- Email: [email protected]
Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation
Pain 111 (2004) 125–135 www.elsevier.com/locate/pain
Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation Hita Adwanikara, Farzana Karima,b, Robert W. Gereau IVa,b,* a Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave, Campus Box 8054, St. Louis, MO 63110, USA
b
Received 16 December 2003; received in revised form 23 April 2004; accepted 7 June 2004
Abstract Group I metabotropic glutamate receptors (mGluRs) and their downstream signaling pathways, which involve the extracellular signalregulated kinases (ERKs), have been implicated as mediators of plasticity in several pain models. In this study, we report that inflammation leads to a long-lasting enhancement of behavioral responses induced by activation of spinal group I mGluRs. Thus, the nocifensive response to intrathecal injection of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG) is significantly potentiated seven days following Complete Freund’s Adjuvant (CFA)-induced inflammation of the hind paw. This potentiation is not associated with increased mGlu1 or mGlu5 receptor expression but is associated with increased levels of phosphorylated ERK in dorsal horn neurons. We also tested whether the increased behavioral response to DHPG following inflammation may be explained by enhanced coupling of the group I mGluRs to ERK activation. DHPG-induced ERK phosphorylation in the dorsal horn is not potentiated following inflammation. However, inhibiting ERK activation using a MEK inhibitor, U0126, following inflammation attenuates the intrathecal DHPG-induced behavioral responses to a greater extent than in control animals. The results from this study indicate that persistent ERK activation is required for the enhanced behavioral responses to spinal group I mGluR activation following inflammation and suggest that tonic modulation of ERK activity may underlie a component of central sensitization in dorsal horn neurons. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: DHPG; Pain; Spinal cord; Metabotropic; CFA; MAPK; Nociception
1. Introduction The mechanisms underlying inflammatory pain are being extensively studied, and a large body of evidence suggests that inflammation sensitizes peripheral afferents and dorsal horn neurons (Millan, 1999). Group I mGluRs (mGlu1 and mGlu5) are present in the dorsal horn of the spinal cord (Alvarez et al., 2000; Berthele et al., 1999; Karim et al., 2001) and have been implicated in inflammatory and neuropathic pain (Fisher and Coderre, 1996; Fundytus et al., 1998; Karim et al., 2001; Neugebauer et al., 1999; Yashpal et al., 2001; Young et al., 1997). Moreover, mGluRs are required for inflammation-evoked hyperexcitability * Corresponding author. Address: Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave, Campus Box 8054, St. Louis, MO 63110, USA. Tel.: þ 1-314-362-8312; fax: þ1-314-362-8571. E-mail address: [email protected] (R.W. Gereau IV).
and sustained nociceptive transmission (Neugebauer et al., 1994; Young et al., 1994). The group I mGluRs play a more prominent role in nociceptive processing in the context of inflammation, suggesting a functional up-regulation (Neugebauer, 2002; Neugebauer et al., 1999; Stanfa and Dickenson, 1998; Varney and Gereau, 2002). Changes in group I mGluR expression in the spinal cord and other regions of the CNS have been observed in a variety of pain models as well as in clinical inflammation (Dolan et al., 2003; Mills et al., 2001; Neugebauer et al., 2003). Several studies have shown a critical role for the activation of ERKs in dorsal horn neurons in various pain models (Hu and Gereau, 2003; Hu et al., 2003; Ji et al., 1999, 2002; Karim et al., 2001; Kominato et al., 2003; Lever et al., 2003; Ma and Quirion, 2002). Group I mGluR activation, as well as inflammation, leads to ERK dependent sensitization of nociception (Galan et al., 2002, 2003; Ji et al., 1999; Karim et al., 2001; Ma and Quirion, 2002).
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.06.009
126
H. Adwanikar et al. / Pain 111 (2004) 125–135
ERKs regulate neuronal excitability in dorsal horn neurons through modulation of A-type potassium currents (Hu and Gereau, 2003; Hu et al., 2003). It has been shown that ERK activation increases following inflammation, for 60 min following formalin injection (Ji et al., 1999; Karim et al., 2001), 65 min in the carrageenan model (Galan et al., 2002), 3 h following noxious visceral stimuli (Galan et al., 2003) and 48 h in CFA-induced inflammation (Ji et al., 2002). However, the role of ERK activation in maintenance of longer lasting forms of inflammatory pain is not clear. The experimental evidence discussed earlier suggests critical roles for group I mGluR signaling and the ERK cascade in nociceptive plasticity. The present study supports the hypothesis that effects of mGlu1/5 activation are enhanced following inflammation, and further that this enhancement is mediated by sustained ERK activation in dorsal horn neurons.
All drugs or their corresponding vehicles were injected intrathecally in a total volume of 5 ml using a 25 ml Hamilton syringe and 30 gauge needles. Mice were pretreated intrathecally for 15 min with the antagonists or appropriate vehicles. 2.3. Behavioral analysis Behavioral tests began with a habituation period, in which mice were placed in Plexiglas cubicles for at least 1 h. For mechanical allodynia, the plantar surface of the hind paw was stimulated with a series of von Frey filaments. The threshold was taken as the lowest force that evoked a brisk withdrawal response. The behavioral response to intrathecal DHPG was measured as the time spent in spontaneous nocifensive behavior for 30 min immediately following a single intrathecal DHPG injection. The spontaneous behaviors measured in the ICR mice include caudally oriented licking, scratching and lifting behaviors.
