A role for extracellular signal-regulated kinases 1 and 2 in the maintenance of persistent mechanical hyperalgesia in ovariectomized mice

A role for extracellular signal-regulated kinases 1 and 2 in the maintenance of persistent mechanical hyperalgesia in ovariectomized mice

Neuroscience 172 (2011) 483– 493 A ROLE FOR EXTRACELLULAR SIGNAL-REGULATED KINASES 1 AND 2 IN THE MAINTENANCE OF PERSISTENT MECHANICAL HYPERALGESIA I...

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Neuroscience 172 (2011) 483– 493

A ROLE FOR EXTRACELLULAR SIGNAL-REGULATED KINASES 1 AND 2 IN THE MAINTENANCE OF PERSISTENT MECHANICAL HYPERALGESIA IN OVARIECTOMIZED MICE M. B. KLINGER,* S. SACKS AND F. CERVERO

the adult (Ji et al., 2009). Activation of MAPK pathways by upstream kinases evokes intracellular responses that include transcriptional as well as non-transcriptional regulation (Chuang and Ng, 1994). These MAPK signaling factors are made of three distinct signaling pathways that include extracellular signal-regulated kinases 1 and 2 (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK) (Derijard et al., 1995). There is substantial experimental evidence that demonstrates that acute noxious stimulation of somatic and visceral tissues activates ERK 1/2 in the spinal cord (Ji et al., 1999, 2002a; Gioia et al., 2001; Galan et al., 2002, 2003; Obata et al., 2004a; Cruz et al., 2005, 2007; Fukuda et al., 2009; Liverman et al., 2009). Inflammation and peripheral neuropathic lesions have also been shown to induce activation of ERK 1/2 (Obata and Noguchi, 2004; Obata et al., 2004b; Cruz et al., 2005; Seino et al., 2006; Cruz and Cruz, 2007). Examples of stimuli that are known to induce ERK activation in the lumbosacral spinal cord include intracolonic capsaicin (Galan et al., 2003) noxious bladder distension (Cruz et al., 2005) and injections in the hind paw of complete Freund’s adjuvant (CFA) (Ji et al., 2002a) carrageenan (Galan et al., 2002) or formalin (Fukuda et al., 2009). The contribution of ERK activation to the development of acute pain states is also demonstrated by the observation that blockade of ERK phosphorylation reduces mechanical hyperalgesia (Sammons et al., 2000; Ji et al., 2002a; Obata et al., 2004b). Current evidence also indicates that other MAP kinases are involved in neuronal plasticity associated with inflammatory and neuropathic pain (Ji et al., 2002b; Ji and Suter, 2007; Hudmon et al., 2008; Wen et al., 2009; Agostini et al., 2010). For instance, p38 is activated in the lumbosacral spinal cord in an abdominal pain model (Agostini et al., 2010) and p38 phosphorylation in spinal microglia has been observed in a model of mechanical allodynia induced by a peripheral incision (Wen et al., 2009). However, the role of MAP kinase activation, and in particular of ERK activation, in the maintenance of chronic pain states not immediately associated with peripheral injury is unknown. Our laboratory has previously described a model of chronic pain characterized by a robust hyperalgesic state in the abdominal region that appears 5– 6 weeks post-ovariectomy and persists for up to 10 weeks after the procedure (Sanoja and Cervero, 2005, 2008, 2010). This chronic hyperalgesic state includes mechanical hyperalgesia and allodynia in the abdomen, hind limbs and proximal tail, and visceral hyperalgesia and can be quickly reversed by the exogenous administration of estro-

Anesthesia Research Unit (Faculty of Medicine), Faculty of Dentistry and the Alan Edwards Centre for Research on Pain, McGill University, Montreal, QC, Canada

Abstract—Mitogen-activated protein kinases (MAPKs) are important signaling factors in many cellular processes including cell proliferation and survival during development and synaptic plasticity induced by acute nociception in the adult. There is extensive evidence for the involvement of members of the MAPK family, the extracellular signal-regulated kinases 1 and 2 (ERKs 1/2), in the development of acute inflammatory somatic and visceral pain, but their role in the maintenance of chronic pain states is unknown. We have previously shown that ovariectomy of adult mice (OVX) generates a persistent and estrogen-dependent abdominal hyperalgesic state that lasts for several months and is not related to a persistent nociceptive afferent input. Here we have used OVX mice to study a possible role of ERK 1/2 in the spinal processing of this form of chronic abdominal hyperalgesia. Eight weeks after OVX the mice showed a robust abdominal hyperalgesia and a significant increase in the activation of ERK1/2 in the lumbosacral spinal cord. This enhanced activation was not seen in control and sham-operated mice or in regions of the cord other than lumbosacral in OVX mice. Also, the increased activation of ERK 1/2 observed in OVX mice matched the time course of the hyperalgesic state as no activation was observed at week 1 after OVX when the hyperalgesic state had not yet developed. Administration of slow-release pellets containing 17␤-estradiol at week 5 post OVX reversed both the development of the hyperalgesia and the enhanced activation of ERK 1/2, suggesting that this activation, like the hyperalgesic state, was estrogen-dependent. Intrathecal injections of the ERK 1/2 inhibitor U0126 successfully rescued the mice from the abdominal hyperalgesia for up to 24 h after the injection and also reversed the enhanced expression of ERK 1/2. Our study shows, for the first time, activation of ERK 1/2 in the spinal cord matching the time course of an estrogen-dependent chronic hyperalgesic state. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: mitogen activated protein kinase, estradiol, pain, spinal cord, p38.

