental cement (Qu et al., 2008; Wang et al., 2015). EEG and EMG recordings were performed by means of a slip ring, designed so that the behavioral movement of the mice would not be restricted (Wang et al., 2015; Xu et al., 2014a). After a 10 d recovery period, the mice were housed individually in transparent barrels and habituated to the recording cable for 3–4 d before polygraphic recording. Sleep–wakefulness states were monitored for a period of 48 h, which comprised baseline and experimental days. Baseline recordings were taken in each animal for 24 h, beginning at 07:00. This served as the control for each individual animal. Cortical EEG and EMG signals were amplified and filtered (EEG, 0.5–30 Hz; EMG, 20–200 Hz) and then digitized at a sampling rate of 128 Hz and recorded by using SleepSign (Kissei Comtec, Nagano, Japan) as described previously (Chen et al., 2013; Huang et al., 2006). When complete, polygraphic recordings were automatically scored off-line by 4 s epochs as wakefulness, rapid eye movement (REM), and non-REM (NREM) sleep by SleepSign according to standard criteria (Huang et al., 2006). As a final step, defined sleep–wake stages were examined visually and corrected if necessary (Xu et al., 2014b).

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To study the antinociceptive effect of pregabalin in PSNL mice, the thermal hyperalgesia and mechanical allodynia were tested before (0) and at 1, 3, 6, and 9 h after pregabalin was administered intragastrically (i.g.). To assess the dose-dependent relationship of the anti-hyperalgesic and anti-allodynic effects of pregabalin under neuropathic-pain-like conditions, increasing doses of pregabalin (6.25, 12.5, or 25 mg/kg) were administered 1 h before assessment of the thermal hyperalgesia and mechanical allodynia.

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framed metal mesh floor and allowed to habituate for 60 min prior to testing. Von Frey monofilaments (bending force range from 0.1 to 8 g) (North Coast Medical, Inc., San Jose, CA, U.S.) were applied and thresholds were measured using the up–down paradigm. At first, a 2 g filament was used. A less strong filament was used in for the next round if the animal responded and a stronger one was used if it did not. Testing consisted of five more stimuli after the first occurrence of a change in response. The threshold of response was calculated using the up–down Excel program provided by Basbaum's laboratory (UCSF, San Francisco, CA, U.S.). Clear paw withdrawal, shaking, and licking were all considered nociceptive-like responses (Dixon, 1980).

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pregabalin mitigates sleep disturbances associated with neuropathic pain and whether therapeutic doses of pregabalin influence the motor coordination and drowsiness in daytime is not yet fully understood. In the present study, the effects of pregabalin on sleep disturbance and neuropathic pain were evaluated in a mouse sciatic nerve ligation (PSNL) model. In addition, recent studies have suggested that the anterior cingulate cortex (ACC) plays an important role in sleep disorders caused by pain (Narita et al., 2011; Plante et al., 2012). It is here speculated that ACC may be an important target of pregabalin. For this reason, changes in c-Fos (a well-known indicator of rapid and transient neuronal activity) expression in neurons of the ACC after pregabalin administration were observed.

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Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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2.9. Statistical analysis

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All results were expressed as means ± SEM. The hourly amounts of each stage for sleep–wake profiles in PSL mice treated with vehicle or pregabalin were compared using repeated measures ANOVA followed by Fisher's probable least-squares difference (PLSD) test. Histograms of the amounts of sleep and wakefulness, the number of transitions between sleep and wakefulness, and the number and duration of bouts of sleep and wakefulness in pregabalin and vehicle groups were built using the non-paired, two-tailed Student's t test. In multifactorial ANOVA, for example, the treatment (vehicle or drug administration) was evaluated as between-group factors.

