Experimental Neurology 247 (2013) 383391

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Experimental Neurology
journal homepage: www.elsevier.com/locate/yexnr

Sigma-1 receptor-mediated increase in spinal p38 MAPK phosphorylation leads to the induction of mechanical allodynia in mice and neuropathic rats
Ji-Young Moon a, Dae-Hyun Roh b, Seo-Yeon Yoon c, Suk-Yun Kang a, Sheu-Ran Choi a, Soon-Gu Kwon a, Hoon-Seong Choi a, Ho-Jae Han a, Alvin J. Beitz d, Jang-Hern Lee a,
a

Department of Veterinary Physiology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea Department of Maxillofacial Tissue Regeneration, School of Dentistry, Kyung Hee University, Seoul, Republic of Korea Laboratory of Molecular Signal Transduction, Center for Neural Science, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea d Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA
b c

a r t i c l e

i n f o

a b s t r a c t
The direct activation of the spinal sigma-1 receptor (Sig-1R) produces mechanical allodynia (MA) and thermal hyperalgesia (TH) in mice. In addition, the blockade of the spinal Sig-1R prevents the induction of MA, but not TH in chronic constriction injury (CCI)-induced neuropathic rats. The present study was designed to investigate whether the increase in spinal p38 MAPK phosphorylation (p-p38 MAPK) mediates Sig-1R-induced MA or TH in mice and the induction of MA in neuropathic rats. MA and TH were evaluated using von Frey laments and a hot-plate apparatus, respectively. Neuropathic pain was produced by CCI of the right sciatic nerve in rats. Western blot assay and immunohistochemistry were performed to determine the changes of p-p38 MAPK expression in the spinal cord. Intrathecal (i.t.) injection of PRE084, a selective Sig-1R agonist, into nave mice time-dependently increased the expression of p-p38 MAPK, which was blocked by pretreatment with BD1047, a Sig-1R antagonist. I.t. pretreatment with SB203580, a p38 MAPK inhibitor also dose-dependently inhibited PRE084-induced MA, whereas TH induction was not affected. In CCI rats, i.t. injection of BD1047 during the induction phase (postoperative days 0 to 5) reduced the CCI-induced increase in p-p38 MAPK. In addition, i.t. SB203580 treatment during the induction phase also suppressed the development of CCI-induced MA, but not TH. Conversely, i.t. SB203580 treatment during the maintenance phase (postoperative days 15 to 20) had no effect on CCI-induced MA or TH. These results demonstrate that the increase in spinal p-p38 MAPK is closely associated with the induction of Sig-1R mediated MA, but not TH. Sigma-1 receptor modulation of p-p38 MAPK also plays an important role in the induction, but not the maintenance, of MA in neuropathic pain. 2013 Elsevier Inc. All rights reserved.

Article history: Received 24 August 2012 Revised 22 December 2012 Accepted 8 January 2013 Available online 15 January 2013 Keywords: p38 MAPK Sigma-1 receptor Mechanical allodynia Neuropathic pain

Introduction Peripheral neuropathic pain, which results from damage or dysfunction of peripheral nerves, is one of the most challenging chronic pain conditions to treat. The development of neuropathic pain, characterized by allodynia (pain produced in response to a nonnociceptive stimulus as dened by the IASP) and hyperalgesia (increased sensitivity to a painful stimuli), is associated with a variety of pathophysiologic changes (Latremoliere and Woolf, 2009; Ueda, 2006). A number of different mechanisms have been suggested to explain the sensory disorders associated with mechanical allodynia (MA) and thermal

Corresponding author at: Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea. Fax: +82 2 885 2732. E-mail address: jhl1101@snu.ac.kr (J.-H. Lee). 0014-4886/$ see front matter 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2013.01.004

hyperalgesia (TH) (Ossipov et al., 1999; Roh et al., 2008a). However, the precise mechanisms underlying the development of MA versus TH particularly within the spinal cord remain to be accurately dened. Sigma-1 receptors (Sig-1Rs) play an important role in a variety of cellular functions via modulation of intracellular Ca2+ concentration (Guitart et al., 2004; Su et al., 2010). It is well recognized that Sig-1Rs are widely distributed in mammalian central nervous system including certain cortical areas, the hypothalamus and the dorsal horn of the spinal cord, but it is less well appreciated that Sig-1Rs are also associated with peripheral tissues including nociceptive nerve endings (Alonso et al., 2000; Ohsawa et al., 2010; Ueda et al., 2001). Thus, systemic administration of Sig-1R agonists or antagonists may have differential effects compared to intrathecal (i.t.) administration because of potential peripheral effects. Recently using Sig-1R knockout mice, it has been shown that Sig-1Rs have a pronociceptive effect in formalin-induced nociception and in nerve injury-induced pain (Cendan et al., 2005; de la Puente et al., 2009). Further support for a pronociceptive role for

