The activation of extracellular signal-regulated protein kinase 5 in spinal cord and dorsal root ganglia contributes to inflammatory pain

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s

Research Report

The activation of extracellular signal-regulated protein kinase 5 in spinal cord and dorsal root ganglia contributes to inflammatory pain Chun Xiao a , Lin Zhang b , Qiu-Ping Cheng a , Li-Cai Zhang a,⁎ a

Research Institute of Anesthesiology, Affiliated Hospital of Xuzhou Medical College, 99 Huaihai West Road, Xuzhou 221002, PR China Department of Anesthesiology, Affiliated Hospital of First Clinical College, China Medical University, 155 Nangjing North Road, Heping District, Shenyang 110001, PR China b

A R T I C LE I N FO

AB S T R A C T

Article history:

Activation of mitogen-activated protein kinases (MAPKs) in dorsal root ganglia (DRG) and

Accepted 27 March 2008

the spinal dorsal horn contributes to inflammatory pain by transcription-dependent and

Available online 6 April 2008

-independent means. In this study, we investigated extracellular signal-regulated protein kinase 5 (ERK5) activation by peripheral inflammation in the spinal cord and DRG of rats and

Keywords:

whether this activation contributes to a heat and mechanical hyperalgesia response.

Extracellular signal-regulated

Injection of complete Freund's adjuvant (CFA) into a hindpaw produced persistent

protein kinase 5

inflammation and sustained ERK5 activation in DRG and the spinal dorsal horn.

Inflammatory pain

Knockdown of the ERK5 by antisense oligonucleotides suppressed the heat and

Spinal cord

mechanical hyperalgesia. In addition, the antisense knockdown of ERK5 reduced CFA-

Dorsal root ganglion

induced phosphorylation of cAMP response-element binding protein (CREB), a downstream

cAMP response-element binding

substrate of the ERK5 pathway, and expression of Fos, a marker for neuronal activation in

protein

the central nervous system. Our study suggests that activation of the ERK5 signaling pathway contributes to persistent hyperalgesia induced by peripheral inflammation. © 2008 Elsevier B.V. All rights reserved.

1.

Introduction

The mitogen-activated protein kinase (MAPK) cascade is a family of serine/threonine kinases activated by phosphorylation of threonine and tyrosine residues (Seger and Krebs, 1995; Widmann et al., 1999). MAPKs transduce a broad range of

extracellular stimuli into diverse intracellular responses by both transcriptional and posttranscriptional mechanisms (Widmann et al., 1999; Sweatt, 2001). In the nervous system, MAPKs are activated by neuronal activity and are involved in neuronal plasticity, including long-term potentiation, learning and memory, and hyperalgesia (Sweatt, 2001; Impey et al.,

⁎ Corresponding author. Fax: +86 516 85708135. E-mail address: [email protected] (L.-C. Zhang). Abbreviations: MAPKs, mitogen-activated protein kinases; ERK5, extracellular signal-regulated protein kinase 5; DRG, dorsal root ganglion; CFA, complete Freund's adjuvant; CREB, cAMP response-element binding protein; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase; BMK1, big MAPK1; CRE, cAMP response element; CGRP, calcitonin gene-related protein; BDNF, brainderived neurotrophic factor; AS-ODN, Antisense oligodeoxynucleotides; MM-ODN, mismatch oligodeoxynucleotides; PWT, pawwithdrawal threshold; PWL, paw-withdrawal latency; CNS, central nervous system; IEG, immediate early gene; NGF, Nerve growth factor; NMDAR, N-methyl-D-aspartate receptor; LVGCC, L-type voltage-gated calcium channel; MEF2, myocyte enhancer factor 2; Sap1a, Ets-domain transcription factor; SGK, serumand glucocorticoid-inducible kinase; RSK, p90 ribosomal S6 kinase 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.03.065

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Fig. 1 – Inflammation induces persistent ERK5 activation in DRG neurons (A and B). ERK5 phosphorylation is increased in DRG neurons 30 min after CFA injection into a hindpaw. Scale bar = 50 μl. (C) Time course of CFA injection induced p-ERK5 increase in the DRG (n = 4). Data are means ± SEM. *p < 0.05 compared to control. (D) Western blot analysis revealed ERK5 activation in the ipsilateral L4 and L5 DRG. Quantification of western blot data is not shown. (E) Frequency histogram of p-ERK5-positive neurons 30 min after CFA injection.