2. Materials and methods 2.4. Assessment of motor function 2.1. Animals The ICR strain of outbred mice (Tac:Icr:Ha(ICR), Taconic, Germantown, NY) were housed in cages with access to food and water ad libitum on a 12 h light/dark cycle. Seven- to ten-week-old male mice weighing 20– 25 g were used for this study. All experiments were done in accordance with ethical guidelines for investigation of pain (Zimmermann, 1983) as well as the guidelines of the National Institutes of Health and The International Association for the Study of Pain and were approved by the Animal Care and Use Committee of Baylor College of Medicine. Mice were allowed to acclimate for at least three days before any behavioral tests. 2.2. Drug administration To induce persistent inflammation, we injected 10 ml of Complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO) into the plantar surface of a hind paw of the mouse. CFA is a commonly used inflammatory agent containing a suspension of heat killed and dried Mycobacterium tuberculosis in paraffin oil. The group I metabotropic Glutamate receptor (mGluR) agonist, (RS)-3,5-Dihydroxyphenylglycine (DHPG; Tocris Cookson Inc, Ellisville, MO) was dissolved in sterile phosphate buffered saline. The MEK inhibitor 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene (U0126; Calbiochem, San Diego, CA) and its inactive analog 1,4-Diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0124; Calbiochem, San Diego, CA) were dissolved in 10% dimethyl sulphoxide (DMSO; Sigma) at 1 mg/ml and then diluted with phosphate buffered saline (PBS) to the appropriate concentrations. The final concentration of DMSO was 0.8% for 0.5 nmol U0126 or U0124.
To test for effects of U0126 and U0124 on motor function, an accelerating rotarod treadmill (Ugo Basile, Comerio, Italy) was used to examine the motor ability of the animals. The mice were placed on the rotating rod at its slowest speed 15 min following intrathecal injection and the time for which the mice stayed on the rod was measured (Malmberg et al., 2003). The mice were not trained in this test prior to the experiment. Each trial was limited to 5 min. The mice were tested for 5 trials with an interval of 15 min between trials. 2.5. Sample preparation Spinal cords were isolated at the indicated time points and ipsilateral and contralateral lumbar spinal cord enlargements (L4 – S1) were homogenized using a Dounce homogenizer in ice-cold homogenization buffer (50 mM Tris– HCl (pH 7.5), 50 mM NaCl, 10 mM EGTA, 5 mM EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 200 mM paranitrophenylphosphate, 1 mM phenylmethylsulfonyl fluoride, 20 mg/ml leupeptin, and 4 mg/ml aprotinin; Sigma). Protein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA). For membrane preparation, the section of the spinal cord was homogenized in a Dounce homogenizer 400 ml of 10% sucrose in 10 mM Tris –HCl (pH 7.4), with 1 mM dithiothreitol (DTT). The homogenate was centrifuged for 10 min at 1000 £ g: All centrifugation steps were done at 4 8C. The supernatant was saved, and the pellet re-homogenized in half the original volume of the same buffer. The homogenate was centrifuged for 10 min at 1000 £ g: The supernatants from both centrifugations were combined and centrifuged for 1 h at 60; 000 £ g: The pellets were re-suspended in 10 mM Tris – HCl (pH 7.4) buffer containing 1 mM DTT
H. Adwanikar et al. / Pain 111 (2004) 125–135
and 5 mM MgCl2. Samples were centrifuged for 30 min at 60; 000 £ g: The neuronal membrane pellets were resuspended in 150 ml of the same buffer and stored at 2 80 8C. 2.6. Immunoblotting for total and phospho-ERK Samples (10 mg protein) were electrophoresed in 10% SDS polyacrylamide gels, electrophoretically transferred onto protein-sensitive nitrocellulose membranes (Criterion blotter, Bio-Rad) and blocked in B-TTBS [3% bovine serum albumin (BSA), 50 mM Tris – HCl (pH 7.5), 150 mM NaCl, 0.02 mM Sodium orthovanadate, 0.05% Tween 20, 1 mM NaF, 1 mM Na4P2O7 and 0.01% thimerosal; Sigma] for 2 h at room temperature. Antibody applications were done in B-TTBS. An anti-phospho-ERK primary antibody that detects ERK1 (44 kDa) as well as ERK2 (42 kDa) phosphorylation at both Thr202 and Tyr204 (1:1000 dilution in B-TTBS; Cell Signaling Technology, Beverly, MA) was used for immunoblotting overnight at 4 8C. An anti-ERK primary antibody (1:1000 dilution in 3% BSA; Upstate Biotechnology, Lake Placid, NY) which detects total ERK1 and ERK2 (44/42 kDa) isoforms was used for immunoblotting for 1 h at room temperature. 2.7. Immunoblotting for mGlu1a and mGlu5 Proteins (10 mg) were electrophoresed in 10% SDS polyacrylamide gels. Proteins were transferred onto proteinsensitive nitrocellulose membranes and blocked in 4% milk in TTBS (50 mM Tris – HCl (pH 7.5), 150 mM NaCl and 0.05% Tween 20] for 1 h at room temperature. The antibody applications were done in 4% milk in TTBS. Anti-mGluRa antibody (1:1000, Upstate), anti-mGlu5 antiserum (1:1000) raised in our lab against a peptide corresponding to the mGlu5 C terminus (CSSPKYDTLIIRDYTNSSSSL) and Anti-Actin antibody (1:1000, Sigma) were used for immunoblotting overnight at 4 8C. The membranes were washed and incubated in HRP-conjugated secondary antibody for 1 h at room temperature. Blots were developed with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL). Densitometric quantification of immunopositive bands for total or phospho-p44/42 ERK was done using computerized image analysis software (Scion Corp., Frederick, MD). To ensure that the bands are in the linear range of the film, band densities were plotted against exposure times for a known amount of protein. Band densities in the linear range of the plot were used for quantification. 2.8. Immunocytochemistry Seven days following CFA injection into the hind paw, mice were anesthetized with Avertin (2,2,2 tribromoethanol, Aldrich, 0.8 –1.2 mg/g, i.p.) and perfused transcardially with warm saline (37 8C, 0.9% NaCl), followed by 250 ml of ice-cold 4% paraformaldehyde solution.