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this receptor comes from a previous study from our laboratories demonstrating that i.t. administration of the Sig-1R antagonists BD1047 or BMY-14802 reduces nociceptive behaviors and spinal Fos expression associated with the formalin test (Kim et al., 2006). In addition, we have shown that the direct activation of spinal cord Sig-1Rs enhances the response to peripheral mechanical and thermal stimuli via Ca 2+-dependent second messenger cascades (Roh et al., 2008b). In addition, i.t. injection of the BD1047 attenuates MA when administered during the induction phase (days 05 after sciatic nerve chronic constriction injury, CCI), but not the maintenance phase (days 1520 after CCI) in CCI rats. On the other hand, BD1047 treatment during either the induction or maintenance phases had no effect on TH (Roh et al., 2008c). These ndings demonstrated that the activation of spinal Sig-1Rs is associated with the induction of MA, but not TH in a rat model of neuropathic pain. p38 mitogen-activated protein kinase (p38 MAPK) is a stressactivated protein kinase which is activated by phosphorylation (Ji and Suter, 2007). It has been reported that p38 MAPK plays an important role in a variety of chronic pain states (Boyle et al., 2006; Jin et al., 2003; Sorkin et al., 2009; Wen et al., 2009; Xu et al., 2007). Moreover, based on experiments performed in a variety of animal pain models, spinal p38 MAPK activation appears to be involved in the pathophysiology of MA, but not TH (Sorkin et al., 2009; Wen et al., 2009). p38 MAPK activation is regulated by elevated concentrations of intracellular Ca 2 + and the activation of Ca 2 + dependent enzymes (Hayashi and Su, 2007; Lee et al., 2000; Trang et al., 2009). Since Sig-1Rs sense endoplasmic reticulum Ca 2 + concentration and the activation of Sig-1R modulate intracellular Ca 2 + signaling via the efux of Ca 2 + into the cytoplasm, we hypothesized that changes in p38 MAPK phosphorylation (p-p38 MAPK) might be regulated by Sig-1R activation, which may ultimately contribute to the development of MA following peripheral nerve injury. The present study was designed to examine whether: (1) i.t. injection of the specic Sig-1R agonist (PRE084) or antagonist (BD1047) could modulate p-p38 MAPK in the spinal cord dorsal horn; (2) i.t. injection of the specic p38 MAPK inhibitor, SB203580 reduces Sig-1R-induced MA and TH in mice; (3) i.t. administration of BD1047, given during the induction phase of neuropathic pain modulates MA as well as the CCI-induced increase in p-p38 MAPK in neuropathic rats; and nally (4) i.t. administration of SB203580 during the induction or the maintenance phase of neuropathic pain could suppress MA or TH in CCI rats. Materials and methods Animal preparation Experiments were performed on male SpragueDawley rats (200250 g) and ICR mice (2025 g). All experimental animals were purchased from the Laboratory Animal Center of Seoul National University (Seoul, Republic of Korea). They had free access to food and water and were maintained in temperature and light controlled rooms (23 2 C, 12/12 h light/dark cycle with lights on at 08:00) for at least 1 week prior to beginning an experiment. The experimental protocols for animal usage were reviewed and approved by the SNU Animal Care and Use Committee and conform to NIH guidelines (NIH publication No. 86-23, revised 1985). A CCI of the common sciatic nerve was performed according to the method described by Bennett and Xie (1988) Briey, rats were anesthetized with 3% isourane in a mixture of N2O/O2 gas. The right sciatic nerve was exposed at the mid-thigh level, and 4 loose ligatures of 40 chromic gut were placed around the dissected nerve with a 1.0- to 1.5-mm interval between each ligature. Sham surgery consisted of exposing the sciatic nerve in the same manner, but without ligating the nerve. Total numbers of mice and rats used in the study were 129 and 45, respectively.

Intrathecal (i.t.) drug injection For i.t. injection, we used the modied method of direct transcutaneous i.t. injection on mice and rats, respectively (Hylden and Wilcox, 1980; Mestre et al., 1994). I.t. injections were made into the L5L6 intervertebral space of animals using a 50 l Hamilton syringe. The ick of the tail was considered indicative of a successful i.t. administration. Rats were briey anesthetized with 3% isourane in a mixture of N2O/O2 gas before i.t. drug injection to prevent any handling-induced stress. Then, while the rats were under anesthesia, drugs were slowly infused. Animals awoke immediately after the i.t. injection procedure and were freely moving within 45 s after injection. All drugs treated in rats were dissolved in 10 l of vehicle. In mice, all drugs were dissolved in 5 l of vehicle and were also administered intrathecally 10 min before PRE084 injection. The control group received an i.t. injection of vehicle. Animals were randomly assigned to experimental groups and subsequent drug treatment and analysis were performed blindly. The following drugs used were: 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE084; PRE, 3 nmol, Tocris Cookson Ltd (Bristol, UK)), a sigma-1 receptor agonist; N-([2-(3,4-dichlorophenyl)ethyl]-Nmethyl-2-(dimethylamino) ethylamine dihydro-bromide (BD1047; BD, 100 nmol, Tocris Cookson Ltd), a sigma-1 receptor antagonist; 4-(4uorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580; SB, 0.1, 0.3, 1, 3, 10 nmol Sigma-Aldrich (St. Louis, MO)), and a p38 MAPK inhibitor. The doses of PRE084 and BD1047 used were based on those used in previous studies from our laboratories showing that these doses produce maximal effects with no detectable side-effects (Roh et al., 2008b, 2008c). The doses of SB203580 used in the present study were selected based on doses previously used in the literature (Lee et al., 2009; Wu et al., 2006). PRE084 and BD1047 were dissolved in physiological saline, while SB203580 was dissolved in 1% DMSO in saline. Mechanical allodynia assay A total of 73 rodents were used in MA assay (mice = 40 and CCI rats = 33). Sensitization to innocuous mechanical stimulation (MA) was examined using von Frey laments (North Coast Medical, Morgan Hill, CA) as described in previous studies from our laboratories (Roh et al., 2008b, 2008c). A 2 g (for CCI rats) and a 0.16 g (for mice) von Frey laments were selected for testing. In each experimental and control mouse, mechanical responses to von Frey laments were measured at the 30, 60, and 120 min time points after treatment with PRE084 (or saline). In CCI rats, mechanical responses to von Frey laments were assessed 1 day before CCI or sham surgery in all animals to obtain normal baseline values and then animals were tested again following CCI or sham surgery for a period of 21 days as described above. These von Frey laments were applied from underneath the metal mesh ooring to each hind paw. The lament was applied 10 times to each paw with each application separated by 10 s intervals. The number of paw withdrawal responses to the 10 von Frey applications was then recorded. The results of the mechanical response testing in each experimental animal were expressed as a percent withdrawal response frequency (PWF, %), which represented the percentage of paw withdrawals out of a maximum of 10 as previously described (Roh et al., 2008c, 2011). Thermal hyperalgesia assay A total of 65 rodents were used in TH assay (mice = 32 and CCI rats = 33). To assess nociceptive responses to heat stimuli in mice, sensitization to noxious heat stimulation (TH) was examined with a hot-plate apparatus (Model-35100, Ugo Basile, Comerio, Italy) (Duman et al., 2006). The temperature of plate was maintained at 55 0.5 C. Animals were placed into an acrylic cylinder (20 cm in