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3.1. Pregabalin decreased tactile allodynia and thermal hyperalgesia caused 256 by the PSNL 257 Partial sciatic nerve ligation caused a remarkable decrease in the mechanical threshold and thermal latency (Fig. 1A and B) in PSNL mice relative to the sham group. Mechanical threshold and thermal latency had decreased 58% (F3,28 = 8.8, P b 0.01) and 41% (F3,28 = 19.7, P b 0.01), respectively on the seventh day after surgery. These results indicated that PSNL resulted in tactile allodynia and thermal hyperalgesia in mice. Previous studies found that this persistent painful state lasted for at least 35 d after PSNL (Liu et al., 2014). To investigate effects of pregabalin on tactile allodynia and thermal hyperalgesia, mechanical threshold and thermal latency were measured 0, 1, 3, 6, and 9 h after administration in PSNL mice. The time-course curve (Fig. 1A, B) showed that intragastric administration of pregabalin at 25 mg/kg significantly increased thermal latency (surgery × treatment interaction, F 1,32 = 23.3, P b0.001; surgery × treatment × hours interaction, F4,120 = 3.6, P b 0.01; Fig. 1C) and prolonged the mechanical threshold for 6 h (surgery × treatment interaction, F1,32 = 15.7, P b0.001; surgery × treatment × hours interaction, F4,120 = 2.9, P b 0.05; Fig. 1D) after administration of pregabalin to PSL mice. The dose-dependency of the antinociceptive effects of pregabalin is summarized in Fig. 1C, D. When 6.25 mg/kg vehicle solution or pregabalin was administered to PSNL mice, no difference from the control group was found in the mechanical threshold or thermal latency. Pregabalin given at 12.5 and 25 mg/kg significantly increased the thermal latency by 1.4- (P b 0.01) and 1.6-fold (P b 0.01) (Fig. 1C) and mechanical threshold by 2.2- and 3.1-fold (Fig. 1D), respectively, in comparison with the vehicle injection in PSNL mice. The antinociceptive effects of pregabalin at 25 mg/kg were no different from the effects of gabapentin at 100 mg/kg. Pregabalin did not change the mechanical threshold or thermal latency in the sham group. Results indicated that pregabalin exerts antiallodynic and antihyperalgesic effects in a dose-dependent manner in PSNL mice.

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To determine whether pregabalin affected motor coordination and equilibrium, a rota-rod test was performed in sham and PSNL mice. As shown in Fig. 2, when the vehicle solution was injected into sham or PSNL mice, no difference was found between rota-rod staying time and the pre-drug. The dose of pregabalin (25 mg/kg), which elicited significant antinociception, did not cause the rota-rod staying time to differ from that of vehicle control. However, 50 mg/kg pregabalin decreased the rota-rod staying time by 32% that of vehicle control (P b 0.05) in PSNL mice 1 h after administration. Gabapentin at 100 mg/kg decreased the rota-rod staying time after administration 1 and 3 h by 42% (P b 0.01) and 28% (P b 0.05) that of vehicle control, respectively, in PSNL mice. These results indicated that pregabalin given at doses of 25 mg/kg or less did not affect motor coordination and equilibrium in PSNL mice.

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Four groups of mice were used: PSNL + vehicle; PSNL + 25 mg/kg pregabalin; sham + vehicle; and sham + 25 mg/kg pregabalin. Each group was given either vehicle or pregabalin intragastrically at 06:30. After 120 min, mechanical allodynia was measured in all animals. All animals were then were sacrificed for immunohistochemical experiments as described previously (Chen et al., 2011). The mice were anesthetized with 10% chloral hydrate and perfused with saline solution followed by ice-cold 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.0) via the heart. The brains were immediately removed and immersed in 4 % PFA in 0.1 M PB (pH 7.4) for 2–4 h. The brains were then transferred to 20% sucrose in PB and kept in the solution until they sank to the bottom. Thereafter, frozen sections were cut at 30 μm in coronal planes by using freezing microtome (Jung Histocut, model820-II, Leica, Nussloch, Germany). The sections were stored in a cryoprotectant solution at − 20 °C for histological analysis. The preparations were washed in PBS for 3–5 min and then preincubated in antiserum solution 1 (10% normal bovine serum albumin (BSA), 0.2% Triton X-100, and 0.4% sodium azide in 0.01 mol/l PBS at pH 7.2) for 30 min followed by incubation with C-FOS antibody (rabbit polyclonal, ABE-457, Millipore, Boston, MA, U.S.). The antibody was diluted 1:10 000 in antiserum solution 2 (1% normal BSA, 0.2% Triton X-100, and 0.4% sodium azide in 0.01 M PBS at pH 7.2) at room temperature overnight. On the second day, the sections were incubated with a 1:1000 dilution of biotinylated goat anti-rabbit secondary antibodies for 1 h followed by a 1:800 dilution of avidin–biotin–peroxidase for 1 h at 37 °C. The peroxidase reaction was visualized with 0.05% DAB in 0.1 M phosphate buffer and 0.01% H2O2. Sections were mounted, dehydrated, and cover slipped. As controls, adjacent sections were incubated without the primary antibody to confirm that no non-specific staining had occurred. The sections were examined under bright-field illumination using a Leica DMLB2 microscope (Leica Microsystems, Wetzlar, Germany). Images were captured with a Cool SNAP-Proof digital camera (SPOT RTKE Diagnostic instruments, Sterling Heights, MI, U.S.).