127
L4 – S1 lumbar spinal cord sections were dissected out and post-fixed at 4 8C for 4 h with paraformaldehyde, followed by overnight cryoprotection at 4 8C in 30% sucrose. After post-fixing, the ventral contralateral side of the spinal cord was marked by a small scalpel incision to identify ipsilateral and contralateral sides following immunocytochemistry. Tissue was embedded in OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN) and stored at 2 80 8C. Coronal sections (30 mM) were cut using a freezing sliding microtome, and free floating sections were kept in PBS (pH 7.4). The sections were rinsed in 10% methanol and 0.3% H2O2 in 0.1 M PBS for 30 min and then blocked in 3% normal goat serum (NGS) with 0.2% Triton X-100 (NGST) two times for 10 min each. All antibodies were diluted in 1% NGST. Sections were then incubated at 4 8C for 36– 48 h in anti-phospho-p44/42 ERK primary antibody (1:1000 dilution in B-TTBS; Cell Signaling Technology. Following the antibody incubation, the sections were rinsed with 1% NGST two times for 10 min each, followed by incubation in a secondary biotinylated anti-rabbit IgG antibody for 90 min (1:200; ABC kit; Vector Laboratories, Burlingame, CA). The sections were again rinsed with 1% NGST two times for 10 min each and incubated in ExtraAvidin peroxidase (1:1000; Sigma) for 1 h at room temperature. They were then rinsed in 0.1 M PBS two times for 10 min each and then in phosphate buffer (two times for 10 min each) and stained with 3,30 -diaminobenzidine tetrahydrochloride (DAB) solution (0.025% DAB in phosphate buffer containing 0.0025% H2O2; Sigma) for 5 – 10 min. The sections were mounted onto gelatin-coated glass slides, air-dried, dehydrated, cleared with Hemo De, coverslipped with DPX mounting medium, and observed for phospho-ERK staining. For laminar distribution analysis, DAB-stained sections were compared to nissl stained sections (4.5 mg/ml Cresyl Violet, Sigma) from a comparable region of the lumbar spinal cord. Different treatments were processed in parallel using the same batch of solutions. The DAB positive cells were identified under a microscope. All cell counts were done while blind to treatment. 2.9. Data presentation and statistical analysis Behavioral responses to intrathecal DHPG are presented as the number of seconds spent in nocifensive behavior. Band densities of mGlu1a and mGlu5 in immunoblotting experiments were plotted relative to density of Actin band for each sample. Band density of phosphorylated ERK was plotted relative to density of total ERK band for each sample. Immunopositive profiles were plotted as average counts per section for each treatment group. Percent attenuation of the DHPG-induced nocifensive response is plotted as the percent decrease in the response of the CFA, saline or naı¨ve animals by U0126 pre-treatment compared to those with vehicle pre-treatment. Behavioral responses induced by intrathecal DHPG were analyzed using one way ANOVA and post-hoc analysis was
128
H. Adwanikar et al. / Pain 111 (2004) 125–135
carried out with Newman-Keuls Multiple Comparison test for pair-wise comparisons using GraphPad Prism 3 software (GraphPad Inc., San Diego, CA) when P , 0:05 for one way ANOVA. Dunnett’s Multiple Comparison test was used when comparing responses to vehicle controls for the dose response. For analysis of band density or ERK activation profiles, the unpaired t-test was used when comparisons were restricted to two means. One way ANOVA followed by post-hoc analysis using NewmanKeuls Multiple Comparison test was used for comparing more than two groups.
3. Results 3.1. Nocifensive behavior caused by activation of spinal group I mGluRs is ERK dependent Activation of Group I mGluRs in the spinal cord has been shown to induce spontaneous nocifensive behaviors and ERK phosphorylation in C57/BL6 mice (Karim et al., 2001). We first tested whether ICR mice used in this study show similar behaviors. Mice were given a single intrathecal injection of vehicle or various doses of DHPG and the time spent in nocifensive behavior was recorded for 5 min. We observed dose-dependent induction of nocifensive behaviors by intrathecal application of the group I mGluR agonist, DHPG, in ICR mice (Fig. 1A). To determine whether ERK activation was necessary for this DHPG-induced nocifensive behavior, we used an inhibitor of the ERK kinase, MEK, to block the activation of ERK. We measured nocifensive behaviors for 30 min following intrathecal injection of 10 nmol DHPG, to examine the acute as well as longer lasting effects of activation of the spinal group I mGluRs. Mice were pre-treated with an intrathecal injection of vehicle (0.8% DMSO), the MEK inhibitor U0126, or its inactive analog U0124, for 15 min before intrathecal injection of (RS)-DHPG (10 nmol), and the time spent in nocifensive behavior was recorded for 30 min. The vehicle DMSO does not itself cause nocifensive behavior (data not shown). Both the low (0.5 nmol) and high (2.6 nmol) doses of U0126 significantly attenuated the nocifensive behavior observed for a 30 min period following intrathecal injection of DHPG (10 nmol), as compared to vehicle-injected controls (Fig. 1B). U0124 (0.5 nmol), a structural analog of U0126 that does not inhibit MEK (Favata et al., 1998), did not affect the nocifensive behavior induced by intrathecal DHPG as compared to vehicle controls. Neither U0126 nor U0124 have any effect on baseline behavior (data not shown). To ensure that the observed reduction in DHPG-induced nocifensive behavior by U0126 was not caused by motor impairment, mice were tested on an accelerating rotarod for motor function. Mice were treated intrathecally with 0.5 or 2.6 nmol of the MEK inhibitor U0126, its inactive analog U0124, or vehicle (0.8% DMSO), 15 min before measuring
Fig. 1. Intrathecal application of the group I mGluR agonist DHPG causes ERK dependent nocifensive behavior. (A) Total time (seconds/5 min) spent in spontaneous nocifensive behaviors after intrathecal injection of (RS)-DHPG. Points represent the mean ^ SEM time spent in nocifensive behaviors. n ¼ 4 – 6 animals per dose. *, indicates a significant increase in nocifensive behaviors induced by intrathecal DHPG injection, relative to vehicle control (P , 0:01; ANOVA followed by Dunnett’s Multiple Comparison test). (B) Effect of the MEK inhibitor U0126 on DHPGinduced spontaneous nocifensive behavior. Points represent the mean ^ SEM time spent in nocifensive behaviors. n ¼ 4 – 6 animals per dose. *, indicates a significant decrease in response relative to the DMSO/DHPG injected group (P , 0:05; ANOVA followed by Newman-Keuls Multiple Comparison test). (C) The effect of U0126 on the DHPG behavioral response is not due to motor impairment. Points represent the mean ^ SEM latency to drop off the rotarod after beginning of test. n ¼ 8 – 9 animals per dose.