1999; Ji and Wolf, 2001). The MAPK family consists of four major members: extracellular signal-regulated kinase 1/2 (ERK1/2), p38, c-Jun N-terminal kinase and extracellular signal-regulated protein kinase 5 (ERK5) (Sturgill and Wu, 1991; Kyriakis and Avruch, 2001; Wang and Tournier, 2006). The amino-terminal half of ERK5 contains the kinase domain, which is similar to that of ERK1/2, and has the Thr-Glu-Tyr activation motif, whereas the carboxy-terminal half is unique (Lee et al., 1995). Increasing evidence shows that activation of ERK1/2 in the spinal cord and primary sensory neurons plays an important role in the induction and maintenance of hyperalgesia induced by inflammation (Ji et al., 2002; Dai et al., 2004). However, these studies relied largely on the pharmacological

inhibitors PD98059 and U0126, which also inhibit the ERK5 signaling pathway (Kamakura et al., 1999; Mody et al., 2001). Thus, some of the functions attributed to ERK1/2 may actually be carried out by ERK5. The activation of ERK5 in DRG and the spinal cord plays a role in the development of neuropathic pain (Obata et al., 2007). However, the effect of ERK5 on persistent inflammatory pain in DRG and the spinal cord has not yet been shown, and the downstream activation of ERK5 in DRG and the spinal cord in response to peripheral inflammation is poorly understood. Activated ERK5 can translocate to the nucleus for regulating the activity of the transcription factor cAMP responseelement binding protein (CREB) in cultured primary sensory neurons (Watson et al., 2001). The phosphorylation of CREB (p-

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Fig. 2 – CFA induces a sustained activation of ERK5. (A) A low magnification image showing induction of ERK phosphorylation in laminae I–II neurons of the ipsilateral spinal cord 30 min after CFA injection into a hindpaw. Scale bar = 100 μm. (B–D) High-magnification image showing ERK5 activation in the superficial dorsal horn of the ipsilateral spinal cord 30 min after CFA injection. Scale bar = 100 μm. (E) Time course of p-ERK5 induction after CFA injection measured by the number of pERK-positive neurons in the superficial layers of the ipsilateral dorsal horn (n = 4). Data are means ± SEM. *p < 0.05 compared to control. (F) Western blot analysis of increased ERK5 phosphorylation dorsal horn compared with control.

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CREB) at serine 133 activates cAMP response-element (CRE)mediated gene expression (Sweatt, 2001; Bonni et al., 1995). Several lines of in vitro and in vivo evidence have indicated that the CREB-dependent gene is required for long-term changes in neuronal plasticity induced by various nociceptive stimuli (Lonze and Ginty, 2002). Peripheral inflammation upregulates p-CREB in DRG and the spinal cord (Ji and Rupp, 1997; Tamura et al., 2005), but whether this upregulation is regulated by ERK5 has not been explored. We aimed to test whether activation of the ERK5 signaling pathway is involved in the heat- and mechanical-induced hyperalgesia response to inflammation in DRG and the spinal cord of rats, in particular the influence of ERK5 on the activity of transcription factor CREB. The hypersensitivity to inflammatory pain induced by intraplantar injection of formalin and capsaicin occurs with such a short latency that it must be mediated by nontranscriptional processing (Ji et al., 1999). Since complete Freund's adjuvant (CFA) injection causes hyperalgesia persisting longer than 1 week, we chose the CFAinjection model.