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diameter, 25 cm high) on the heated surface, and the time (in seconds) between placement and shaking or licking or lifting of their hind paws or jumping (whichever occurred rst), was recorded as the response latency (sec). Baseline response latency (812 s) were determined before experimental treatment. The response latency were then measured 30, 60, and 120 min after treatment with PRE084 (or saline) in each experimental mice. To assess nociceptive responses to heat stimuli in CCI rats, we measured paw withdrawal response latency (PWL) by using the plantar paw-ick latency test as previously described by Hargreaves et al. (1988). Briey, rats were placed in a plastic chamber with a glass oor and were allowed to acclimatize for 30 min before testing. A radiant heat source was positioned under the glass oor beneath the hind paw to be tested, and withdrawal latency was measured using a plantar analgesia meter (IITC Life Science Inc., Woodland Hills, CA). The test was repeated in the ipsilateral hind paw of each animal, and the mean withdrawal latency was calculated. Cutoff time in the absence of a response was set at 20 s. TH assay was performed 1 day before CCI surgery on all animals to obtain normal baseline values of thermal stimuli. Western blotting analysis A total of 37 rodents were used in western blotting analysis (mice = 25 and CCI rats = 12). Mice were sacriced at several time points (60 and 120 min) after i.t. injection of PRE084 (3 nmol) to determine the time-dependent effect of Sig-1R activation on changes in p-p38 MAPK expression (n = 5 at each time point group). Five days post-CCI or post-sham surgery, the spinal cords were collected from CCI and sham animals (n= 4 at each time point group) to examine the effect of BD1047 on the CCI-induced increase in p-p38 MAPK expression. Rats and mice were rst deeply anesthetized using combination of Zoletil 50, Rompun and saline (a ratio of 2:1:2, respectively) and the location of the L4L6 spinal cord segments were then veried by identifying the attachment site of each lumbar spinal nerve in the anesthetized animals. The spinal cord was extracted by pressure expulsion with air into an ice-cooled, saline-lled glass dish. Subsequently the spinal cord was separated into left and right halves under a neuro-surgical microscope. The spinal cord was further subdivided into dorsal and ventral halves by cutting straight across from the central canal laterally to a midpoint in the white matter. The ipsilateral and contralateral dorsal quadrants of each CCI or sham rat spinal cord were separated and each half containing the ipsilateral or contralateral spinal cord dorsal horn, respectively, was subsequently processed for Western blot analysis. In mice, the individual right and left spinal cord dorsal horns were used for Western blot analysis. The L46 spinal cord dorsal segments were homogenized in buffer containing 1 M Tris (pH 7.5), 1% NP-40, 0.5 M EDTA (pH 7.5), 50 mM EGTA, 1 M dithiothreitol, 1 M benzamidine and 0.1 M PMSF. The total amount of protein in each sample was determined using the Bradford dye assay prior to loading on polyacrylamide gels. Spinal cord homogenates (20 g protein) were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. After the blots had been washed with TBST (10 mM TrisHCl (pH 7.6), 150 mM NaCl, 0.05% Tween-20), the membranes were blocked with 5% skim milk for 1 h and incubated at 4 C overnight with a primary antibody specic for p38 MAPK (1:500, cat# 9212, Cell Signaling Technology, Beverly, Massachusetts) and p-p38 MAPK (1:1000, cat# 9211, Cell Signaling Technology). After washing with TBST, membranes were incubated for 4 h with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (1:2000, Santa Cruz Biotechnology Inc.) to detect p38 MAPK and p-p38 MAPK. The bands were visualized with enhanced chemiluminescence (Amersham Biosciences; Buckinghamshire, UK). The positive pixel area of specic bands was measured with a computer-assisted image analysis system and normalized against the