their motor ability on an accelerating rotarod. The performance of mice treated with U0126 or U0124 was not significantly different from that of vehicle-injected controls, suggesting that the effects of U0126 on the DHPG-induced
H. Adwanikar et al. / Pain 111 (2004) 125–135
behavioral response were specific to nociception (Fig. 1C). Although not reaching significance, the higher dose of U0126 showed a trend towards decreased locomotor ability at later time points. Therefore, in all further experiments, the 0.5 nmol dose of U0126 or U0124 was used. 3.2. CFA induced inflammation potentiates nocifensive behaviors induced by activation of spinal group I mGluRs The results described earlier suggest that nociception induced by intrathecal DHPG involves ERK activation. As aforementioned, many forms of inflammatory pain involve ERK activation and are associated with functional upregulation of group I mGluRs. However, it is not known whether chronic inflammatory states change the behavioral responses induced by activating group I mGluRs. Consistent with previous observations, injection of 10 ml CFA into the plantar surface of a hind paw produced an area of localized swelling and hypersensitivity to mechanical stimuli that lasted for at least seven days. Normal withdrawal threshold force is in the range of 2.44 –3.84 g. Following inflammation, thresholds are reduced to as little as 0.008 g in the ipsilateral paw. There was a significant (, 55%) decrease in the ipsilateral threshold of force required to elicit paw withdrawal as compared to salineinjected controls (data not shown). We measured the nocifensive response to intrathecal DHPG at seven days following intraplantar injection of 10 ml CFA. The intermediate dose of 10 nmol DHPG was used to allow measurement of increases or decreases in nocifensive responses. The behavioral response to intrathecal DHPG was dramatically potentiated following inflammation as compared to naı¨ve or saline-injected controls (Fig. 2).
129
The response to intrathecal DHPG in saline-injected animals was similar to that in naı¨ve controls. It may be noted that in the naı¨ve group which received no intraplantar injection, the behavioral response to DHPG was greater in animals when pre-injected intrathecally with vehicle (DMSO, Fig. 1B) compared to animals with no pretreatments (Fig. 2). However, DMSO itself did not induce nocifensive behaviors (not shown). Therefore, this increase could be an effect of the presence of DMSO or of the act of giving two intrathecal injections (Fig. 1B). Increased responses to intrathecal DHPG following inflammation compared to naı¨ve or saline-injected controls were observed in both injection protocols. To ensure that any differences observed in the experiments looking at effects of intraplantar CFA are due to inflammation and not due to an intrathecal pre-injection effect similar to that described earlier, every experiment included a group of naı¨ve animals with no intraplantar injections and the same intrathecal manipulations. Thus, every drug effect is compared to a control with identical intrathecal treatments other than the application of vehicle instead of the drug being tested. These results indicate that inflammation potentiates the effect of group I mGluR activation in the spinal cord. To test whether up-regulation of mGlu1/5 accounts for the increased response to DHPG following inflammation, we analyzed expression of mGlu1/5 in the spinal cord. Mice were given a single intraplantar injection of CFA or saline in the hind paw. Seven days after the intraplantar injection, membrane preparations were made from ipsilateral and contralateral segments of the lumbar spinal cord, and these samples were immunoblotted with mGlu1a and mGlu5 antibodies. Actin blots from the same samples were used as loading controls. Consistent with previous reports, band intensities of mGlu5 were much higher than the mGlu1a bands. However, there was no significant change in the expression levels of mGlu1a or mGlu5 at 7 days following inflammation (Fig. 3). 3.3. CFA-induced inflammation causes persistent activation of ERK in the dorsal horn
Fig. 2. CFA induced inflammation potentiates responses to intrathecal DHPG injection. Points represent the mean ^ SEM time spent in nocifensive behaviors. n ¼ 9 – 10 animals. *, indicates a significant increase in response compared to naı¨ve or saline-injected controls (P , 0:01; ANOVA followed by Newman-Keuls Multiple Comparison test).
The ERK signaling cascade is important in generating acute pain hypersensitivity as well as the altered pain sensitivity following inflammation. Since Group I mGluR expression was not significantly increased following CFAinduced inflammation, we wanted to identify downstream components of the group I mGluR signaling cascade which may be influenced by inflammation. We tested the longterm effects of peripheral inflammation on ERK signaling in the dorsal horn. Mice were injected with intraplantar CFA and homogenates of the lumbar spinal cord were collected 7 days later. These homogenates were probed for phosphorylated ERK as well as total ERK. Analysis of band intensities from immunoblotting of the homogenates indicated that there was a significant increase in both ERK1 and ERK2 phosphorylation in the spinal cord 7 days following CFA
130
H. Adwanikar et al. / Pain 111 (2004) 125–135
Fig. 4. Persistent increases in phosphorylation of ERK1 and ERK2 in the dorsal horn following CFA induced inflammation. (A) Representative Western blot of phosphorylated ERK1 and ERK2 bands in mouse spinal cord homogenates seven days following the injection of CFA in one hind paw, using a phospho-ERK1/2 antibody (top) or total ERK1/2 antibody (bottom). The arrows indicate position of the 44 kDa (ERK1) and 42 kDa (ERK2) isoforms. (B) Densitometric analysis of phospho-ERK1 and phospho-ERK2 at seven days following CFA induced inflammation. Points represent the mean ^ SEM densities of phospho-ERK1 and phosphoERK2 bands normalized to total ERK for each sample. n ¼ 6 animals. *, indicates a significant increase in density as compared to saline-injected controls (P , 0:05; unpaired t-test). Fig. 3. Expression of group I mGluRs in the spinal cord dorsal horn 7 days following CFA induced inflammation. (A) Representative Western blot and densitometric analysis of mGluR1a expression in spinal cord membrane preparations. Points represent the mean ^ SEM densities of mGluR1a bands normalized to actin for each sample. n ¼ 5 – 6 animals. (B) Representative blot and densitometric analysis of mGluR5 expression in spinal cord membrane preparations. Points represent the mean ^ SEM densities of mGluR5 bands normalized to actin for each sample. n ¼ 5 – 6 animals.