2.

Results

2.1. ERK5 is activated in DRG following peripheral inflammation Use of a phospho-specific ERK5 antibody revealed a basal activation of ERK5 (p-ERK5) in nonstimulated DRG slices (Fig. 1A). CFA injected into the plantar surface of the left hindpaw produced localized swelling and erythema, and the hyperalgesia response to mechanical and thermal stimuli in rats persisted for the duration of the experiment (96 h). With the inflammation, the number of p-ERK5-immunoreactive DRG rapidly increased (Fig. 1B), peaked at 0.5 h and was maintained at a high level for 4 days (Figs. 1C, D). The increase in p-ERK5 was seen mainly in small- to medium-diameter DRG neurons (Fig. 1E). The changes in ERK5 phosphorylation in ipsilateral DRG were confirmed by western blot analysis (Fig. 1D).

2.2. ERK5 is activated in the spinal cord following peripheral inflammation To investigate the activation of ERK5 in the spinal cord of rats, L4–L5 spinal cord sections underwent immunohistochemistry analysis. Very few p-ERK5-irradiated cells were found in the spinal dorsal horn of control rats (Fig. 2B); however, inflammation produced by CFA injection induced p-ERK5 in the superficial dorsal horn at the ipsilateral side of the lumbar enlargement (laminae I–II; Figs. 2A, B) but not in the contralateral spinal cord (Figs. 2A, C). Only a few p-ERK5-labeled neurons were in the deep dorsal horn (Fig. 2B). The number of p-ERK5-labeled neurons peaked at 30 min and remained elevated for 24 h, but the total protein level of ERK5 did not alter (Figs. 2E, F).

2.3. Antisense knockdown of ERK5 reverses the hyperalgesia response to peripheral inflammation To examine the functional consequences of ERK5 activation, we tested whether inhibition of ERK5 activation modified the

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hyperalgesia response to inflammation. Intrathecal administration of ERK5 antisense oligodeoxynucleotides (AS-ODN), started before the CFA injection, significantly reduced the inflammation-induced heat and mechanical hypersensitivity in rats; the threshold of the mismatch-ODN (MM-ODN) group did not differ from that of vehicle-treated group (Figs. 3A, B). Western blot assay confirmed that intrathecal administration of ERK5 AS-ODN, not vehicle or MM-ODN, reduced the level of total ERK5 protein in DRG and the spinal cord (Figs. 3C, D, E, F). The increased expression of p-ERK5 in DRG and the spinal cord was also inhibited by intrathecal administration of ERK5 ASODN (Figs. 3G, H, I, J, K, L).

2.4. Antisense knockdown of ERK5 reduces peripheral inflammation-induced p-CREB expression In agreement with previous studies, the immunohistochemical results revealed that the number of p-CREB-positive neurons was increased significantly in the ipsilateral DRG at 0.5 h after CFA injection and reached a plateau at 12 h after the injection, which was maintained until 96 h (Figs. 4A, B, D). The p-CREB-positive neurons were distributed in all of the bilateral spinal cord, and the time course of p-CREB expression was similar in the spinal cord as in DRG (Fig. 4H). Intrathecal injection of ERK5 AS-ODN markedly inhibited p-CREB activity in both DRG and the spinal cord (Figs. 4C, F, G, H).

2.5. Antisense knockdown of ERK5 reduces peripheral inflammation-induced c-fos expression The immunohistochemical results indicated that peripheral inflammation markedly increased Fos expression, which was distributed in all the laminae of the ipsilateral spinal cord (Figs. 5A, B). After CFA injection, the increase in number of Fos-positive neurons reached a plateau at 12 h, which was maintained until 96 h (Fig. 5D). Intrathecal injection of ERK5 AS-ODN significantly reduced the Fos expression in the spinal cord (Figs. 5C, D).

3.