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Dunnett's test was used for post hoc comparisons. One-way ANOVA was used to assess the number of c-Fos immunoreactive neurons, followed by PLSD test. In all cases, P values under 0.05 were considered statistically significant.

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To evaluate possible non-specific muscle-relaxant or sedative effects of pregabalin, the average time for which mice remained on the rotarod was measured at 1, 3, 6, and 9 h after pregabalin at 25 mg/kg, gabapentin 100 mg/kg (reference drug) or vehicle was intragastrically (i.g.) administered. To study the dose-dependent relationship of the muscle-relaxant effects of pregabalin under neuropathic pain-like conditions, increasing doses of pregabalin (12.5, 25, and 50 mg/kg) were administered 1 h before assessment of the rota-rod test. The dosage of pregabalin that elicited significant antinociception and sleep promotion was selected. To study the effect of pregabalin on sleep disturbance under neuropathic-pain-like conditions, pregabalin was administered intragastrically (i.g.) at 06:30 on the day of the experiment at a dose of 6.25, 12.5, or 25 mg/kg in PSNL mice. Pregabalin was administered intragastrically (12.5, 25, and 50 mg/kg) at 20:30 to estimate possible drowsiness effects of pregabalin on the active stage of normal and PSNL mice.

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3.3. Hypnotic effect of pregabalin given during the light phase under a 304 neuropathic pain-like state 305 To determine whether pregabalin exerted a hypnotic effect in the neuropathic pain-like state during the light phase, pregabalin at a dose of 6.25, 12.5, or 25 mg/kg was administered at 06:30 on the day of the experiment in PSNL mice. Typical examples of polygraphic recording

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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a decrease in wakefulness during the first 4 h after the administration of pregabalin. No subsequent disruption of sleep architecture was observed during the subsequent period. The total amounts of time spent in NREM, REM sleep, and wakefulness during the 4 h following treatment with pregabalin or gabapentin in PSNL mice are summarized in Fig. 3C. When pregabalin at 6.25 mg/kg was injected into PSNL mice, no difference from vehicle control was observed with respect to the sleep–wake cycle. Compared with the vehicle control, pretreatment of PSNL mice with pregabalin at 12.5 and 25 mg/kg significantly increased NREM sleep by 1.3- (P b 0.01) and 1.4- (P b 0.01) fold and increased REM sleep by 1.7- (P b 0.05) and 1.7- (P b 0.01) fold and decreased wakefulness by 28% (P b 0.01) and 38% (P b 0.01), respectively. Treatment with 100 mg/kg gabapentin

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and corresponding hypnograms from a PSNL mouse given vehicle or pregabalin at a dose of 25 mg/kg are shown in Fig. 3A. The pregabalin-treated mice spent more time in NREM sleep than the control mouse. As shown in Fig. 3B, Time course changes revealed that pregabalin at 25 mg/kg significantly increased NREM sleep (F1,42 = 52, P b 0.05) and decreased wakefulness (F1,42 = 73, P b 0.05) in PSNL mice, compared with vehicle-treated mice. Pregabalin increased the hourly NREM sleep time by 3.0- (P b 0.01), 1.5- (P N 0.05), 1.3- (P N 0.05), and 1.2(P b 0.05) fold relative to vehicle control during the first, second, third, and fourth hours after administration, respectively. The duration of REM sleep was increased 5.7- and 3-fold during the first and second hour. The enhancement of NREM and REM sleep was concomitant with