corresponding p38 MAPK loading control bands. The mean value of the ratio of p38 MAPK to p-p38 MAPK expression in animals prior to PRE injection (0 min) or CCI injury was set at 100%. Thus, the % change in the ratio of p-p38 MAPK to p38 MAPK expression at each time-point in each group was calculated. Immunohistochemistry A total of 32 mice were used in immunohistochemical assay. In a separate set of experiments, mice were anesthetized using combination of Zoletil 50, Rompun and saline (a ratio of 2:1:2, respectively) at several time points (60 and 120 min) after i.t. injection of PRE084 (3 nmol). Mice were perfused transcardially with calcium-free Tyrode's solution followed by a xative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 6.9). The spinal cords were removed immediately after perfusion, post-xed in the identical xative for 12 h and then placed in 30% sucrose in PBS (pH 7.4) overnight at 4 C. Serial transverse sections (40 m) of the spinal cord were cut using a cryostat (Microm, Germany). Spinal L4L6 tissue sections were processed for p-p38 MAPK (1:1000; cat# 4511; Cell Signaling Technology) immunohistochemistry using the avidinbiotin peroxidase complex (ABC) procedure as previously described (Osuka et al., 2007). Visualization of the ABC complex was performed using 3,3-diaminobenzidine (DAB; Sigma) and the DAB reaction was intensied with 0.2% nickel chloride. Tissue sections were examined under a brighteld microscope (Zeiss Axioscope, Hallbergmoos, Germany) and ve spinal cord sections from the L46 lumbar spinal cord segments were randomly selected from each animal, and subsequently scanned. Individual sections were digitized with 4096 gray levels using a cooled CCD camera (Micromax Kodak 1317; Princeton Instruments, AZ) connected to a computer-assisted image analysis system (Metamorph version 6.3r2; Molecular Devices Corporation, PA). To maintain a constant threshold for each image and to compensate for subtle variability of the immunostaining, we only counted cells that were at least 70% darker than the average gray level of each image after background subtraction and shading correction. The average number of p-p38 MAPK-immunoreactive (ir) cells per section from each animal was obtained and these values were averaged across each group and presented as group data. The expression of p-p38 MAPK was quantied in the following three dorsal horn regions: 1) the supercial dorsal horn (SDH, laminae I and II); 2) the nucleus proprius (NP, laminae III and IV); and 3) the neck region (NECK, laminae V and VI). All analytical procedures described above were performed blindly without knowledge of the experimental conditions. Statistical analysis All values are expressed as the mean SEM. Statistical analysis was performed using Prism 5.0 (Graph Pad Software, San Diego, USA). Repeated measures two-way ANOVA was performed to determine overall differences in the time-course of all nociceptive behavioral tests. One-way ANOVA was used to determine differences in the number of spinal p-p38 MAPK-ir cells (immunohistochemistry) and in the ratio of p-p38 MAPK to p38 MAPK expression (Western blot assay) between experimental groups. Post-hoc analysis was performed using the Bonferroni's multiple comparison test in order to determine the P value among experimental groups. In all cases, P b 0.05 was considered statistically signicant. Results Effect of intrathecal PRE084 injection on p-p38 MAPK expression in the mouse spinal cord dorsal horn An anti-p-p38 MAPK antibody was used to examine changes in p38 MAPK activation. To determine whether p-p38 MAPK was activated by

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Sig-1R, we performed immunohistochemistry (Figs. 1A and B) and Western blot analysis (Fig. 1C). The expression of p-p38 MAPK was examined initially in the spinal cord of nave mice that received administration of the Sig-1R agonist PRE084. The i.t. administration of PRE084 (3 nmol, VEH + PRE), signicantly increased the number of p-p38 MAPK-ir cells in the spinal dorsal horn at the 60 min post-injection

A
40

VEH+VEH 60min (n=11) VEH+PRE 60min (n=9) VEH+PRE 120min (n=4) BD + PRE 60 min(n=4) BD+ VEH 60min (n=4)

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35 30 25 20 15 10 5 0 SDH NP NECK

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VEH+VEH SDH

Spinal Regions
VEH+PRE

NP

time point as compared with that of a vehicle-treated group (Figs. 1A, B; **P b 0.01 and ***P b 0.001 as compared with those of the VEH + VEH group). To further determine whether this increase in the number of p-p38 MAPK-ir cells was a direct and specic result of activation of spinal Sig-1R, we next pretreated a separate group of mice with a Sig-1R antagonist prior to i.t. treatment with PRE084. As illustrated in Fig. 1A, i.t. pretreatment with the BD1047 (100 nmol, BD+ PRE), completely blocked the PRE084-induced increase in the number of p-p38 MAPK-ir cells at the 60 min post-injection time point (Fig. 1A; #P b 0.05, ##P b 0.01 and ###P b 0.001 as compared with those of the VEH+ PRE group). The i.t. injection of this inhibitor alone (BD + VEH), in the absence of PRE084, did not affect the number of p-p38 MAPK-ir cells in comparison to the VEH + VEH group. Western blot analysis also conrmed the effects of i.t. administration of PRE084 on p-p38 MAPK levels in the spinal cord of nave mice (Fig. 1C). Thus, the relative pixel area (%) of p-p38 MAPK expression in Western blots was signicantly increased by PRE084 treatment (Fig. 1C; *P b 0.05 as compared with those of the VEH + VEH group), but there was no change in p38 MAPK levels, in spinal cord homogenates 60 min after i.t. injection of PRE084 as compared with the vehicle-treated group. In addition, i.t. pretreatment with the BD1047 (100 nmol, BD + PRE), completely blocked the PRE084-induced increase in p-p38 MAPK expression at the 60 min post-injection time point (##P b 0.01 as compared with those of the VEH + PRE group), which con rms the immunocytochemical data. Effect of intrathecal pretreatment with a p38 MAPK inhibitor on PRE084 induced pain hypersensitivity in nave mice To determine whether the PRE084-induced pain behaviors involve the activation of p38 MAPK, we examined the effect of i.t. pretreatment with the p38 MAPK inhibitor, SB203580 on PRE084-induced pain hypersensitivity in mice. The i.t. administration of the Sig-1R agonist, PRE084 (3 nmol, VEH+PRE), signicantly increased time-dependent PWF (%) to innocuous mechanical stimuli (MA, Fig. 2A) and decreased response latency (sec) to noxious heat stimuli (TH, Fig. 2B) as compared with those of the vehicle-treated group (Fig. 2; *P b 0.05, **P b 0.01 and ***P b 0.001 as compared with those of the VEH+VEH group). I.t. pretreatment with SB203580 (0.3, 1, 3 nmol, Fig. 2A), dose-dependently suppressed this PRE084-induced increase in PWF (###P b 0.001 as compared with those of the VEH+PRE group). On the other hand, i.t. pretreatment with SB203580 (3, 10 nmol, Fig. 2B) did not affect the PRE084-induced decrease in thermal response latency. The i.t. injection of this inhibitor alone (SB+VEH), in the absence of PRE, did not affect PWF or thermal response latency in comparison with the VEH+VEH group. Effects of intrathecal BD1047 administration on the development of pain behaviors and p38 MAPK phosphorylation in CCI rats Previous reports have demonstrated that nerve injury activates the p38 MAPK cascade and the activation of p38 MAPK in the spinal cord contributes to the generation of neuropathic pain (Jin et al., 2003; Xu et al., 2007). We rst conrmed that i.t. administration of the BD1047 (100 nmol, CCI + BD) on postoperative days 05 signicantly attenuated MA, but not TH, as previously reported from our laboratory (Figs. 3A and B) (Roh et al., 2008c). MA and TH did not develop on the contralateral side following CCI surgery (data not shown). Next we performed a Western blot analysis to examine whether p38 p-p38 MAPK was regulated by Sig-1 R activation (Figs. 3C and D). Rats were euthanized on day 5 (vehicle and BD1047, n = 4, respectively) following CCI surgery. Sham surgery animals (n= 4) were euthanized at the same time point for comparison. CCI induced a signicant increase in the phosphorylation of p38 MAPK in the ipsilateral dorsal horn (Fig. 3C; *P b 0.05 as compared with those of the SHAM group). I.t. administration of the BD1047 signicantly decreased the level of