Discussion

In the present study, persistent peripheral inflammation increased ERK5 activation in small- to medium-diameter DRG, most of which are nociceptors (Snider and McMahon, 1998), and laminae I–II neurons of the ipsilateral superficial dorsal horn. Thus, activation of ERK5 in DRG and the spinal cord is not a universal response but is induced by a specific signal in a particular subset of neurons. Katsura et al. found that CFA injection induced the activation of ERK5 in small DRG neurons but not in the spinal cord (Katsura et al., 2007); the probable reasons for the different results may be the different detection times and the different CFA dilutions. Inflammatory mediators released from damaged tissue not only increase the excitability of primary sensory neurons but also lead to hyperexcitability in the central nervous system, and both peripheral and central elements contribute to pain hypersensitivity (Meyer et al., 2006; LaMotte et al., 1991). In primary sensory and dorsal horn neurons, inflammatory

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mediators and aberrant neuronal activity activate several signaling pathways, including protein kinase A and C, calcium/ calmodulin-dependent protein kinase and MAPKs, which mediate the induction and maintenance of pain hypersensitivity induced by peripheral inflammation through both posttranslational and transcriptional mechanisms (Ji and Strichartz, 2004). Mizushima et al. showed that ERK5 activation in DRG contributes to heat hyperalgesia and peripheral sensitization (Mizushima et al., 2007). We found that knockdown of ERK5 reduced both heat and mechanical pain hypersensitivity. Furthermore, intrathecal injection of ERK5 AS-ODN significantly reduced the Fos expression in the spinal cord in rats with inflammation. Fos protein, the product of c-fos immediate early gene (IEG), has been used as a marker for neuronal activation in the CNS (Coggeshall, 2005). Therefore, ERK5 activation in the spinal cord could be involved in central sensitization. Nerve growth factor (NGF) has been implicated in the activation of ERK5 in the nervous system (Watson et al., 2001; Wang et al., 2006). However, NGF is upregulated in inflamed tissue and needs to be retrogradely transported to the cell body of sensory neurons in DRG, where it regulates gene expression (Miller and Kalan, 2001); however, we found that the time course of phosphorylation of ERK5 did not match that required for the retrograde transport of NGF from inflamed paws to DRG. The activation of ERK5 can be mediated by neurotrophin as well as Nmethyl-D-aspartate receptor (NMDAR) and L-type voltage-gated calcium channel (LVGCC) (Wang et al., 2004), which leads to increased intracellular Ca2+ concentration and therefore activates the specific intracellular signal pathway (Fields et al., 1997). Furthermore, heat and cold stimulation of the receptive field at different intensities results in changes in the number of p-ERK5labeled neurons (Mizushima et al., 2007). Thus, increased intracellular Ca2+ induced by activation of NMDAR and LVGCC may activate the ERK5 signal pathway in DRG and the spinal cord. Nociceptive input can activate transcription factors and evoke a change in gene expression in primary sensory and dorsal horn neurons to produce long-lasting changes in function that underlie some of the persistent changes in sensitivity found under chronic pain conditions (Ji and Strichartz, 2004). Activated ERK5 translocates to the nucleus, where it directly or indirectly phosphorylates several nuclear factors. Activation of p90 ribosomal S6 kinase (RSK), a substrate of ERK5 (Wang and Tournier, 2006), phosphorylates the transcription factor CREB, and CREB constitutively binds with high affinity to the cAMP response element (CRE) in the promoter regions of genes; the phosphorylation of CREB at serine 133 is required for CREB-mediated transcription (Xing et al., 1996). In the current study, CFA injection significantly increased the expression of p-CREB in DRG and the spinal cord, whereas knockdown of ERK5 markedly