Mitogen-activated protein kinases (MAPKs) are important signaling factors in a multitude of cellular processes, including cell proliferation and survival during development, nociception, learning and memory and synaptic plasticity in *Corresponding author. Tel: ⫹1-514-398-4565; fax: ⫹1-514-398-8241. E-mail address: [email protected] (M. B. Klinger). Abbreviations: aCSF, artificial cerebrospinal fluid; DMSO, dimethyl sulfoxide; ERK 1/2, extracellular signal-regulated kinases 1 and 2; MAPK, mitogen-activated protein kinase; OVX, ovariectomy.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2010.10.043

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gen. Women suffer from a variety of chronic painful conditions with a greater prevalence than men, including migraine, fibromyalgia, rheumatoid arthritis, temporomandibular disorder and irritable bowel syndrome (Fillingim and Ness, 2000; Sanoja and Cervero, 2010) which points to a potential relationship between sex hormones and chronic pain states. The mechanisms that mediate the development and maintenance of ovariectomy-induced hyperalgesia are yet unknown. In the present study we have investigated if activation of ERK 1/2 in the spinal cord could be involved in the generation of this persistent pain state and, by extension, if spinal ERKs participate in persistent pain states that are hormonally-dependent and not immediately associated with acute noxious input from the periphery.

EXPERIMENTAL PROCEDURES Animals Adult female C57/Bl6 mice were used (21–26 weeks old and 20 –25 g body weight at the start of the experiments). All animals were purchased from Charles River (Boucherville, Canada) including ovariectomized and sham-operated controls. All of the surgical procedures were performed prior to purchase. Animals were received at the McGill Animal Holding Facility 4 days after ovariectomy (OVX) or sham operation. For each experimental procedure we used three groups of matching animals: mice that were ovariectomized bilaterally (OVX), mice that received the same surgical procedure without removing the ovaries (sham surgery) and control animals without any surgical procedure. Both OVX and sham-operated animals received minimal surgery via a dorsal approach. The mice were housed in cages (n⫽5 per cage) with water and food ad libitum on a 12/12 h light/dark cycle. After the conclusion of each experiment the animals were euthanized with an overdose of isoflurane (Baxter Corporation, ON, Canada) and necropsies were performed to confirm the OVX or the sham status. The guidelines of the Canadian Council for Animal Care were rigorously followed and all efforts were made to minimize the number of animals used and their suffering. All protocols were reviewed and approved by the McGill University Animal Care Committee.

Spinal cord extraction and Western blotting Mice were anesthetized with isoflurane (5%), decapitated and the whole spinal cord was extracted by pressure expulsion with icecold saline into an ice-cooled glass dish. The lumbosacral, thoracic and cervical regions were identified as required, excised and snap-frozen in liquid nitrogen. Tissue samples were homogenized in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris– HCl, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, pH 7.4), sonicated for 15 min at 4 °C and centrifuged 5 min at 7500 rpm. Supernatants were collected and total protein amounts measured using Bradford assay (BioRad, Mississauga, ON, Canada). Spinal cord extracts were fractionated by SDS-polyacrylamide gel electrophoresis in 4 –10% polyacrylamide gels and transferred to Immun-blot PVDF transfer membranes (BioRad, Mississauga, ON, Canada). Gels were stained with GelCode Blue Stain Reagent (Pierce, Rockford, IL, USA) following the instructions of the manufacturer to ensure that proteins had transferred correctly to the membrane. The membranes were blocked with 1% bovine serum albumin (BSA) in 1⫻ TTBS buffer (0.1% v/v; Tween 20, 25 mM Tris, 150 mM NaCl, pH 7.5). Membranes were then incubated overnight with the following primary antibodies: rabbit anti-phospho-p44/42 MAPK pAb (1:2,000), rabbit anti-p44/p42

MAPK pAb (1:2,000), rabbit anti-phospho-p38 MAPK mAb (1: 1,000), rabbit anti-p38 MAPK pAb (1:1,000; Cell Signaling Technology, Danvers, MA, USA) or mouse anti-GAPDH (1:20,000; Sigma, Oakville, ON, Canada). Membranes were washed in TTBS and incubated for 1 h at room temperature with the secondary antibody (1:10,000; goat anti-rabbit or donkey anti-mouse horseradish peroxidase-conjugated antibody, Jackon Immunoresearch, Inc., West Grove, PA, USA). After washing with TTBS, the immune complexes were detected by chemiluminescence (Immobilon Western, Millipore, Billerica, MA, USA). Densitometric quantification of immunopositive bands was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For phosphorylated ERK and ERK measurements, the mean value of the two bands was calculated. When multiple blots were required, the data for each lane are represented normalized to the control mean to which we assigned the arbitrary value of 1. Western blot quantification is shown normalized to levels detected in control tissue, run on the same gel. Data are expressed as the ratio of phosphorylated p38, ERK, or total p38, against total GAPDH or total ERK for each sample. Statistical analysis (analysis of variance with post hoc Newman– Keuls test) was performed on the raw data.

Behavioral testing and intrathecal injections All behavioral experiments were performed blind to the surgical treatment of the animals in a room in which the temperature and humidity were controlled (21⫾1 °C, 45⫾5%). The animals were habituated to the environment for at least 30 min before the test started. Eight or nine animals were used per treatment group. The frequency of withdrawal responses to the application of von Frey filaments to the abdomen was examined, as a test of referred mechanical hyperalgesia (see; Sanoja and Cervero, 2005 for details). We used a plastic box divided into 10 small chambers (12.5⫻18⫻15 cm3 each) with a wire mesh floor. A mouse was placed in each of the chambers so that 10 mice were tested sequentially. The animals were allowed to adapt to the chamber for at least 30 min before testing began. Five von Frey filaments with increasing exponential forces of 1, 4, 8, 16 and 32 mN were applied five times each in ascending order of force and the number and intensity of withdrawal responses was noted. The filaments were applied for 1–2 s with an inter-stimulus interval of 5 s. Care was taken not to stimulate the same point twice in succession to avoid learning or sensitization. Only a sharp withdrawal from the filament was considered to be a positive response. The ERK 1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene monoethanolate (U0126; Sigma-Aldrich, Oakville, ON, Canada) was dissolved in 99.9% dimethyl sulfoxide (DMSO) at 40 mg/ml and then diluted in artificial cerebrospinal fluid (aCSF) for a final concentration of 2 ␮g/␮l and a final DMSO concentration of 5%. Five percent DMSO in aCSF was administered as a vehicle control. Thirty min before the start of the behavioral test, the animals were briefly anesthetized with isoflurane (2%). U0126 (2 ␮g/␮l; 23.4 nmol; (Adwanikar et al., 2004; Karim et al., 2006; Seino et al., 2006) or vehicle (5% DMSO in aCSF) were injected intrathecally as previously described (Pitcher et al., 2007). Intrathecal injections were made as a volume of 5 ␮l on lightly anesthetized mice by lumbar puncture at the L4 –L5 level with a 30-gauge needle on a 50 ␮l Hamilton syringe. U0126 was dissolved in aCSF comprised of (in mM) 1.3 CaCl2 2H2O, 2.6 KCl, 0.9 MgCl, 21.0 NaHCO3, 2.5 Na2HPO47H2O, 25.0 NaCl, and 3.5 dextrose (pH 7.2–7.4). The same batch of animals was used for a single test at all time points between 30 min and 24 h post-injection.