NECK

C
relative pixed area (%) (p-p38/p38)
200 150 100 50 0

No. of p-p38-ir cells

VEH+VEH 60min (n=5) VEH+PRE 60min (n=5) VEH+PRE 120min (n=5) BD+PRE 60min (n=5) BD+VEH 60min (n=5)
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p-p38

t-p38

Fig. 1. Graphs illustrating immunocytochemical and Western blot data showing the effect of i.t. administration of the Sig-1R agonist, PRE084 on p-p38 MAPK in the mouse spinal cord dorsal horn. Quantitative graphs (A and C) and representative photomicrographs (B) illustrating the effect of i.t. injection of PRE084 (VEH+PRE, 3 nmol) on the number of p-p38 MAPK-immunoreactive (ir) cells in the spinal cord dorsal horn. (A) The number of p-p38-ir cells in the supercial dorsal horn (SDH, laminas III), in the nucleus proprius (NP, laminas IIIIV) and in the neck region (NECK, laminas VVI) of the spinal dorsal horn is depicted graphically. I.t. administration of PRE084 (VEH+PRE), signicantly increased the number of p-p38 MAPK-ir cells in the dorsal horn as compared with that of the vehicle-treated group (VEH+VEH) at the 60 min post-injection time point (**P b 0.01 and ***P b 0.001 as compared with those of the VEH+VEH group). Moreover, pretreatment with the Sig-1R antagonist BD1047 (BD+PRE, 100 nmol) before Sig-1R agonist injection completely blocked the effect of PRE084 on p-p38 MAPK expression. (#P b 0.05, ##P b 0.01 and ###P b 0.001 as compared with those of the VEH+PRE group). (B) Representative photomicrographs depicting p-p38 MAPK-ir cells in the SDH, NP, and NEC of the spinal dorsal horn from vehicle and PRE084 treated mice. Arrows indicate representative p-p38 MAPK-ir cells. Scale bar=200 m. (C) Western blot analysis illustrating the effect of i.t. administration of the PRE084 on p-p38 MAPK. A graph depicting the change in the p-p38 MAPK is shown in the upper portion, and the representative bands of p-p38 MAPK and p38 MAPK are presented in the lower portion. Representative Western blots showing an increase in p-p38 MAPK (top), but no change in p38 MAPK (bottom). *P b 0.05 as compared with those of the VEH+VEH group and ##P b 0.01 as compared with those of the VEH+PRE group.

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A
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VEH+VEH (n=6) VEH+PRE (n=10) SB 0.3nmol+PRE (n=6) SB 1nmol+PRE (n=6) SB 3nmol+PRE (n=6)

with SB203580 (at doses ranging from 1 to 10 nmol) throughout the 21-day testing period (Fig. 4B). Effects of intrathecal SB203580 on the maintenance phase of neuropathic pain behaviors To investigate the contribution of p38 MAPK activation to the CCI-induced altered behavior during the maintenance phase of neuropathic pain, we intrathecally administered the SB203580 on postoperative days 1520. Daily treatment with SB203580 (10 nmol) had no effect on the CCI-induced increase in PWF (%) to innocuous mechanical stimuli (Fig. 5A) nor the CCI-induced decrease in response latency (seconds) as compared with those in the vehicletreated group (Fig. 5B).

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SB 3nmol+VEH (n=6)

PWF (%)

30 20 10 0 0 30 60 90 120 VEH+VEH (n=4) VEH+PRE (n=8) 14 12 10 8 6 4 0 SB 3nmol+PRE (n=10) SB 10nmol+PRE (n=5) SB 10nmol+VEH (n=5)

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Time after PRE084 inj. (min)