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inhibited the increased p-CREB expression. Thus, activation of ERK5 contributes to the increased p-CREB expression induced by peripheral inflammation, and the function of ERK5 occurs via CREB-dependent gene expression in part. The CREB-binding site CRE has been identified in the promoter regions of many “pain genes,” including c-fos, zif 268, COX-2, NK-1, dynorphin, calcitonin gene-related peptide and brain-derived neurotrophic factor (BDNF) (Wisden et al., 1990; Mannion et al., 1999; Samad et al., 2001; Ji et al., 2002; Lonze and Ginty 2002). A growing body of evidence indicates that CREB-dependent gene expression is required for neuronal plasticity induced by various nociceptive stimuli (Wang et al., 2006; Hoeger-Bement and Sluka, 2003). Peripheral inflammation induces ipsilateral p-ERK5 and c-fos expression but bilateral p-CREB expression in the dorsal horn. p-ERK5 is predominantly induced in the superficial dorsal horn DRG, whereas p-CREB and c-fos are also induced in the deep dorsal horn. Therefore, a direct coupling of p-ERK5, p-CREB, and c-fos appears to be restricted to the superficial dorsal horn, with other protein kinases playing a role in CREB phosphorylation and c-fos expression (Lonze and Ginty, 2002). Furthermore, we found the duration of expression of p-CREB, c-fos and hyperalgesia longer than that of p-ERK5 in the spinal cord. In contrast to the restricted pattern of ERK5 activation in the dorsal horn, the activation of p-ERK5 in DRG correlates with the activation of CREB and hyperalgesia. Primary afferent inputs also play an important role in central sensitization (LaMotte et al., 1991; Ibrahim et al., 2003; Porreca et al., 1999). Recently, Obata et al. demonstrated the expression of BDNF regulated by ERK5 (Obata et al, 2007). Under inflammatory pain, BDNF is upregulated in DRG and transported to the central terminals of primary afferents in the dorsal spinal horn, which further modifies the excitability of target neurons and participates in central sensitization (Michael et al., 1997; Obata et al., 2003). Therefore, activation of the ERK5-CREB signal pathway in DRG may indirectly mediate CREB phosphorylation and central sensitization through synaptic transmission. In conclusion, we found that activation of the ERK5-CREB signal pathway in DRG and the spinal cord of rats contributes to the hyperalgesia induced by peripheral inflammation.

4.

Experimental procedures

4.1.

Animals

Adult (3-month-old) male Sprague–Dawley rats (200–250 g) were kept under a 12-h light/12-h dark cycle regime, with free access to food and water. The animals were provided by the Experimental Animal Center of Xuzhou Medical College. All

Fig. 3 – Intrathecal injection of antisense (AS)-oligodeoxynucleotide (ODN) against ERK5 reduces CFA-induced inflammatory pain. (A and B) Pretreatment with ERK5 AS-ODN prevented heat- and mechanical-induced hypersensitivity with peripheral inflammation, measured by paw-withdrawal latency and paw-withdrawal threshold respectively (n = 8). Data are means ± SEM. *p < 0.05 compared to MM-ODN control. (C and D) The expression of total ERK5 in the rat DRG and spinal cord after intrathecal injection of ERK5 AS-ODN, by western blot analysis. Quantification of the western blot data is shown in panels E and F. The expression of p-ERK5 in DRG (G and H) and spinal cord (I and J) in the MM-ODN and AS-ODN groups 30 min after CFA injection. Quantification of the immunohistochemistry data is shown in panels K and L. Scale bar = 50 μl. Data are means ± SEM (n = 4). *p < 0.05 compared to vehicle control (saline).

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Fig. 5 – ERK5 activation regulates Fos expression. (A, B, C) Immunohistochemical staining showing inhibition of the CFA-induced increase in Fos-labeled neurons in the dorsal horn by ERK5 AS-ODN 30 min after CFA injection. Scale bar, 100 μm. (D) Quantitative data indicating the number of Fos-positive neurons in the spinal cord of rats in each group. Data are means ± SEM (n = 4). *p < 0.05 compared to control. #p < 0.05 compared with the MM-ODN group.

experimental protocols were approved by the Animal Care and Use Committee of the college and were in accordance with the European Communities Council Directive of November 1986.