Estrogen replacement To test the estrogen dependence of the OVX-induced changes in the lumbosacral spinal cord, groups of OVX animals were im-

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Fig. 1. ERK 1/2 activation and total ERK expression in the spinal cord after OVX. Each panel shows representative Western blots (top) and quantification (bottom) of the phosphorylation of spinal ERK 1/2 (panel A) or the total ERK expression (panel B). (A) Lumbosacral spinal cord 8 wk after OVX, when the hyperalgesic state is robust and fully developed. Note the increase in ERK activation in OVX but not sham operated or control mice. (B) Lumbosacral spinal cord 8 wk after OVX. Note that there is no increase in total ERK expression in OVX mice versus sham operated or control mice. Blot was probed for GAPDH expression as a loading control. In all cases, the two bands shown represent ERK 1 (p44) and ERK 2 (p42). Quantification of ERK 1/2 activation is shown normalized to levels of control tissue run on the same gel. * Pⱕ0.01, n⫽3–7.

planted with pellets of 0.18 mg of 17␤-estradiol or with placebo pellets (Innovative Research of America, Sarasota, FL, USA). These pellets produce a slow release of the active drug over 60 days (3 ␮g per day) and have been shown to maintain estrogen plasma levels of around 125 pg/ml for the duration of the pellet (Lee et al., 2004). The pellets were implanted 5 weeks after OVX once the hyperalgesic state of the animals was fully developed. Pellets were implanted s.c. in the back, between the scapula, under 3% isoflurane using a stainless steel reusable precision trochar (10 gauge, Innovative Research of America). All subsequent tests were carried out blind to the type of pellet implanted (active compound or placebo). The animals were marked individually and a key as to their status was kept separately and only opened at the end of each experimental series after the death of the animals and the postmortem verification of the OVX status.

Statistical analysis All results are expressed as mean⫾SEM. Behavioral data (von Frey) were analyzed by two-way ANOVA with Bonferroni post hoc test; Western blot data were analyzed by one-way ANOVA with Newman–Keuls multiple comparison test. The level of statistical significance was set at P⬍0.05 in all tests.

RESULTS Activation of ERK 1/2 ERK 1/2 activation was measured by Western blotting using a specific antibody. A time point of 8 weeks post-ovariectomy was chosen as the most suitable time for analysis. We have previously shown that the hyperalgesia starts at about 5

weeks post-OVX and that estrogen-replacement therapy takes about 1-week to reverse it (Sanoja and Cervero, 2005, 2008, 2010). We were interested to see if there were changes in ERK activation in the absence of acute stimuli and at a time when the hyperalgesia was well developed and had been established for a couple of weeks. We therefore selected a time point of 8 weeks post-OVX as the most suitable time to test for ERK activation and possible effects of estrogen reversal. As shown in Fig. 1A, at 8 weeks post-ovariectomy surgery we observed a clear activation of ERK 1/2 in the lumbosacral spinal cord (Pⱕ0.05). This is the area of processing of somatic and visceral input from the hyperalgesic region including projections from the hind limbs and the abdomen and visceral afferents from the colon, bladder and uterus (Morgan et al., 1981). No change in total ERK expression was detected in the lumbosacral cord at this time point (8 weeks post-ovariectomy) normalized against total GAPDH (Fig. 1B). No activation of ERK 1/2 was observed at week 1 post-ovariectomy, when the hyperalgesic state is not yet developed (Fig. 2A). Also, no ERK activation was detected in regions of the cord outside the lumbosacral region, like the cervical cord sample shown in Fig. 2B. A separate quantification of ERK-1 and ERK-2 was also carried out. There was a significant 1.5 fold increase (Pⱕ0.05) in the activation of ERK-1 and no statistically significant change in the activation of ERK-2 (Fig. 2C). It therefore appears that the increase in ERK activation was mostly due to activation of ERK-1.

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Fig. 2. ERK 1/2 activation in the spinal cord after OVX. Each panel shows representative Western blots (top) and quantification (bottom) of the phosphorylation of spinal ERK 1/2. (A) Lumbosacral spinal cord 1 wk after OVX when the hyperalgesic state is not yet developed. Blots were probed for total ERK 1/2 expression as a loading control. (B) Cervical spinal cord 8 wk after OVX surgery, showing that the ERK activation is restricted to the lumbosacral spinal cord. (C) Activation of ERK-1 and ERK-2 were analyzed separately. We demonstrate a significant 1.5-fold increase in activation of ERK-1 and no significant increase in the activation of ERK-2. Blots were probed for total ERK-1 or total ERK-2 as a loading control. In all cases, the two bands shown represent ERK 1 (p44) and ERK 2 (p42). Quantification of ERK 1/2 activation is shown normalized to levels of control tissue run on the same gel. * Pⱕ0.01, n⫽3–7.