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Fig. 1. Effects of pregabalin on thermal latency and mechanical threshold. Time course of pregabalin (25 mg/kg) treatment for (A) thermal hyperalgesia and (B) and mechanical allodynia in sham and PSNL mice. Dose–response effect of pregabalin on (C) thermal latency and (D) mechanical threshold in PSNL mice. Mice were treated with vehicle or pregabalin 3 h before starting threshold evaluations. Gabapentin was used as the reference drug. Values are means ± SEM (n = 7). *P b 0.05, **P b 0.01 indicate significant differences from vehicle value in PSNL group, ##P b 0.01 indicates significant differences among doses of pregabalin, as assessed by multifactor ANOVA followed by (A, B) Dunnett's test and (C, D) one-way ANOVA followed by the PLSD test.

Fig. 2. Effect of pregabalin on the motor performance of mice in the rota-rod test. (A) Time course of pregabalin and gabapentin effect for rota-rod staying time in PSNL mice. (B) Dose–response effect of pregabalin on rota-rod staying time in PSNL mice. Mice were treated with vehicle or pregabalin 1 h before the beginning of the rota-rod test. Gabapentin was used as the reference drug. Values are the means ± SEM (n = 7–8). *P b 0.05 indicates significant differences form vehicle value of the PSNL group as assessed by one-way ANOVA followed by PLSD test.

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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Fig. 3. Sleep–wake profiles produced by administration of pregabalin in PSNL mice. (A) Typical examples of polygraphic recording and corresponding hypnograms in PSNL mice treated with vehicle (up panel) or pregabalin (low panel) at a dose of 25 mg/kg. (B) Changes in NREM, REM, sleep, and wakefulness over time in PSNL mice treated with pregabalin. Each circle represents the hourly mean amount of each stage. Open and closed circles represent profiles of vehicle and pregabalin treatments, respectively. The horizontal open and filled bars on the X-axes indicate the 12 h light and 12 h dark periods, respectively. Values are means ± SEM (n = 6–8). *P b 0.05 or **P b 0.01 indicates significant differences compared to vehicle control group as assessed by repeated measures ANOVA followed by the PLSD test. (C) Dose–response effect on total time spent in NREM sleep, REM sleep, and wakefulness for 4 h after administration of vehicle and pregabalin in PSNL mice. Open and filled bars show the profiles of vehicle and pregabalin or gabapentin treatments, respectively. Values are the mean ± SEM (n = 6–8). *P b 0.05 or **P b 0.01 indicates significant differences from vehicle value in PSNL group, ##P b 0.01 indicates significant differences among dose of pregabain as assessed by one-way ANOVA followed by the PLSD test.

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increased the total amount of NREM sleep and REM sleep by 1.5(P b 0.01) and 2.3- (P b 0.01) fold that of vehicle control, respectively, and decreased wakefulness by 44% (P b 0.01) during the 4 h period.

Pregabalin at 25 mg/kg did not change the amounts of NREM, REM 339 sleep, or wakefulness in the sham mice (data not shown). These results 340 indicate that pregabalin increased NREM sleep in PSNL mice. 341

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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To determine the effects of pregabalin on sleep–wake profiles at night, pregabalin was administered intragastrically into normal mice at 20:30 at a dose of 12.5, 25, or 50 mg/kg. From 21:00 to 00:00, the mice treated with pregabalin at 50 mg/kg spent more time in sleep than control mice. Pregabalin given at 50 mg/kg increased the amount of NREM sleep during the second and fourth by 1.8- (P b 0.05) and 1.7- (P b 0.05) fold, respectively, relative to vehicle control, as shown in Fig. 5A. This enhancement of NREM and REM sleep was concomitant with a decrease in wakefulness during the second and fourth hours after the administration of pregabalin. Pregabalin at 12.5 and 25 mg/kg did not affect sleep profiles (data has not shown). The other dosages did not affect the amount of sleep. Fig. 5B summarizes the total time mice spent in NREM and REM sleep and wakefulness in the first 4 h after pretreatment with vehicle or different doses of pregabalin in normal and PSNL mice. When pregabalin at 12.5 or 25 mg/kg was given into normal and PSNL mice, no difference was found between the sleep–wake cycle and vehicle

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3.5. Drowsiness effects of pregabalin on the dark phase in normal mice.