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Discussion The present study demonstrates that i.t. administration of the Sig-1R agonist, PRE084 signicantly increased spinal p-p38 MAPK in the spinal cord dorsal horn and this increase was blocked by pretreatment with the Sig-1R antagonist, BD1047. In addition, in a rat chronic neuropathic pain model, i.t. treatment with BD1047 administered during the induction phase following nerve injury prevented the CCI-induced early increase in p-p38 MAPK in the spinal cord dorsal horn. These results indicate that p38 MAPK activation, i.e. an increase in the phosphorylated form (p-p38 MAPK) is affected by direct activation of spinal Sig-1Rs. A potential relationship between Sig-1R activation and p-p38 MAPK has been previously reported by Nishimura et al. (2008). They demonstrated that stimulation of Sig-1Rs potentiates the nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells through the interaction with IP3 receptors and several subsequent signaling molecules including p38 MAPK (Nishimura et al., 2008). In addition, Sig-1Rs have been shown to modulate intracellular Ca 2+ signaling and activate Ca2+-dependent enzymes, which lead to p-p38 MAPK in dorsal horn neurons (Hayashi and Su, 2007; Lee et al., 2000; Roh et al., 2008b; Trang et al., 2009). Recently, we have also demonstrated that the facilitatory effects of a Sig-1R agonist in pain perception are mediated by nitric oxide (NO) signaling via neuronal nitric oxide (nNOS) activation (Roh et al., 2011), which is well recognized to be closely linked to p38 MAPK signaling. In addition, NO has been reported to serve as an upstream signaling molecule of p38 MAPK that mediates hypoxia/ reoxygenation-induced neuronal cell death (Chen et al., 2009). Moreover, the inhibition of nNOS has been shown to attenuate morphine antinociceptive tolerance by reducing p38 MAPK activation in spinal microglia (Liu et al., 2006). These ndings together with those of the present study suggest that Sig-1Rs can modulate the phosphorylation of p38 MAPK via a Ca 2+ dependent cascade and/or a NO signaling pathway. Interestingly, the role of Sig-1R in modulating central sensitization associated with the development of neuropathic pain and the potential role of another MAPK family member, extracellular signal-regulated kinase (ERK) has recently been investigated in Sig-1R knockout mice (de la Puente et al., 2009). De la Puente et al. reported that cold and mechanical allodynia, but not TH, is suppressed in Sig-1R knockout mice exposed to partial sciatic nerve injury. In addition, in contrast to wild-type mice, Sig-1R knockout mice did not show phosphorylation of ERK (pERK) in the spinal cord 14 days after surgery. These results are in agreement with the observation reported here that MA, but not TH, is associated with Sig-1Rs and subsequently p38 MAPK activation. De la Puente et al. (2009) used a partial sciatic nerve ligation model, which differs from the CCI model used here. It is important that the pattern of p-p38 and pERK expression related to Sig-1R activation be investigated in different models of neuropathic pain, which may have relevance related to the efcacy of Sig-1R antagonists in treating neuropathic pain of different origins.

Response latency (sec)

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60 90 120

Time after PRE084 inj. (min)

Fig. 2. Graphs illustrating the effect of i.t. administration of the p38 MAPK inhibitor, SB203580 (SB + PRE) on the Sig-1R agonist PRE084-induced changes in the paw withdrawal frequency over time (PWF; %, A), and in the thermal response latency (seconds, B) of mice. SB203580 was applied 10 min before PRE084 injection. (A) I.t. pretreatment with SB203580 (1, 3 nmol) blocked the increase in PWF that occurred in VEH + PRE group (###P b 0.001 as compared with those of the VEH + VEH group). (B) However, the decrease in response latency to heat stimuli was unaffected by i.t. pretreatment with even the highest dose of SB203580 tested (10 nmol).

CCI-induced p-p38 MAPK expression as compared with the vehicle treated group (#P b 0.05 as compared with those of the CCI + VEH injected group). There was no signicant increase in p-p38 MAPK expression in the contralateral dorsal horn as compared to sham rats at the same time point (Fig. 3D). Similarly spinal cord p38 MAPK expression was not signicantly changed following nerve injury as compared with sham surgery animals.

Effects of intrathecal SB203580 on the induction phase of neuropathic pain behaviors CCI-induced MA and TH were present on day 3 post-surgery and were maintained for more than 3 weeks. To investigate the contribution of p38 MAPK activation to this altered behavior during the induction phase, we intrathecally administered the p38 MAPK inhibitor SB203580 on postoperative days 05. Repeated daily, i.t. treatment with SB203580 (1, 3, or 10 nmol) dramatically reduced the CCI-induced increase in PWF (%) to innocuous mechanical stimuli, as compared with vehicle-treated CCI rats (Fig. 4A). After the termination of repeated SB203580 injection on day 5, this suppressive anti-allodynia effect of SB203580 was sustained throughout the 21-day experimental period following CCI surgery (*P b 0.05 and **P b 0.01 as compared with those of the CCI+VEH group). On the other hand, the CCI-induced decrease in response latency (seconds) to heat stimuli (TH) was not inuenced by repeated i.t. treatment

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A
PWF (%)

80 60 40

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20 15 10 5 0 CCI+VEH CCI+BD CCI+ VEH CCI+BD day 0 day 5

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20 0 CCI+VEH CCI+BD CCI+ VEH CCI+BD day 0 day 5
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CCI+VEH (n=4) CCI+BD (n=4)

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p-p38 t-p38

Fig. 3. Graphs illustrating the effect of i.t. administration of the Sig-1R antagonist BD1047 (CCI + BD, 100 nmol, administrated from days 0 to 5 after surgery) on the development of MA, TH and p-p38 MAPK in rats with chronic constriction injury (CCI)-induced neuropathic pain. (A) Repeated daily treatment with BD1047 blocked the increase in paw withdrawal frequency (%) that occurred in vehicle-treated CCI-rats (CCI + VEH). (**P b 0.01 as compared with those of the CCI + VEH group on day 0, and #P b 0.05 as compared with those of the CCI + VEH group on day 5) (B) However, the decrease in response latency (seconds) to heat stimuli was unaffected by repeated i.t. injection with BD1047 (*P b 0.05 and **P b 0.01 as compared with those of the CCI + VEH and CCI + BD group on day 0, respectively). (C and D) Western blot analysis illustrating the effect of i.t. administration of the BD1047 on p38 MAPK and p-p38 MAPK expression in the ipsilateral and contralateral dorsal horn. A graph depicting the change in the p-p38 MAPK is shown in the upper portion, and the representative bands of p-p38 MAPK and p38 MAPK expression are presented in the lower portion. Representative western blots showing an expression of p-p38 MAPK (top) and p38 MAPK (bottom). (C) I.t. administration of the Sig-1R antagonist BD1047 signicantly decreased the level of CCI-induced p-p38 MAPK expression as compared with the vehicle-treated group (*P b 0.05 as compared with those of the SHAM group, and #P b 0.05 as compared with those of the CCI + VEH group). (D) There was no substantial increase in p-p38 MAPK expression in the contralateral dorsal horn compared to sham rats at same time point. p38 MAPK expression was not signicantly changed after nerve injury compared with sham surgery animals in both ipsilateral and contralateral dorsal horn.