Treatment with 17␤-estradiol reversed the activation of ERK 1/2 OVX mice received either slow-release estrogen pellets or placebo pellets at week 5 post-surgery, once the hyperalgesic state had already developed. The active pellets used in the study release 17␤-estradiol over 60 days and maintain plasma levels of estrogen around 125 pg/ml for the duration of the pellet. This method of estrogen replacement has previously been shown by our laboratory to reverse the hyperalgesic state of OVX mice (Sanoja and Cervero, 2005) As shown in Fig. 3, control and sham operated mice did not

show activation of ERK 1/2 whereas OVX mice receiving placebo pellets did show a significant ERK activation. Mice that were OVX but received estrogen replacement pellets showed levels of ERK activation similar to those of control and sham mice and significantly smaller than those of OVX mice with no exogenous estrogen (Fig. 3). No change in the activation or expression of p38 in OVX mice To test the possibility that other MAP kinases were also activated during the OVX-induced hyperalgesic state, the

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DISCUSSION

Fig. 3. The activation of ERK 1/2 is estrogen-dependent. (A) Representative Western blots of lumbosacral spinal cord of control (C) and sham operated (S) mice, as well as of OVX mice that received placebo (OVX⫹P) and OVX mice that received 17␤-estradiol pellets (OVX⫹E). Cords were extracted 8 wk after OVX or sham surgery. Placebo and 17␤-estradiol pellets were implanted 5 wk after OVX. (B) quantification of the phospho-ERK 1/2 activation. The levels of phospho-ERK 1/2 activation in OVX⫹P mice are significantly higher than those of control, sham or estrogen treated animals. Blots were probed for total ERK 1/2 expression as a loading control. In all cases, the two bands shown represent ERK 1 (p44) and ERK 2 (p42). Quantification of ERK 1/2 activation is shown normalized to levels of control tissue run on the same gel. * Pⱕ0.01, n⫽9.

activation and expression of p38 was also studied in the lumbosacral spinal cord. As shown in Fig. 4, no change was observed in p38 expression or activation in control, sham or OVX mice at week 8 post-OVX, a time point when OVX mice have a fully developed hyperalgesia. This demonstrates some specificity in the activation of ERK 1/2 in the OVX-induced hyperalgesic state. The MEK inhibitor U0126 reverses ERK activation and rescues the hyperalgesic state A series of behavioral tests were carried out at week 8 post OVX in the presence of the MEK inhibitor U0126. Fig 5 shows the results of such tests on control and sham animals. It can be seen that the MEK inhibitor did not change the baseline behavioral responses to von Frey stimulation in these two groups of animals. However, OVX mice showed enhanced behavioral responses consistent with their hyperalgesic state (Fig. 6A). Intrathecal injection of U0126 (2 ␮g/␮l) reversed the hyperalgesia by significantly decreasing the responses of OVX mice to von Frey hairs 90 min, 3 h, 6 h and 24 h post-injection (* Pⱕ0.05, ** Pⱕ0.01, *** Pⱕ0.001; Fig. 6A). U0126 treatment also reduced the activation of ERK 1/2 in OVX mice as measured by Western blotting (Fig. 6B).

In the present study, we have demonstrated a clear activation of ERK 1/2 in the lumbosacral spinal cord of female mice at 8 weeks post-OVX. We have previously shown that OVX generates a slow developing and persistent hyperalgesic state localized to the abdomen, lower limbs and abdominal viscera. This hyperalgesic state does not appear to be linked to a maintained nociceptive input from the periphery but can be quickly reversed by the exogenous administration of estrogen. In the current study, treatment of hyperalgesic mice with 17␤-estradiol at week 5 postOVX reversed the hyperalgesia and reduced the ERK activation, which was also shown to be restricted to the lumbosacral cord. We also examined the expression in OVX mice of p38, another member of the MAPK family, but failed to find significant changes. Pharmacological blockade of the phosphorylation of ERK resulted in a significant decrease in the hyperalgesic response of OVX mice and a reduced activation of ERK. Taken together, these results indicate a role of spinal ERK 1/2 in the maintenance of the persistent hyperalgesic state induced by OVX which seems to be the consequence of reduced estrogen levels. The spinal activation of ERK has previously been implicated in the development of acute pain, as well as inflammatory and neuropathic pain (Ji et al., 1999, 2002a; Galan et al., 2002, 2003; Choi et al., 2005; Cruz et al., 2005, 2006, 2007). Our study offers the first evidence that ERK activation could also have a role in persistent pain states that are expressed in the absence of an obvious pathology or of continuous nociceptive input from a defined peripheral source. The OVX model produces a slowly developing, chronic hyperalgesic process that is hormonally dependent and mimics some of the pathophysiology of chronic functional pain states in women (Sanoja and Cervero, 2010). However, the mechanism by which estrogen deficiency leads to ERK activation, which in turn maintains a chronic pain state, remains unknown. While there is still much to be studied, recent evidence has pointed to the potential for estrogen receptor influence on nociceptor activity. Estrogen receptor-␣ is expressed in nociceptive-sensitive neurons in the CNS (Amandusson and Blomqvist, 2010), and estrogen is co-expressed with VR1 in bladder-projecting dorsal root ganglion neurons (Bennett et al., 2003). Furthermore, estrogen receptor-␣ is co-expressed with dynorphin in the spinal cord, providing a potential avenue for an estrogen influence in anti-nociceptive activity as well (Gintzler et al., 2008). ERK phosphorylation has been described in various acute pain states including inflammatory and neuropathic. The activation of ERK 1/2 in lumbosacral spinal cord contributes to several downstream effects (Cruz and Cruz, 2007). Activated ERK is involved in transcriptional as well as non-transcriptional processes, and is associated with many factors and plasticity that are relevant to pain processing. ERK activation is required for BDNF activation in DRG in an inflammatory pain model (Obata et al., 2003, 2004b; Obata and Noguchi, 2004), can lead to an increase in neuropeptide Y in DRG neurons (Obata et al., 2004a),