induced inflammation, whereas the total ERK levels were unchanged (Fig. 4). To study the laminar distribution of CFA-induced persistent ERK activation, we carried out immunostaining for phosphorylated ERK in the spinal cord 7 days following induction of inflammation. ERK activation was enhanced both ipsilaterally and contralaterally in the dorsal horn (Fig. 5A), although there was significantly more activation ipsilaterally. Nissl staining of similar sections was used to estimate the positions of different laminae in the dorsal horn. Naı¨ve animals had no intraplantar injection. The cells showing pERK activation appear to be morphologically consistent with neurons under high magnification. However, it is formally possible that some of these cells are of glial
origin. Laminar quantification of the phospho-ERK positive profiles indicated that there was a significant increase in the number of phospho-ERK positive profiles in the superficial laminae of the ipsilateral dorsal horn, whereas the significant increases in the contralateral dorsal horn were in the deep laminae (Fig. 5B). In spinal cords taken from mice seven days after saline injected into the paw, there was no change in ERK activation as compared to naı¨ve controls. 3.4. Laminar distribution of DHPG-induced activation of ERK in the dorsal horn following inflammation To determine whether enhanced behavioral responses to intrathecal DHPG injection following inflammation are associated with an altered pattern of ERK activation in the cord, we measured phospho-ERK immunostaining induced by intrathecal injection of 10 nmol DHPG following intraplantar injection of saline or CFA. Seven days following an intraplantar saline injection, animals were intrathecally injected with DHPG and then perfused to collect spinal cord samples for immunocytochemistry.
H. Adwanikar et al. / Pain 111 (2004) 125–135
131
Fig. 5. Laminar distribution of phospho-ERK positive neurons in the spinal cord following CFA induced inflammation. (A) Immunocytochemistry showing phospho-ERK immunoreactivity in the dorsal horn 7 days following intraplantar CFA or saline injection. (B) Analysis of laminar distribution of phospho-ERK positive profiles in the spinal cord. Data represent the mean ^ SEM ERK phosphorylation profiles of 9 sections taken from 3 animals. *, indicates a significant increase in intrathecal DHPG-induced ERK activation profiles in each lamina following CFA-induced inflammation compared to profiles from saline-injected animals (P , 0:05; ANOVA followed by Newman-Keuls Multiple Comparison test).
Intrathecal DHPG caused bilateral increases in phosphoERK positive profiles in the saline injected animals. In animals with CFA-induced inflammation, however, there was no significant difference in the number of phospho-ERK positive profiles on the side ipsilateral to the inflamed paw in animals injected with DHPG compared to animals injected with vehicle (Fig. 6C). This indicates that DHPG activation of ERK may be occurring in the same subset of neurons, which are already activated following sustained inflammation. In the dorsal horn on the side contralateral to the inflamed paw, however, DHPG caused an increase in profiles with ERK activation in the superficial laminae similar to controls, which received a saline injection in the hind paw (Fig. 6D). The deep laminae did not show such an increase. These data suggest that the subset
of neurons in the spinal cord which are involved in transmitting group I mGluR mediated responses show a persistent increase in ERK activation following inflammation. 3.5. U0126 is significantly more effective in inhibiting DHPG-induced nocifensive behaviors following inflammation We observed persistent ERK activation and an increased behavioral response to spinal group I mGluR activation following inflammation. However, group I mGluR-mediated activation of ERK was not significantly enhanced following inflammation compared to controls. To elucidate the role of persistent ERK activation following inflammation in
132
H. Adwanikar et al. / Pain 111 (2004) 125–135
Fig. 6. Laminar distribution of ERK activation due to DHPG following CFA induced inflammation. (A,B) Comparison of laminar distribution of phospho-ERK positive neuron profiles induced by DHPG in the ipsilateral (A) and contralateral (B) dorsal horn in mice without inflammation. (C,D) Comparison of laminar distribution of phospho-ERK positive neuron profiles induced by DHPG in the ipsilateral (C) and contralateral (D) dorsal horn, 7 days following CFA-induced inflammation. Data represent the mean ^ SEM of 12 sections taken from 4 animals. *, indicates a significant increase in ERK activation profiles in each lamina of intrathecal DHPG-injected animals compared to PBS-injected controls (P , 0:05; Unpaired t-test).
processing Group I mGluR-induced behavioral responses, we compared the effects of inhibiting ERK activation in animals with inflammation to control animals. Seven days following CFA or saline injection, mice were pre-treated with an intrathecal injection of the MEK inhibitor U0126 15 min prior to the intrathecal DHPG injection. MEK inhibition leads to an attenuation of the behavioral response to intrathecal DHPG even in naı¨ve animals (Time spent in nocifensive responses 499.5 ^ 95 s in U0126 injected animals as compared to 777.5 ^ 84 s in vehicle injected controls). We compared the percent attenuation of the DHPG-induced nocifensive response by intrathecal U0126 pre-treatment for each intraplantar treatment to a group of animals with the same intraplantar treatment but intrathecal vehicle pre-treatment. U0126 inhibited the DHPG response to a significantly greater extent in animals with CFA induced inflammation (Fig. 7).
Inhibition of the DHPG-induced nocifensive behavior in animals with CFA induced inflammation (time spent in nocifensive responses 281.6 ^ 51.1 s in U0126 injected animals as compared to 685 ^ 70.5 s in vehicle injected controls) was greater than the inhibition in control animals that received an intraplantar saline injection (time spent in nocifensive responses 593.3 ^ 20 s in U0126 injected animals as compared to 813.3 ^ 69.6 s in vehicle injected controls) or naı¨ve animals. In each set of animals, the nocifensive response to intrathecal DHPG was increased due to the effect of vehicle pre-injection, as mentioned earlier. It is possible that a ceiling to the observed behavioral effect was reached. However, the percent attenuation of the DHPG-induced behavioral response by U0126 pre-treatment was significantly greater following inflammation (Fig. 7). The attenuation of the response in saline injected animals was the same as in naı¨ve animals. Thus, the contribution of
H. Adwanikar et al. / Pain 111 (2004) 125–135
Fig. 7. Increase in U0126 inhibition of intrathecal DHPG-induced nocifensive response following inflammation. Points represent the mean ^ SEM percent attenuation of the DHPG response by intrathecal pre-injection of U0126 compared to vehicle/DHPG injected controls. n ¼ 5 – 6 animals. *, indicates a significant increase in percent inhibition compared to naı¨ve or saline-injected animals (P , 0:001; ANOVA followed by Newman-Keuls Multiple Comparison test).