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To better understand the sleep–wake profile caused by pregabalin, the distribution of bouts of NREM and REM sleep was determined as a function of duration of the bout or episode (Fig. 4A). Pregabalin at 25 mg/kg increased the number of bouts of NREM sleep that had durations of 256–512 s. Similarly, pregabalin increased the number of REM sleep bouts with durations of 32–64 s. As shown in Fig. 4B, pregabalin changed the total number of episodes of wakefulness, but there was no difference in the number of episodes of NREM and REM sleep. Pregabalin (25 mg/kg) decreased the number of wake bouts by 43%. However, the mean duration of wakefulness, NREM, and REM showed no change during the 4 h immediately following administration of pregabalin at 25 mg/kg (Fig. 4C). Pregabalin at 25 mg/kg decreased the number of state transitions from NREM sleep to wakefulness and from NREM sleep to REM sleep (Fig. 4D). Neither a change in the number of transitions from wakefulness to NREM sleep nor in that from REM to wakefulness was observed. EEG power spectra were then gathered during NREM sleep in PSNL mice. The power of each 0.5 Hz bin was first averaged across the sleep stages individually and then normalized by calculating the relative duration of each bin from the total power (0–24.5 Hz) for each individual

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animal. As shown in Fig. 4E, there were no significant differences in EEG power density of NREM sleep between the pregabalin treatment and the vehicle control. These results suggest that pregabalin increased the duration of NREM sleep but did not decrease the depth of sleep.

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3.4. Changes in the number of bouts, mean duration of episodes, stage transition, and power density of NREM sleep induced by pregabalin in PSNL mice

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Fig. 4. Characteristics of sleep–wake episodes and transitions during the 4 h after administration of pregabalin at 25 mg/kg. (A) Number of NREM sleep and REM sleep bouts, (B) number of total episode numbers, (C) mean durations, (D) stage transitions, (E) EEG power density of NREM sleep. Open and filled bars show the profiles of vehicle and pregabalin treatments, respectively. Values are the means ± SEM (n = 6–8). *P b 0.05 indicates significant differences from vehicle value in the PSNL group, assessed by two-tailed unpaired Student's t test.

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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3.6. Effects of pregabalin on c-Fos expression in the ACC of PSNL and sham mice

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control. Pregabalin at 50 mg/kg significantly increased NREM sleep and REM sleep by 1.7- (P b 0.01) and 1.5- (P b 0.01) fold in normal mice, respectively.

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Fig. 5. (A) Sleep-stage distributions in normal mice and PSNL mice by i.g. administration of pregabalin. (B) Time course changes in NREM sleep, REM sleep, and wakefulness in normal mice treated with pregabalin 50 mg/kg at 20:30. Each circle represents the hourly mean amount of each stage. Open and filled circles indicate profiles of vehicle and pregabalin treatments, respectively. Dose–response effect on total time spent in NREM sleep, REM sleep, and wakefulness 4 h after administration of pregabalin in normal mice and PSNL mice. Open and filled bars show the profiles of vehicle and pregabalin and gabapentin treatments, respectively. Values are the means ± SEM (n = 6–7). *P b 0.05 or **P b 0.01 indicates significant differences from vehicle value in the normal group, assessed using one-way ANOVA followed by PLSD test.

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In the present study, PSNL caused marked thermal hyperalgesia and tactile allodynia in mice, and PSNL mice exhibited sleep disturbance during the light phase recorded from the 10th day after surgery. This