To date, four different p38 MAPK isoforms, , , and , have been identied (Kumar et al., 2003). In the spinal cord, only p38 and p38 are constitutively expressed (Fitzsimmons et al., 2010). It has been also shown that these isoforms are distinctly expressed in the spinal cord dorsal horn: p38 in neurons and p38 in microglia (Gwak et al., 2009; Svensson et al., 2005). Accumulating evidence now suggests that both spinal p38 and p38 play an important role in nociceptive processing (Fitzsimmons et al., 2010; Svensson et al., 2005). Since SB203580 non-selectively inhibits both p38 and p382 (Barone et al., 2001), it is not clear whether i.t. SB203580 acts on glial p38 MAPK or neuronal p38 MAPK or both in its suppressive effect on MA. However, it is well-established that p38 MAPK activation is involved in the production and release of proinammatory cytokines, such as IL-1 , IL-6 and TNF-, which is essential for neuropathic pain development (Gao and Ji, 2010; Gosselin et al., 2010; Ji and Suter, 2007). Thus the current data suggest that spinal cord p38 MAPK activation regulated by Sig-1Rs following sciatic nerve injury may be a valuable therapeutic target for neuropathic pain management. Our previous studies have demonstrated that direct activation of spinal Sig-1Rs induces both mechanical allodynic and thermal hyperalgesic behaviors in mice (Roh et al., 2008b). Remarkably, the present study shows that i.t. pretreatment with a p38 MAPK inhibitor only attenuated the MA, but not the TH produced by i.t. administration of a Sig-1R agonist. In addition, the inhibition of spinal p38 MAPK activation during the induction phase of neuropathic pain

reduces the development of MA, but not TH in CCI rats. These results corroborate those of our previous study, which examined the effect of i.t. treatment with a Sig-1R antagonist (Roh et al., 2008c). The present results indicate that the activation of spinal p38 MAPK is closely involved with the induction of Sig-1R-mediated MA, but not TH, in a model of neuropathic pain. Recently several studies have also reported that spinal p38 MAPK activation plays an important role in the pathophysiological mechanism of MA in a variety of experimental pain models. p38 MAPK phosphorylation is immediately increased by plantar incision, which is coincident with the development of incisional pain (Wen et al., 2009). Single i.t. of another p38 MAPK inhibitor, FR167653 potently attenuated incision-induced MA for 2 days after pretreatment incision, whereas a higher dose of FR167653 only resulted in a very brief inhibition on TH. In addition, i.t. pretreatment of SB203580 dose-dependently blocked the development of tactile allodynia induced by a rst-degree burn in the rat (Sorkin et al., 2009). These results imply that the activation of spinal Sig-1Rs and the subsequent signaling of p38 MAPK are more closely associated with the development of MA, but not TH in chronic pain conditions. Moreover, Romero et al. recently reported that mice receiving systemic administration of S1RA, a new Sig-1R antagonist exhibit antinociceptive effects on MA as well as TH (Romero et al., 2012). In contrast, we demonstrate in the present study that inhibition of spinal Sig-1Rs only inhibits the development of MA, but not TH. This discrepancy may be due to the difference in administration

J.-Y. Moon et al. / Experimental Neurology 247 (2013) 383391

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VEH (n=9)

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SB 1 nmol (n=4) SB 3 nmol (n=4) SB 10nmol (n=7)

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PWF (%)

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0 0 5

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VEH (n=9) SB 1 nmol (n=4) SB 3 nmol (n=4) SB 10nmol (n=7) VEH (n=4) SB 10nmol (n=5)

Tx.
16 14 12 10 8 6 0 5 10 15

Response latency (sec)

B
18

Tx.
16 14 12 10 8 6 0 5 10 15 20

20

Days after surgery

Fig. 4. Graphs illustrating the effect of i.t. treatment with the p38 MAPK inhibitor SB203580 (SB, administered from days 0 to 5 after surgery) on the development of mechanical allodynia and thermal hyperalgesia in rats with chronic constriction injury (CCI)-induced neuropathic pain. (A) Repeated daily treatment with SB203580 (1, 3, 10 nmol) blocked the increase in response frequency (%) that occurred in vehicle-treated CCI rats (*P b 0.05 and **P b 0.01 as compared with that of the CCI+VEH group). (B) However, the decrease in response latency (seconds) to heat stimuli was unaffected by repeated i.t. injection at even the highest dose of SB203580 tested.

Response latency (sec)

Days after surgery

Fig. 5. Graphs illustrating the effect of intrathecal (i.t.) treatment with the p38 MAPK inhibitor SB203580 (SB, administered from days 15 to 20 after surgery) on the maintenance of mechanical allodynia and thermal hyperalgesia in rats with chronic constriction injury (CCI)-induced neuropathic pain. Repeated daily treatment with SB203580 (10 nmol) neither suppressed the CCI-induced increase in paw withdrawal frequency (%) to mechanical stimuli (A) nor reversed the CCI-induced decrease in response latency (seconds) as compared with the vehicle-treated group (B).