ERK activation to DHPG-induced behavioral responses is greater in animals with inflammation compared to naı¨ve and saline-injected groups.
4. Discussion In summary, the studies described here have resulted in three main findings. First, CFA-induced inflammation of the hind paw causes a sustained increase in ERK activation in dorsal horn neurons. Second, this sustained increase in ERK activation is associated with a persistent increase in the behavioral response to intrathecally applied DHPG, a group I mGluR agonist. This behavior was shown to be dependent on ERK signaling. Finally, the behavioral response to intrathecal DHPG shows a greater ERK dependence following inflammation. A number of mechanisms could account for the potentiation of the behavioral response to intrathecal DHPG following CFA-induced inflammation. Among the possible mechanisms are increases in group I mGluR expression, increases in coupling of the receptors to intracellular signaling cascades, and increased excitability of dorsal horn neurons. We tested whether a change in mGluR expression in the dorsal horn mediates the potentiation of mGlu1/5 agonistinduced responses after inflammation. Such increases have been shown in a clinical model of persistent inflammation (Dolan et al., 2003) and a recent report shows an increase in the group I mGluR modulation of afferent evoked neurotransmission in substantia gelatinosa neurons seven days following CFA-induced inflammation (Giles et al., 2003). However, our results indicate that there is no
133
measurable change in mGlu1a or mGlu5 protein expression in the dorsal horn following inflammation. Thus, the enhanced sensitivity to group I mGluR activation does not appear to be mediated by increased receptor expression in the dorsal horn. It is possible that small but biologically significant changes in group I mGluR expression may remain undetected due to dilution of signal. The agonist sensitivity could also be enhanced due to changes in expression of receptor splice variants not detected by the anti-mGlu1a antibody, such as mGlu1b or mGlu1d, which are also present in the dorsal horn (Karim et al., 2001). Changes in these or other splice variants could be responsible for the observed effects. Another possibility is that while there is no change in the total expression of the group I mGluRs, more receptors could be inserted into or retained in the cell surface membrane following inflammation. It is also possible that feedback regulation by ERK of group I mGluR coupling to other signal transduction pathways mediates this enhanced sensitivity. These mechanisms cannot be ruled out by our experiments. We also tested whether there is an increase in coupling of the spinal group I mGluRs to ERK activation following CFA-induced inflammation. Inflammation did not potentiate the DHPG-induced activation of ERK. However, we find that basal levels of ERK activation in the dorsal horn are increased following inflammation. It is possible that this increased basal ERK activation underlies the enhanced response of the dorsal horn neurons to group I mGluR activation. ERK activation in the dorsal horn has been shown to occur transiently following intraplantar capsaicin, formalin and CFA (Ji et al., 1999, 2002; Karim et al., 2001). This observation has also been extended to models of neuritis and neuropathic pain (Kominato et al., 2003; Ma and Quirion, 2002). We find that at seven days following CFA-induced inflammation, enhanced ERK phosphorylation is still present. This suggests a more persistent activation of ERK than has previously been reported. Previous studies have indicated genotypic variability of nocifensive behaviors (Mogil et al., 1999), and thus it is possible that there is a genetic component to these differences in ERK activation. Analysis of the laminar distribution of phospho-ERK immunopositive cells showed a sustained enhancement of ERK activation in both the ipsilateral and contralateral segments of the dorsal horn as compared to saline injected controls or naı¨ve mice, although the activation is significantly higher ipsilaterally than contralaterally. In contrast, previous studies show increases only ipsilaterally (Ji et al., 2002). It is possible that the longer lasting ERK activation reflects a central sensitization effect, which is bilaterally mediated rather than acute activation in ipsilateral neurons. We further find that the DHPG-induced increase in the number of pERK positive neurons in the ipsilateral dorsal horn is occluded by inflammation, compared to its effect on the contralateral dorsal horn. This suggests that the DHPGinduced nocifensive response could be mediated by the same
134
H. Adwanikar et al. / Pain 111 (2004) 125–135
subset of neurons which show an increase in ERK activation following inflammation. Dorsal horn neurons can operate in several functional states with different capabilities for information transfer that can be modulated by the group I metabotropic glutamate receptors (Derjean et al., 2003). These receptors act through ERK to modulate A-type potassium currents and neuronal excitability (Gereau and Hu, 2003; Hu and Gereau, 2003; Hu et al., 2003). Thus, regulation of the tonic level of ERK activation in dorsal horn neurons may be an important mechanism by which nociceptive plasticity is mediated. Inflammation leads to an enhancement of ERK activity in dorsal horn neurons, and it is plausible that this regulates plasticity in the spinal cord and underlies a component of central sensitization. The results from the present study indicate that there is indeed a change in the tonic level of ERK activation in dorsal horn neurons associated with inflammation. Moreover, this change correlates with a change in the behavioral response to group I mGluR activation. There is a significant body of evidence supporting increased involvement of the group I mGluRs in nociceptive processing following inflammation (Dolan et al., 2003; Neugebauer et al., 2003; Varney and Gereau, 2002). However, whether the observed change in ERK activity underlies enhanced excitability due to inflammation still remains to be tested. Noxious stimulation and inflammation can lead to activation of CREB and the induction of CRE-containing genes in dorsal horn neurons (Ji et al., 2002, 2004; Seybold et al., 2003). Blocking ERK activation reverses both the transcriptional changes and the development of a late-onset component of inflammatory pain hypersensitivity (Ji et al., 2002). Thus, it is believed that the maintenance of latephase central sensitization involves ERK mediated activation of CREB and subsequent transcriptional changes in dorsal horn neurons (Ji et al., 2004). Our data suggest that the persistence of ERK activity itself is also important for the enhanced group I mGluR mediated signaling in dorsal horn neurons following inflammation. Whether this sustained ERK activity serves to drive gene transcription, regulate ion channel function, or both, remains to be determined. In conclusion, our data show that inflammation induces a persistent ERK activation as well as ERK-dependent potentiation of group I mGluR-mediated nociceptive signaling. These results suggest that modulation of the level of ERK activity in dorsal horns neurons may be an important mechanism by which central sensitization is mediated.