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Fig. 4. The activation and expression of p38 MAP kinase does not change in hyperalgesic OVX mice. (A) Representative Western blots of lumbosacral spinal cord tissue 8 wk after OVX from control (C), sham operated (S) and OVX (OVX) mice. (B) Quantification of p38 activation (phosphorylation) in control, sham and OVX lumbosacral spinal cord tissue. (C) Quantification of total p38 expression in control, sham and OVX lumbosacral spinal cord tissue. Blots were probed for total GAPDH expression as a loading control for both phospho-p38 and p38 expression. Quantification of p38 activation and p38 total expression is shown normalized to levels of control tissue run on the same gel. n⫽3.

and interacts with TRPV1 mediated currents (Bron et al., 2003). In spinal cord, ERK 1/2 activation has been found to increase expression of the NR1 subunit of NMDA after spinal cord injury (Yu and Yezierski, 2005), can phosphorylate the potassium channel Kv 4.2 (Adams et al., 2000; Hu et al., 2006), is required for upregulation of NK1 and prodynorphin seen with hind paw inflammation (Ji et al., 2002a), and can regulate the pain-evoked immediate early gene c-fos (Johnson et al., 1997; Cruz et al., 2007). Yet, these are not the only specific target genes regulated by ERK (Obata and Noguchi, 2004). Intrathecal blockade of ERK phosphorylation by blockade of its upstream kinase, MEK, has been used to attenuate experimentally induced mechanical allodynia in inflammatory and neuropathic pain, heat hypersensitivity, and mechanical hyperalgesia (Galan et al., 2003; Obata et al., 2003, 2004a,b; Adwanikar et al., 2004; Dai et al., 2004; Kawasaki et al., 2004; Obata and Noguchi, 2004; Choi et al., 2005; Song et al., 2005; Zhuang et al., 2005; Daulhac et al., 2006; Karim et al., 2006; Seino et al., 2006; Komatsu et al., 2009). It also reduced frequency and pressure of bladder contractions in animals with bladder inflammation (Cruz et al., 2006). Experimental evidence demonstrates several potential upstream effectors of ERK 1/2 activation in spinal pathways in pain models, including the activation of ionotropic and metabotropic glutamate receptors, brain derived neurotrophic factor binding its specific receptor TrkB, and involvement of the protein kinase C (PKC), protein kinase A (PKA) and phosphoinositide 3-kinase

(PI3K) pathways (Ji et al., 2002a; Lever et al., 2003; Kawasaki et al., 2004; Slack et al., 2005; Karim et al., 2006). NGF and the ATP receptor, P2X3 play a regulatory role in ERK phosphorylation in spinal cord during peripheral inflammation (Averill et al., 2001; Obata et al., 2003). There is also evidence that supraspinal serotonergic input could influence ERK activation in spinal cord in paw formalin model (Svensson et al., 2006). In our study we have used intrathecal injection of U0126, thus avoiding the blood– brain barrier and our injections were at a dose comparable to that of other published studies (Obata et al., 2003; Adwanikar et al., 2004; Karim et al., 2006; Komatsu et al., 2009). Control experiments were performed to ensure that spinal ERK activation was indeed inhibited with the dose used. The overall effect of ERK blockade was significant at 90 min post-injection and persisted for up to 6 h postinjection, suggesting that both transcriptional regulation and post-translational events could be involved in the activation of ERK 1/2. There is also strong evidence in the literature for ERK 1/2 activation localized to the spinal dorsal horn. Immunohistochemical staining has shown activation in the superficial laminae I–II of the spinal dorsal horn, as well as select supraspinal structures, induced by cutaneous stimuli, hind paw injection of Complete Freund’s Adjuvant (Ji et al., 1999; Ji et al., 2002a; Karim et al., 2006), colorectal distension (Zhang et al., 2009), muscle pain (Liu et al., 2004), bladder distension (Cruz et al., 2005), and the formalin test (Fukuda et al., 2009). The activation of ERK 1/2 in our OVX

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Fig. 5. Behavioral responses of control and sham operated mice to von Frey stimulation of their abdomens and effects of administration of the ERK inhibitor U0126. The graphs show the percent responses to abdominal stimulation with five von Frey hair intensities (1, 4, 8, 16, 32 mN). Data are expressed as mean percent response frequency ⫾SEM. Administration of the inhibitor does not change the behavioral responses in either control or sham operated mice.

mice was specific to the lumbosacral cord, as Western blots in the cervical spinal cord did not show a change in activation of phospho-ERK. Since the OVX-induced hyperalgesia is localized to hind limbs, abdomen and the base of the tail, as well as the abdominal viscera, this observation suggests that the activation is specific to the region of the spinal cord that receives afferents from the hyperalgesic areas. This could be due to a persistent and yet unidentified afferent input from these regions or to the sensitization of lumbosacral spinal neurons by mechanisms linked to estrogen deficiency. The abdominal localization of the

OVX-induced hyperalgesia and its estrogen dependency have been puzzling factors of this model. If the cause of the hyperalgesic state was a hormonal imbalance, a more generalized hyperalgesic state would be expected. On the other hand the easy reversal of the hyperalgesia by systemic estrogen seems to rule out a single peripheral focus as the cause of the painful state. The current observation of a persistent activation of lumbosacral ERKs that matches the timeline and estrogen dependency of the hyperalgesic state, points to the existence of some kind of maintained peripheral drive that still needs to be identified.