4.2.

Implantation of intrathecal catheter

For intrathecal drug administration, rats were implanted with catheters. The protocol is similar to that described by Yaksh and Rudy (1976). In brief, under anesthesia with pentobarbital sodium (40 mg/kg, i.p.), the cisternal membrane was exposed. A polyethylene catheter (PE-10) was inserted via an incision in the cisterna magna, and advanced 7.0–7.5 cm caudally to the level of the lumbar enlargement. The catheter was judged to be intrathecal if paralysis and dragging of bilateral hind limbs occurred within 30 s of 10 μl 2% lidocaine injected through the catheter. Animals with signs of motor dysfunction were excluded from the experiment. The rats were housed individually after surgery and allowed to recover 5–7 days before inducing inflammation.

4.3.

Inflammatory pain model

For inducing peripheral inflammation, animals received a subcutaneous injection of 100 μl of CFA (Sigma, USA) into the plantar surface of the left hindpaw. All injections were performed under ether anesthesia. The injections produced classical signs of inflammation, including redness, edema, and hyperalgesia at the left hindpaw. Animals were allowed to survive for 0.5, 12, 24, 48 or 96 h after injection (n = 4 for immunohistochemistry and n = 4 for western blotting for each time point). Rats without surgery (n = 8) were used as naive controls for immunohistochemistry and western blotting.

4.4.

Antisense knockdown of ERK5 expression

The sequences of ERK5 oligodeoxynucleotides (ODN) were adopted from Nadruz et al. (2003): antisense ODN (AS-ODN: 5′GAGACTCAATGTCAGCG-3′), and mismatch-ODN (MM-ODN: 5′ACTACTACACTAGACTAC-3′), ODN was synthesized by

Fig. 4 – ERK5 activation regulates p-CREB expression. (A, B, C, E, F, G) Immunohistochemical staining of p-CREB in the ipsilateral DRG and spinal cord of rats in the MM-ODN and AS-OND groups 30 min after CFA injection. Scale bar = 100 μm. Quantitative data indicating the number of p-CREB-positive neurons in the DRG (D) and spinal cord (H) of rats in each group. Data are means ± SEM (n = 4). *p < 0.05 compared to control. #p < 0.05 compared with the MM-ODN group.

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Shanghai Sangon (Shanghai, China) modified with full phosphorothioate. To investigate the possible role of ERK5 in response to peripheral inflammation, 10 nmol of ERK5 AS-ODN in 10 μl physiological saline was intrathecally administrated to the rats at 36, 24 and 12 h before the establishment of the inflammatory pain model (Cao et al. 2005). The same dose of physiological saline and MM-ODN was injected as controls. The intrathecal drug delivery was accomplished by using a microinjection syringe connected to the intrathecal catheter in awake, briefly restrained rats. The injection was performed manually over a 30-s period in a single injection volume of 10 μl followed by a flush with 10 μl physiological saline. The efficacy of the ASODN in inhibiting ERK5 expression was confirmed at the protein level by western blotting.

4.5.

Behavioral assessment of hyperalgesia

Mechanical hyperalgesia was assessed by the plantar surface of the hindpaw being stimulated with a series of von Frey hairs. The paw-withdrawal threshold (PWT) was determined by sequentially increasing and decreasing the stimulus strength (the “up-and-down” method), and the data were analyzed by the nonparametric method of Dixon, as described by Chaplan et al. (1994). Thermal hyperalgesia was assessed by the paw-withdrawal latency (PWL) to radiant heat according to the protocol of Hargreaves et al. (1998). To avoid tissue damage, a cut-off time of 30 s was used. Each rat underwent 3 trials, with 5-min intervals between trials, and withdrawal latency was averaged over the three trials. All testing was performed blind. The tests were performed 1 day before and 0.5, 12, 24, 48, and 96 h after CFA injection. Eight animals per group were used for testing.