Acknowledgements These studies were supported by National Institutes of Health Grant R01 MH-60230 to R.W.G., and by funds from the Arthritis Foundation to F.K.
References Alvarez FJ, Villalba RM, Carr PA, Grandes P, Somohano PM. Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord. J Comp Neurol 2000;422:464 –87. Berthele A, Boxall SJ, Urban A, Anneser JM, Zieglgansberger W, Urban L, Tolle TR. Distribution and developmental changes in metabotropic glutamate receptor messenger RNA expression in the rat lumbar spinal cord. Brain Res Dev Brain Res 1999;112:39–53. Derjean D, Bertrand S, Le Masson G, Landry M, Morisset V, Nagy F. Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states. Nat Neurosci 2003;6:274 –81. Dolan S, Kelly JG, Monteiro AM, Nolan AM. Up-regulation of metabotropic glutamate receptor subtypes 3 and 5 in spinal cord in a clinical model of persistent inflammation and hyperalgesia. Pain 2003; 106:501– 12. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM. Identification of a novel inhibitor of mitogenactivated protein kinase kinase. J Biol Chem 1998;273:18623–32. Fisher K, Coderre TJ. Comparison of nociceptive effects produced by intrathecal administration of mGluR agonists. Neuroreport 1996;7: 2743–7. Fundytus ME, Fisher K, Dray A, Henry JL, Coderre TJ. In vivo antinociceptive activity of anti-rat mGluR1 and mGluR5 antibodies in rats. Neuroreport 1998;9:731 –5. Galan A, Lopez-Garcia JA, Cervero F, Laird JM. Activation of spinal extracellular signaling-regulated kinase-1 and -2 by intraplantar carrageenan in rodents. Neurosci Lett 2002;322:37 –40. Galan A, Cervero F, Laird JM. 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 2003;116:126–34. Gereau RW, Hu HJ. Metabotropic glutamate receptor 5 modulates the potassium channel Kv4.2 subunit via the ERK signaling pathway in isolated spinal cord dorsal horn neurons. Society for Neuroscience, 33rd Annual Meeting; 2003. pp. 382– 16. Giles PA, Davies CH, King AE. Synaptic modulation by group I metabotropic gluramate receptor (mGluR) agonists in rat substantia gelatinos in vitro: a comparision between normal and arthritic rats. Society for Neuroscience, 33rd Annual Meeting; 2003. pp. 695–7. Hu HJ, Gereau RW. ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. II. Modulation of neuronal excitability. J Neurophysiol 2003;90:1680–8. Hu HJ, Glauner KS, Gereau RW. ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. I. Modulation of A-Type K þ currents. J Neurophysiol 2003;90:1671–9. Ji RR, Baba H, Brenner GJ, Woolf CJ. Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 1999;2:1114–9. Ji RR, Befort K, Brenner GJ, Woolf CJ. ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J Neurosci 2002;22:478 –85. Ji RR, Kohno T, Moore KA, Woolf CJ. Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci 2003;26: 696 –705. Karim F, Wang CC, Gereau RW. Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice. J Neurosci 2001;21:3771–9. Kominato Y, Tachibana T, Dai Y, Tsujino H, Maruo S, Noguchi K. Changes in phosphorylation of ERK and Fos expression in dorsal horn neurons following noxious stimulation in a rat model of neuritis of the nerve root. Brain Res 2003;967:89– 97. Lever IJ, Pezet S, McMahon SB, Malcangio M. The signaling components of sensory fiber transmission involved in the activation of ERK MAP kinase in the mouse dorsal horn. Mol Cell Neurosci 2003;24:259–70.
H. Adwanikar et al. / Pain 111 (2004) 125–135 Ma W, Quirion R. Partial sciatic nerve ligation induces increase in the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus. Pain 2002;99:175–84. Malmberg AB, Gilbert H, McCabe RT, Basbaum AI. Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T. Pain 2003;101: 109–16. Millan MJ. The induction of pain: an integrative review. Prog Neurobiol 1999;57:1– 164. Mills CD, Fullwood SD, Hulsebosch CE. Changes in metabotropic glutamate receptor expression following spinal cord injury. Exp Neurol 2001;170:244 –57. Mogil JS, Wilson SG, Bon K, Lee SE, Chung K, Raber P, Pieper JO, Hain HS, Belknap JK, Hubert L, Elmer GI, Chung JM, Devor M. Heritability of nociception I: responses of 11 inbred mouse strains on 12 measures of nociception. Pain 1999;80:67–82. Neugebauer V. Metabotropic glutamate receptors–important modulators of nociception and pain behavior. Pain 2002;98:1–8. Neugebauer V, Lucke T, Schaible HG. Requirement of metabotropic glutamate receptors for the generation of inflammation-evoked hyperexcitability in rat spinal cord neurons. Eur J Neurosci 1994;6: 1179–86. Neugebauer V, Chen PS, Willis WD. Role of metabotropic glutamate receptor subtype mGluR1 in brief nociception and central sensitization of primate STT cells. J Neurophysiol 1999;82:272–82.
135
Neugebauer V, Li W, Bird GC, Bhave G, Gereau RW. Synaptic plasticity in the amygdala in a model of arthritic pain: differential roles of metabotropic glutamate receptors 1 and 5. J Neurosci 2003;23:52–63. Seybold VS, McCarson KE, Mermelstein PG, Groth RD, Abrahams LG. Calcitonin gene-related peptide regulates expression of neurokinin1 receptors by rat spinal neurons. J Neurosci 2003;23:1816–24. Stanfa LC, Dickenson AH. Inflammation alters the effects of mGlu receptor agonists on spinal nociceptive neurones. Eur J Pharmacol 1998;347: 165– 72. Varney MA, Gereau RW. Metabotropic glutamate receptor involvement in models of acute and persistent pain: prospects for the development of novel analgesics. Curr Drug Target CNS Neurol Disord 2002;1: 283– 96. Yashpal K, Fisher K, Chabot JG, Coderre TJ. Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats. Pain 2001;94:17–29. Young MR, Fleetwood-Walker SM, Mitchell R, Munro FE. Evidence for a role of metabotropic glutamate receptors in sustained nociceptive inputs to rat dorsal horn neurons. Neuropharmacology 1994;33:141– 4. Young MR, Fleetwood-Walker SM, Dickinson T, Blackburn-Munro G, Sparrow H, Birch PJ, Bountra C. Behavioural and electrophysiological evidence supporting a role for group I metabotropic glutamate receptors in the mediation of nociceptive inputs to the rat spinal cord. Brain Res 1997;777:161–9. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10.