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Fig. 6. Intrathecal injection of the MEK inhibitor U0126 (2 ␮g/␮l) reduces the abdominal hyperalgesia induced by OVX and the increased ERK activation in OVX mice. (A) Graphs showing a significant reduction in percent response to abdominal stimulation with five von Frey hair intensities (1, 4, 8, 16, 32 mN). Data are expressed as mean percent response frequency ⫾SEM. Asterisks indicate significant difference in response frequency between OVX⫹vehicle and OVX⫹U0126. * Pⱕ0.05, ** Pⱕ0.01, *** Pⱕ0.001, n⫽8 –9 for all groups. (B) Western blot of lumbosacral spinal cord 24 h after intrathecal injection of U0126 demonstrating a reduction in ERK 1/2 activation in mice that received intrathecal U0126 (two lanes) as compared to mice that received intrathecal vehicle (5% DMSO in aCSF). As a loading control, blots were probed a second time for total ERK 1/2 expression. Quantified data shown below the blots (n⫽9, * Pⱕ0.01).

ERK activation occurs in both glial cells and neurons in the CNS. Our Western blot studies include homogenates of the lumbosacral spinal cord in its entirety, so it is pos-

sible that the effect we see of ERK 1/2 activation is in part due to glial as well as neuronal activation (Obata and Noguchi, 2004; Wang et al., 2004, 2009). ERK activation

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could influence prostaglandin expression, inducible nitric oxide synthase, and the expression of cytokines interleukin-1␤, tumor necrosis factor-␣, interleukin-6 in glia in the spinal cord (Marchand et al., 2005; Wang et al., 2009). The ERK1 and ERK2 isoforms can be differentially activated, often demonstrating different roles for each (Xu et al., 2008; Cheng and Keast, 2009; Alter et al., 2010). While Xu et al. (Xu et al., 2008) have demonstrated a role for ERK2 specifically in the development of complete Freund’s adjuvant-induced hyperalgesia, we have shown exclusive activation of ERK1 in the lumbosacral spinal cord of ovariectomized mice as compared to control and sham. This fits with a study by Cheng and Keast (2009), who have shown an increase in ERK1 activation in the dorsal root ganglia of ovariectomized rats (Cheng and Keast, 2009). The p38 MAPK pathway has been implicated as an underlying factor in neuropathic pain conditions, and to affect expression of TRPV1 in primary sensory neurons, and phosphorylation and current density the sodium channel Nav 1.8 (Ji and Suter, 2007; Hudmon et al., 2008). This kinase has been described to be activated in a visceral hyperalgesia model of narcotic bowel syndrome (Agostini et al., 2010). Even more relevant to our study, p38 has been shown to be activated in DRG cultures following 17␤-estradiol treatment, and p38 total expression and activation was increased in lumbosacral DRG of OVX rats 4 weeks post-surgery (Cheng and Keast, 2009). However, in our study, we saw no activation or change in expression of p38 MAP kinase. It is possible that a spinal cord homogenate is not sensitive enough to detect changes that might have occurred in expression of specific cell types, including afferents. Also, the possibility exists that p38 is not involved in longer-term molecular changes such as those expected to operate in the maintenance of the persistent hyperalgesia induced by OVX.

CONCLUSION This study shows, for the first time, activation of ERK 1/2 in spinal cord in a chronic model of pain involving no clear and demonstrable sustained peripheral input. This activation matches the time course and estrogen dependency of the hyperalgesic state induced by OVX. Further study is required to determine the details of the interaction between the hormonal imbalance, the activation of ERK 1/2 and the generation of the persistent pain state. Acknowledgments—This study was supported by the Canadian Foundation for Innovation (CFI) and the Canadian Institutes of Health Research (CIHR). MBK received the 2008 Ronald Melzack Postdoctoral Fellowship from the Louise and Alan Edwards Foundation. FC is the holder of a CIHR Research Chair. The authors are grateful to Lisa Krawec and Albena Davidova for their expert technical support.

REFERENCES Adams JP, Anderson AE, Varga AW, Dineley KT, Cook RG, Pfaffinger PJ, Sweatt JD (2000) The A-type potassium channel Kv4.2 is a

491

substrate for the mitogen-activated protein kinase ERK. J Neurochem 75:2277–2287. Adwanikar H, Karim F, Gereau R 4th (2004) Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation. Pain 111:125–135. Agostini S, Eutamene H, Cartier C, Broccardo M, Improta G, Houdeau E, Petrella C, Ferrier L, Theodorou V, Bueno L (2010) Evidence of central and peripheral sensitization in a rat model of narcotic bowel like-syndrome. Gastroenterology 139:553–563, 563.e1–5. Alter BJ, Zhao C, Karim F, Landreth GE, Gereau R 4th (2010) Genetic targeting of ERK1 suggests a predominant role for ERK2 in murine pain models. J Neurosci 30:11537–11547. Amandusson A, Blomqvist A (2010) Estrogen receptor-alpha expression in nociceptive-responsive neurons in the medullary dorsal horn of the female rat. Eur J Pain 14:245–248. Averill S, Delcroix JD, Michael GJ, Tomlinson DR, Fernyhough P, Priestley JV (2001) Nerve growth factor modulates the activation status and fast axonal transport of ERK 1/2 in adult nociceptive neurones. Mol Cell Neurosci 18:183–196. Bennett HL, Gustafsson JA, Keast JR (2003) Estrogen receptor expression in lumbosacral dorsal root ganglion cells innervating the female rat urinary bladder. Auton Neurosci 105:90 –100. Bron R, Klesse LJ, Shah K, Parada LF, Winter J (2003) Activation of Ras is necessary and sufficient for upregulation of vanilloid receptor type 1 in sensory neurons by neurotrophic factors. Mol Cell Neurosci 22:118 –132. Cheng Y, Keast JR (2009) Effects of estrogens and bladder inflammation on mitogen-activated protein kinases in lumbosacral dorsal root ganglia from adult female rats. BMC Neurosci 10:156. Choi SS, Seo YJ, Kwon MS, Shim EJ, Lee JY, Ham YO, Park SH, Suh HW (2005) Involvement of phosphorylated extracellular signalregulated kinase in the mouse substance P pain model. Brain Res Mol Brain Res 137:152–158. Chuang CF, Ng SY (1994) Functional divergence of the MAP kinase pathway. ERK1 and ERK2 activate specific transcription factors. FEBS Lett 346:229 –234. Cruz CD, Avelino A, McMahon SB, Cruz F (2005) Increased spinal cord phosphorylation of extracellular signal-regulated kinases mediates micturition overactivity in rats with chronic bladder inflammation. Eur J Neurosci 21:773–781. Cruz CD, Cruz F (2007) The ERK 1 and 2 pathway in the nervous system: from basic aspects to possible clinical applications in pain and visceral dysfunction. Curr Neuropharmacol 5:244 –252. Cruz CD, Ferreira D, McMahon SB, Cruz F (2007) The activation of the ERK pathway contributes to the spinal c-fos expression observed after noxious bladder stimulation. Somatosens Mot Res 24:15–20. Cruz CD, McMahon SB, Cruz F (2006) Spinal ERK activation contributes to the regulation of bladder function in spinal cord injured rats. Exp Neurol 200:66 –73. Dai Y, Fukuoka T, Wang H, Yamanaka H, Obata K, Tokunaga A, Noguchi K (2004) Contribution of sensitized P2X receptors in inflamed tissue to the mechanical hypersensitivity revealed by phosphorylated ERK in DRG neurons. Pain 108:258 –266. Daulhac L, Mallet C, Courteix C, Etienne M, Duroux E, Privat AM, Eschalier A, Fialip J (2006) Diabetes-induced mechanical hyperalgesia involves spinal mitogen-activated protein kinase activation in neurons and microglia via N-methyl-D-aspartate-dependent mechanisms. Mol Pharmacol 70:1246 –1254. Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis RJ (1995) Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267: 682– 685. Fillingim RB, Ness TJ (2000) Sex-related hormonal influences on pain and analgesic responses. Neurosci Biobehav Rev 24:485–501. Fukuda T, Hisano S, Tanaka M (2009) Licking decreases phosphorylation of extracellular signal-regulated kinase in the dorsal horn of the spinal cord after a formalin test. Anesth Analg 109:1318 –1322.