4.6.

Immunohistochemistry

Rats were anesthetized with sodium pentobarbital and perfused transcardially with 0.9% sodium chloride, followed by 4% paraformaldehyde in 0.1 M PB, 0.5, 12, 24, 48, and 96 h (n = 4 at each time point) after surgery to remove L5 spinal cord segments and L4/5 DRG, which were post-fixed in the same fixative for 6 h, then 25% sucrose overnight. Transverse spinal cord and DRG sections (25 µm) were cut in a cryostat and processed for p-ERK5, p-CREB, and c-Fos immunostaining. Spinal cord and DRG sections were processed for immunohistochemistry using the ABC method. Briefly, after a washing in phosphate buffer saline (PBS), sections were incubated for 2 h in a solution containing 0.01% sodium azide and 0.1% H2O2 in PBS to block endogenous peroxidase activity. To reduce nonspecific staining, sections were incubated for 2 h in a blocking solution containing 1% bovine serum albumin (BSA), 2% normal goat serum, 0.3% Triton X-100, and 5% nonfat dry milk in PBS followed by p-ERK5 antibody (goat-anti 1:200 Santa Cruz Biotechnology, Santa Cruz, CA), ser133-p-CREB antibody (rabbit-anti 1:200) or Fos antibody (rabbit-anti 1:400; both Cell Signaling Technology) at 4 °C overnight. The sections were then incubated in biotinylated goat anti-rabbit or rabbit anti-goat IgG (1:200) at room temperature for 2 h and in avidin–biotin–peroxidase complex (1:100) at 37 °C for 0.5 h. Finally, the sections underwent reaction with 0.05% diaminobenzidine for 15 min. Sections were rinsed in PBS, then mounted on gelatin-coated slides, air-dried, dehydrated with 70–100% alcohol, cleared with xylene, and

cover-slipped for microscopy examination. In control experiments, the primary antibodies were omitted or replaced with normal immunoglobulin G or serum. To quantify the positive-cell profile, five spinal cords and DRG sections per animal were randomly selected. For each animal, we recorded the total number of positive neurons in the bilateral spinal cord and DRG sections. All positive neurons were counted without considering the intensity of the staining. An assistant, who was unaware of the treatment group of the tissue sections, performed all counting.

4.7.

Western blot analysis

Tissue samples from the left L4/5 DRG and L5 spinal cord segments were lysed by homogenizing in 200 μl lysis buffer containing 20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 2 mM Na3VO4, 0.5 mM DTT, 1 mM PMSF, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 9 μg/ml aprotinin, and 10% glycerol. Lysates were centrifuged at 14,400 g for 60 min, and the concentration of protein in each sample (supernatant) was determined by the Lowry method. Samples with equal amounts of protein were then separated by 10–20% PAGE, and the resolved proteins were electrotransferred to nitrocellulose membrane. Membranes were incubated with 5% nonfat milk in Tris buffer containing Tween 20 (TBST; 10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.2% Tween 20) for at least 10 min at room temperature and incubated with rabbit anti-total ERK5 polyclonal antibody (1:500; Cell Signaling Technology), goat anti-p-ERK5 antibody (1:200), and antibody for β-actin (1: 1000; Sigma) at 4 °C overnight. The membranes were extensively washed with TBST and incubated for 2 h with the secondary antibody conjugated with alkaline phosphatase (1:1000) at room temperature. The immune complexes were detected by using a NBT/BCIP assay kit (Sigma). The scanned images were imported into Adobe Photoshop software (Adobe, CA, USA). Scanning densitometry was used for semiquantitative analysis.

4.8.

Statistical analysis

Data are presented as mean±SD. Comparisons were performed by a one-way analysis of variance (ANOVA) with multiple comparisons or Student's t-test. A P<0.05 was considered significant.

Acknowledgments We thank Dr. Jun-Li Cao for the support and mentorship. The study was financed by the National Science Foundation (30570974).

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