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To investigate the effects of pregabalin on the ACC in PSNL and sham mice, the number of c-Fos positive neurons in the ACC was counted. The structure has been reported to be associated with pain and sleep regulation (Brown et al., 2012; Xu et al., 2008; Zhao et al., 2006). Fig. 6 (A–D, a–d) shows representative photomicrographs of c-Fos expression in the ACC of vehicle and pregabalin 25 mg/kg treated PSNL or sham mice. As shown in Fig. 6E, compared with the control group, administration of pregabalin (25 mg/kg) significantly increased mechanical threshold in PSNL group but had no effect on the sham mice. These results certified that pregabalin exerted analgesic effects in the PSNL group in the current experiment. Analysis of the number of c-Fos immunoreactive nuclei showed that PSNL increased expression of c-Fos in the ACC 2.1-fold relative to the sham group (Fig. 6F), and pregabalin at 25 mg/kg decreased the number of c-Fos-immunoreactive nuclei in the ACC of PSNL mice by 64% (F3,20 = 13.0, P b 0.01), but this effect did not appear in the sham mice.

result is consistent with previous reports that chronic pain induced by PSNL caused a significant increase in wakefulness and a decrease in NREM sleep at 7 d, which lasted for at least 28 d after PSNL (Narita et al., 2011; Takemura et al., 2011). A recent study reported that the PSNL caused sleep discontinuity and increased NREM sleep fragmentation during the light phase (Liu et al., 2014). This is consistent with reports that chronic pain disturbs sleep continuity and efficiency in humans (Argoff, 2007). These results suggest that the clinical symptoms of neuropathic pain in patients can be simulated in the PSNL mice. In the present study, 25 mg/kg pregabalin exerted antinociceptive effects in PSNL mice but did not change mechanical threshold or thermal latency in sham mice. This suggested that the analgesic effects of pregabalin are expressed under pathological conditions. In addition, pregabalin at a dose of 25 mg/kg did not alter motor performance, indicating that pregabalin can control pain effectively without impairing motor function. Desipramine and amitriptyline are classical tricyclic antidepressants used to treat neuropathic pain. They have been shown to alleviate pain in patients with diabetic neuropathy (Boyle et al., 2012; Sindrup et al., 1990), but they cause sleep fragmentation (Mayers and Baldwin, 2005), and an analgesic dose of amitriptyline doesn't improve sleep (Boyle et al., 2012). Selective serotonin reuptake inhibitors (SSRIs) such as venlafaxine and duloxetine, have also been used to treat patients with neuropathic pain (Goldstein et al., 2005; Sumpton and Moulin, 2001). However, they increased wakefulness and suppressed sleep (Thase, 1999). In PSNL mice, we found that pregabalin (25 mg/kg) significantly improved insomnia during the light phase (“sleeping period” for mice). In contrast, administration of pregabalin (25 mg/kg) did not affect sleep patterns during the dark phase (waking

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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period for mice) in PSNL and normal mice. However, a higher dose of pregabalin, 50 mg/kg, significantly increased the duration of NREM and REM sleep and decreased that of wakefulness in normal mice. The effects of pregabalin (25 mg/kg) on sleep disturbance took place mainly through the analgesic effect, but high doses of pregabalin (more than 50 mg/kg) also had a hypnotic effect in normal mice. Some clinical studies have shown administration of 150 mg pregabalin twice a day to be efficacious and safe in patients with neuropathic pain (Rosenstock et al., 2004; Sabatowski et al., 2004). However, other studies have found 300 mg pregabalin twice a day to be more suitable to the treatment of neuropathic pain (Richter et al., 2005). In the present study, we found that pregabalin at 25 mg/kg is an appropriate dose to treat the pain and sleep disturbance, without motor impairment and drowsiness during the active phase in mice. Based on the U.S. Food and Drug Administration guidelines for calculation of human equivalent dose, the dose of 25 mg/kg in mice roughly equals to that of 2.3 mg/kg in human. If the body weight is 70 kg in adult human, it is expected that pregabalin at 160 mg/kg should be effective enough. These results suggest that the lower dose of pregabalin may mitigate the chronic pain and sleep disturbance without causing drowsiness or ataxia during the day. Neuropathic pain is caused by abnormal discharge of neurons in the central nervous system attributed to nerve injury. Increased input induces central sensitization through synaptic strengthening and amplifying nociceptive processing. Sensitization of rat dorsal horn neurons is notable after peripheral nerve injury (Ohnami et al., 2011) Pregabalin, an antiepileptic drug, is considered to have effects on analgesic in diabetic peripheral neuropathic pain, post-herpetic neuralgia and fibromyalgia syndrome patients (Boomershine, 2010; Boyle et al., 2012; Dworkin et al., 2003). Further research showed that pregabalin