492

M. B. Klinger et al. / Neuroscience 172 (2011) 483– 493

Galan A, Cervero F, Laird JM (2003) Extracellular signaling-regulated kinase-1 and -2 (ERK 1/2) mediate referred hyperalgesia in a murine model of visceral pain. Brain Res Mol Brain Res 116: 126 –134. Galan A, Lopez-Garcia JA, Cervero F, Laird JM (2002) Activation of spinal extracellular signaling-regulated kinase-1 and -2 by intraplantar carrageenan in rodents. Neurosci Lett 322:37– 40. Gintzler AR, Schnell SA, Gupta DS, Liu NJ, Wessendorf MW (2008) Relationship of spinal dynorphin neurons to delta-opioid receptors and estrogen receptor alpha: anatomical basis for ovarian sex steroid opioid antinociception. J Pharmacol Exp Ther 326:725– 731. Gioia M, Galbiati S, Rigamonti L, Moscheni C, Gagliano N (2001) Extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the tele- and diencephalon of rat after visceral pain stimulation: an immunocytochemical study. Neurosci Lett 308:177–180. Hu HJ, Carrasquillo Y, Karim F, Jung WE, Nerbonne JM, Schwarz TL, Gereau R 4th (2006) The kv4.2 potassium channel subunit is required for pain plasticity. Neuron 50:89 –100. Hudmon A, Choi JS, Tyrrell L, Black JA, Rush AM, Waxman SG, Dib-Hajj SD (2008) Phosphorylation of sodium channel Na(v)1.8 by p38 mitogen-activated protein kinase increases current density in dorsal root ganglion neurons. J Neurosci 28:3190 –3201. Ji RR, Baba H, Brenner GJ, Woolf CJ (1999) Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 2:1114 –1119. Ji RR, Befort K, Brenner GJ, Woolf CJ (2002a) ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J Neurosci 22:478 – 485. Ji RR, Gereau R 4th, Malcangio M, Strichartz GR (2009) MAP kinase and pain. Brain Res Rev 60:135–148. Ji RR, Samad TA, Jin SX, Schmoll R, Woolf CJ (2002b) p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 36:57– 68. Ji RR, Suter MR (2007) p38 MAPK, microglial signaling, and neuropathic pain. Mol Pain 3:33. Johnson ML, Redmer DA, Reynolds LP (1997) Uterine growth, cell proliferation, and c-fos proto-oncogene expression throughout the estrous cycle in ewes. Biol Reprod 56:393– 401. Karim F, Hu HJ, Adwanikar H, Kaplan D, Gereau R 4th (2006) Impaired inflammatory pain and thermal hyperalgesia in mice expressing neuron-specific dominant negative mitogen activated protein kinase kinase (MEK). Mol Pain 2:2. Kawasaki Y, Kohno T, Zhuang ZY, Brenner GJ, Wang H, Van Der Meer C, Befort K, Woolf CJ, Ji RR (2004) Ionotropic and metabotropic receptors, protein kinase A, protein kinase C, and Src contribute to C-fiber-induced ERK activation and cAMP response element-binding protein phosphorylation in dorsal horn neurons, leading to central sensitization. J Neurosci 24:8310 – 8321. Komatsu T, Sakurada S, Kohno K, Shiohira H, Katsuyama S, Sakurada C, Tsuzuki M, Sakurada T (2009) Spinal ERK activation via NO-cGMP pathway contributes to nociceptive behavior induced by morphine-3-glucuronide. Biochem Pharmacol 78: 1026 –1034. Lee JY, Kim JH, Hong SH, Cherny RA, Bush AI, Palmiter RD, Koh JY (2004) Estrogen decreases zinc transporter 3 expression and synaptic vesicle zinc levels in mouse brain. J Biol Chem 279:8602– 8607. Lever IJ, Pezet S, McMahon SB, Malcangio M (2003) The signaling components of sensory fiber transmission involved in the activation of ERK MAP kinase in the mouse dorsal horn. Mol Cell Neurosci 24:259 –270. Liu Y, Obata K, Yamanaka H, Dai Y, Fukuoka T, Tokunaga A, Noguchi K (2004) Activation of extracellular signal-regulated protein kinase in dorsal horn neurons in the rat neuropathic intermittent claudication model. Pain 109:64 –72.