of the Sig-1R antagonist between the two studies such that the reduction in CCI-induced TH produced by systemic Sig-1R antagonism is mediated by the inhibition of Sig-1Rs located either in supra-spinal brain regions or in the periphery, where Sig-1Rs have been shown to modulate some ion channels like Kv 1.4 potassium channels in dorsal root ganglion cells (Aydar et al., 2002; Matsuyoshi et al., 2012). Thus, spinal Sig-1R activation may be more important for the development of CCI-induced MA rather than TH. We found that i.t. treatment with SB203580 during the induction phase of neuropathic pain signicantly suppresses CCI-induced MA, while SB203580 treatment during the maintenance phase has no effect on MA. In this regard we have previously reported that the early blockade of spinal Sig-1Rs inhibits both the development of CCI-induced MA and the CCI-induced increase in spinal NMDA receptor subunit 1 expression and phosphorylation (pNR1). In contrast, BD1047 treatment during the maintenance phase of neuropathic pain had no effect on MA or pNR1 expression (Roh et al., 2008c). In addition, there is a corresponding up-regulation of spinal Sig-1R expression during the induction phase, but not the maintenance phase (Roh et al., 2008c). These results suggest that the activation of spinal Sig-1R plays a critical role in the induction of MA rather than the maintenance of MA in CCI-induced neuropathic pain rats. Recently it has been reported that pretreatment or early treatment with the p38 MAPK inhibitor, SB203580 inhibits p38 MAPK activity, which results in a signicant reduction in TNF- synthesis and subsequent

attenuation of MA. On the other hand, post-treatment with SB203580 starting on day 7 produces no effect on MA or TNF- levels in CCI rats (Xu et al., 2007). Collectively it appears that p38 MAPK activation is regulated by Sig-1R stimulation, which ultimately leads to the induction of MA, but not to the maintenance of MA in CCI rats. However, administration of the p38 MAPK inhibitor was found to only affect the development phase, but not the maintenance phase in our model. This result differs with respect to the effect of p38 MAPK inhibitors reported using other neuropathic pain models. In this regard Cheng et al. reported that increased levels of p-p38 MAPK were detected in db/db mice with diabetic neuropathy when compared to db + mice at 5, 8 and 10 weeks of age and SB203580 treatment inhibited MA in db/db mice following one week of daily administration beginning at week 7 (Cheng et al., 2010). Thus, the p38 MAPK inhibitor appeared to inhibit the maintenance phase of diabetic neuropathy, although dening the development phase versus the maintenance phase in this model remains to be clearly dened. Importantly diabetic neuropathy involves hyperglycemia-mediated nerve damage and the pathways regulated in the db/db nerve include lipid metabolism, carbohydrate metabolism, energy metabolism, peroxisome proliferator-activated receptor signaling, apoptosis, and axon guidance (Pande et al., 2011). Thus, we would argue that the mechanisms underlying diabetic neuropathy are very different from those involved in CCI-induce neuropathy and this may explain the differences in our results. Similarly, i.t. administration of SB203580 reduced MA during the maintenance phase of

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J.-Y. Moon et al. / Experimental Neurology 247 (2013) 383391 D., Vela, J.M., 2009. Sigma-1 receptors regulate activity-induced spinal sensitization and neuropathic pain after peripheral nerve injury. Pain 145, 294303. Duman, E.N., Kesim, M., Kadioglu, M., Ulku, C., Kalyoncu, N.I., Yaris, E., 2006. Effect of gender on antinociceptive effect of paroxetine in hot plate test in mice. Prog. Neuropsychopharmacol. Biol. Psychiatry 30, 292296. Fitzsimmons, B.L., Zattoni, M., Svensson, C.I., Steinauer, J., Hua, X.Y., Yaksh, T.L., 2010. Role of spinal p38alpha and beta MAPK in inammatory hyperalgesia and spinal COX-2 expression. NeuroReport 21, 313317. Gao, Y.J., Ji, R.R., 2010. Chemokines, neuronalglial interactions, and central processing of neuropathic pain. Pharmacol. Ther. 126, 5668. Gosselin, R.D., Suter, M.R., Ji, R.R., Decosterd, I., 2010. Glial cells and chronic pain. Neuroscientist 16, 519531. Guitart, X., Codony, X., Monroy, X., 2004. 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neuropathic pain in two different spinal cord injury (SCI) models of neuropathic pain (Crown et al., 2008; Gwak et al., 2009). Crown et al. showed that increases in p38 MAPK activation occurred in astrocytes, microglia, and dorsal horn neurons in the spinal cord rostral to the site of injury and that inhibiting the enzymatic activity of p38 MAPK dose dependently reverses the behavioral expression of at-level MA after moderate SCI. There are clear differences between CCI-induced and SCI-induced models of neuropathic pain. For instance, CCI-induced neuropathy is associated with a signicant increase in spinal cord dorsal horn microglia, but not astrocytes (Ikeda et al., 2012), while SCI-induced neuropathic pain is associated with an upregulation of both astrocytes and microglia as indicated above (Crown et al., 2008). Moreover, spinal astrocytes, but not microglia contribute to the pathogenesis of paclitaxel-induced painful neuropathy (Zhang et al., 2012). In addition, TNF- is upregulated in the spinal cord above the site of SCI, but not in the sciatic nerve, while TNF- is upregulated in the sciatic nerve following CCI-injury (Hama et al., 2012). Thus, the differences observed between our results and those of other studies regarding the effect of p38 MAPK inhibitors on the development versus the maintenance of neuropathic pain is probably a reection of the different models used and the variations that occur in the spinal mechanisms responsible for the development and maintenance of pain across these various models. This is an area that is clearly deserving of further study and begs the question of whether there are different mechanisms underlying neuropathic pain of different origins in human patients. In conclusion, the current study shows that spinal Sig-1Rs can modulate the activation of p38 MAPK in the spinal cord dorsal horn. Furthermore, Sig-1R-mediated activation of p38 MAPK is important for the induction of MA, but not TH, in neuropathic pain. These results demonstrate that spinal Sig-1Rs play an important role in the activation of p38 MAPK signaling and through this signaling cascade participate in the development of MA during the early (induction) phase of neuropathic pain. Acknowledgments This research was supported by a grant (2012K001118) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Education, Science and Technology, the Republic of Korea. This research was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (no. 2012-0005436). References
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