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Fig. 6. Effects of pregabalin on c-Fos expression in the ACC in PSNL and sham mice. (A, a) Representative photomicrographs of c-Fos expression in the ACC of sham + vehicle; (B, b) 25 mg/kg pregabalin treated sham mice; (C, c) PSNL + vehicle; and (D, d) 25 mg/kg pregabalin treated PSNL mice. (a–d) High-magnification views of the rectangular areas marked in a–d from A–D; the arrowheads indicate Fos-positive cells in the ACC region. Scale bars: upper panel, 100 μm; lower panel, 50 μm. (E) Mechanical allodynia was measured 120 min after treatment with vehicle or pregabalin in PSNL and Sham mice. (F) Amount of c-Fos expression in the ACC of PSNL and sham mice 120 min after treatment with vehicle or pregabalin. Values are expressed as means ± SEM (n = 5–6), **P b 0.01 indicates significant differences from vehicle value in the sham group, ##P b 0.01 indicates significant differences from vehicle value in PSNL group assessed by one-way ANOVA followed by the PLSD test.

is preferentially active in states of facilitated nociceptive processes, which are probably associated with central sensitization (Tuchman et al., 2010), Pregabalin also reduced the rate of formalin-induced flinching behavior in rats (Kumar et al., 2010). In this way, this research suggests that pregabalin can reduce enhanced pain responses from facilitated pain processing and central sensitization in the level of the spinal cord and probably at other sites in the central nervous system, which is the main mechanism for the analgesia effect of pregabalin (Tuchman et al., 2010). The binding of pregabalin at α2-δ subtype 1 (α2-δ-1) is suggested to be necessary and likely to be sufficient to modulate pharmacological activities (Field et al., 2006; Lotarski et al., 2011). The α2-δ-1 subunits densely distribute in the neocortex, hippocampus, amygdala, and spinal cord (Li et al., 2011; Taylor and Garrido, 2008). Among the abovementioned areas, the anterior cingulate cortex (ACC) is the cortical area, which appears to be involved in the emotional reaction to pain rather than to the perception of pain itself (Ossipov, 2012). Recently, many studies have shown the ACC region to be closely linked to the chronic pain and sleep disorders. A human study showed that the concentration of GABA relative to total creatine (measure GABA levels) was lower in the ACC region in patient of primary insomnia (Plante et al., 2012). A similar phenomenon was observed in animal models of neuropathic pain. A recently research demonstrated that neuropathic pain may decrease the level of release of GABA at the synaptic cleft of ACC region in the PSNL mice (Narita et al., 2011). Xu et al. reported that peripheral nerve injury triggered long-term changes in excitatory synaptic transmission in ACC (Xu et al., 2008). The present study showed that pregabalin (25 mg/kg i.g.) decreased expression of c-Fos in the ACC of PSNL mice. These results indicate that inhibition of ACC might play important roles in the analgesic and hypnotic effects of pregabalin in PSNL mice.

Please cite this article as: Wang, T.-X., et al., Antinociceptive and hypnotic activities of pregabalin in a neuropathic pain-like model in mice, Pharmacol Biochem Behav (2015), http://dx.doi.org/10.1016/j.pbb.2015.05.007

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Pregabalin increases NREM and REM sleep during the first 4 h after administration in a chronic pain insomnia model of mice, and the effect may be attributed to the inhibition of ACC hyperactive neurons.

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We sincerely thank Prof Jun Lu, Dr. Xin-Hong Xu, Dr. Yi-Qun Wang, Dr. Hui-Jing Wang and Mr. Hui Dong for their valuable comments. This study was supported by the National Basic Research Program of China (2015CB856401, 2011CB711000), the National Natural Science Foundation of China (81171255, 81301135, 81420108015, 31421091, 31471064, 81471344, 31171010, 31171049, 31271164, J1210041), a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005), the Shanghai Committee of Science and Technology (14JC1400900, 13dz2260700, 13140903100, 13ZR1403200), the Shanghai Leading Academic Discipline Project (B119), and Shanghai Postdoctoral Scientific Program (14R21410100).

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