Liverman CS, Brown JW, Sandhir R, Klein RM, McCarson K, Berman NE (2009) Oestrogen increases nociception through ERK activation in the trigeminal ganglion: evidence for a peripheral mechanism of allodynia. Cephalalgia 29:520 –531. Marchand F, Perretti M, McMahon SB (2005) Role of the immune system in chronic pain. Nat Rev Neurosci 6:521–532. Morgan C, Nadelhaft I, de Groat WC (1981) The distribution of visceral primary afferents from the pelvic nerve to Lissauer’s tract and the spinal gray matter and its relationship to the sacral parasympathetic nucleus. J Comp Neurol 201:415– 440. Obata K, Noguchi K (2004) MAPK activation in nociceptive neurons and pain hypersensitivity. Life Sci 74:2643–2653. Obata K, Yamanaka H, Dai Y, Mizushima T, Fukuoka T, Tokunaga A, Noguchi K (2004a) Differential activation of MAPK in injured and uninjured DRG neurons following chronic constriction injury of the sciatic nerve in rats. Eur J Neurosci 20:2881–2895. Obata K, Yamanaka H, Dai Y, Tachibana T, Fukuoka T, Tokunaga A, Yoshikawa H, Noguchi K (2003) Differential activation of extracellular signal-regulated protein kinase in primary afferent neurons regulates brain-derived neurotrophic factor expression after peripheral inflammation and nerve injury. J Neurosci 23:4117– 4126. Obata K, Yamanaka H, Kobayashi K, Dai Y, Mizushima T, Katsura H, Fukuoka T, Tokunaga A, Noguchi K (2004b) Role of mitogenactivated protein kinase activation in injured and intact primary afferent neurons for mechanical and heat hypersensitivity after spinal nerve ligation. J Neurosci 24:10211–10222. Pitcher MH, Price TJ, Entrena JM, Cervero F (2007) Spinal NKCC1 blockade inhibits TRPV1-dependent referred allodynia. Mol Pain 3:17. Sammons MJ, Raval P, Davey PT, Rogers D, Parsons AA, Bingham S (2000) Carrageenan-induced thermal hyperalgesia in the mouse: role of nerve growth factor and the mitogen-activated protein kinase pathway. Brain Res 876:48 –54. Sanoja R, Cervero F (2005) Estrogen-dependent abdominal hyperalgesia induced by ovariectomy in adult mice: a model of functional abdominal pain. Pain 118:243–253. Sanoja R, Cervero F (2008) Estrogen modulation of ovariectomyinduced hyperalgesia in adult mice. Eur J Pain 12:573–581. Sanoja R, Cervero F (2010) Estrogen-dependent changes in visceral afferent sensitivity. Auton Neurosci 153:84 – 89. Seino D, Tokunaga A, Tachibana T, Yoshiya S, Dai Y, Obata K, Yamanaka H, Kobayashi K, Noguchi K (2006) The role of ERK signaling and the P2X receptor on mechanical pain evoked by movement of inflamed knee joint. Pain 123:193–203. Slack SE, Grist J, Mac Q, McMahon SB, Pezet S (2005) TrkB expression and phospho-ERK activation by brain-derived neurotrophic factor in rat spinothalamic tract neurons. J Comp Neurol 489: 59 – 68. Song XS, Cao JL, Xu YB, He JH, Zhang LC, Zeng YM (2005) Activation of ERK/CREB pathway in spinal cord contributes to chronic constrictive injury-induced neuropathic pain in rats. Acta Pharmacol Sin 26:789 –798. Svensson CI, Tran TK, Fitzsimmons B, Yaksh TL, Hua XY (2006) Descending serotonergic facilitation of spinal ERK activation and pain behavior. FEBS Lett 580:6629 – 6634. Wang H, Dai Y, Fukuoka T, Yamanaka H, Obata K, Tokunaga A, Noguchi K (2004) Enhancement of stimulation-induced ERK activation in the spinal dorsal horn and gracile nucleus neurons in rats with peripheral nerve injury. Eur J Neurosci 19:884 – 890. Wang W, Mei X, Huang J, Wei Y, Wang Y, Wu S, Li Y (2009) Crosstalk between spinal astrocytes and neurons in nerve injury-induced neuropathic pain. PLoS One 4:e6973. Wen YR, Suter MR, Ji RR, Yeh GC, Wu YS, Wang KC, Kohno T, Sun WZ, Wang CC (2009) Activation of p38 mitogen-activated protein kinase in spinal microglia contributes to incision-induced mechanical allodynia. Anesthesiology 110:155–165. Xu Q, Garraway SM, Weyerbacher AR, Shin SJ, Inturrisi CE (2008) Activation of the neuronal extracellular signal-regulated kinase 2 in

M. B. Klinger et al. / Neuroscience 172 (2011) 483– 493 the spinal cord dorsal horn is required for complete Freund’s adjuvant-induced pain hypersensitivity. J Neurosci 28:14087– 14096. Yu CG, Yezierski RP (2005) Activation of the ERK1/2 signaling cascade by excitotoxic spinal cord injury. Brain Res Mol Brain Res 138:244 –255. Zhang XJ, Li Z, Chung EK, Zhang HQ, Xu HX, Sung JJ, Bian ZX (2009) Activation of extracellular signal-regulated protein kinase is

493

associated with colorectal distension-induced spinal and supraspinal neuronal response and neonatal maternal separation-induced visceral hyperalgesia in rats. J Mol Neurosci 37:274 –287. Zhuang ZY, Gerner P, Woolf CJ, Ji RR (2005) ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 114:149 –159.

(Accepted 15 October 2010) (Available online 31 October 2010)