Inflammation persistently enhances nocifensive behaviors mediated by spinal group I mGluRs through sustained ERK activation Hita Adwanikara, Farzana Karima,b, Robert W. Gereau IVa,b,* a Division of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave, Campus Box 8054, St. Louis, MO 63110, USA
b
Received 16 December 2003; received in revised form 23 April 2004; accepted 7 June 2004
Abstract Group I metabotropic glutamate receptors (mGluRs) and their downstream signaling pathways, which involve the extracellular signalregulated kinases (ERKs), have been implicated as mediators of plasticity in several pain models. In this study, we report that inflammation leads to a long-lasting enhancement of behavioral responses induced by activation of spinal group I mGluRs. Thus, the nocifensive response to intrathecal injection of the group I mGluR agonist (RS)-3,5-Dihydroxyphenylglycine (DHPG) is significantly potentiated seven days following Complete Freund’s Adjuvant (CFA)-induced inflammation of the hind paw. This potentiation is not associated with increased mGlu1 or mGlu5 receptor expression but is associated with increased levels of phosphorylated ERK in dorsal horn neurons. We also tested whether the increased behavioral response to DHPG following inflammation may be explained by enhanced coupling of the group I mGluRs to ERK activation. DHPG-induced ERK phosphorylation in the dorsal horn is not potentiated following inflammation. However, inhibiting ERK activation using a MEK inhibitor, U0126, following inflammation attenuates the intrathecal DHPG-induced behavioral responses to a greater extent than in control animals. The results from this study indicate that persistent ERK activation is required for the enhanced behavioral responses to spinal group I mGluR activation following inflammation and suggest that tonic modulation of ERK activity may underlie a component of central sensitization in dorsal horn neurons. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: DHPG; Pain; Spinal cord; Metabotropic; CFA; MAPK; Nociception
1. Introduction The mechanisms underlying inflammatory pain are being extensively studied, and a large body of evidence suggests that inflammation sensitizes peripheral afferents and dorsal horn neurons (Millan, 1999). Group I mGluRs (mGlu1 and mGlu5) are present in the dorsal horn of the spinal cord (Alvarez et al., 2000; Berthele et al., 1999; Karim et al., 2001) and have been implicated in inflammatory and neuropathic pain (Fisher and Coderre, 1996; Fundytus et al., 1998; Karim et al., 2001; Neugebauer et al., 1999; Yashpal et al., 2001; Young et al., 1997). Moreover, mGluRs are required for inflammation-evoked hyperexcitability * Corresponding author. Address: Washington University Pain Center, Department of Anesthesiology, Washington University School of Medicine, 660 S. Euclid Ave, Campus Box 8054, St. Louis, MO 63110, USA. Tel.: þ 1-314-362-8312; fax: þ1-314-362-8571. E-mail address: [email protected] (R.W. Gereau IV).
and sustained nociceptive transmission (Neugebauer et al., 1994; Young et al., 1994). The group I mGluRs play a more prominent role in nociceptive processing in the context of inflammation, suggesting a functional up-regulation (Neugebauer, 2002; Neugebauer et al., 1999; Stanfa and Dickenson, 1998; Varney and Gereau, 2002). Changes in group I mGluR expression in the spinal cord and other regions of the CNS have been observed in a variety of pain models as well as in clinical inflammation (Dolan et al., 2003; Mills et al., 2001; Neugebauer et al., 2003). Several studies have shown a critical role for the activation of ERKs in dorsal horn neurons in various pain models (Hu and Gereau, 2003; Hu et al., 2003; Ji et al., 1999, 2002; Karim et al., 2001; Kominato et al., 2003; Lever et al., 2003; Ma and Quirion, 2002). Group I mGluR activation, as well as inflammation, leads to ERK dependent sensitization of nociception (Galan et al., 2002, 2003; Ji et al., 1999; Karim et al., 2001; Ma and Quirion, 2002).
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.06.009
126
H. Adwanikar et al. / Pain 111 (2004) 125–135
ERKs regulate neuronal excitability in dorsal horn neurons through modulation of A-type potassium currents (Hu and Gereau, 2003; Hu et al., 2003). It has been shown that ERK activation increases following inflammation, for 60 min following formalin injection (Ji et al., 1999; Karim et al., 2001), 65 min in the carrageenan model (Galan et al., 2002), 3 h following noxious visceral stimuli (Galan et al., 2003) and 48 h in CFA-induced inflammation (Ji et al., 2002). However, the role of ERK activation in maintenance of longer lasting forms of inflammatory pain is not clear. The experimental evidence discussed earlier suggests critical roles for group I mGluR signaling and the ERK cascade in nociceptive plasticity. The present study supports the hypothesis that effects of mGlu1/5 activation are enhanced following inflammation, and further that this enhancement is mediated by sustained ERK activation in dorsal horn neurons.
All drugs or their corresponding vehicles were injected intrathecally in a total volume of 5 ml using a 25 ml Hamilton syringe and 30 gauge needles. Mice were pretreated intrathecally for 15 min with the antagonists or appropriate vehicles. 2.3. Behavioral analysis Behavioral tests began with a habituation period, in which mice were placed in Plexiglas cubicles for at least 1 h. For mechanical allodynia, the plantar surface of the hind paw was stimulated with a series of von Frey filaments. The threshold was taken as the lowest force that evoked a brisk withdrawal response. The behavioral response to intrathecal DHPG was measured as the time spent in spontaneous nocifensive behavior for 30 min immediately following a single intrathecal DHPG injection. The spontaneous behaviors measured in the ICR mice include caudally oriented licking, scratching and lifting behaviors.
2. Materials and methods 2.4. Assessment of motor function 2.1. Animals The ICR strain of outbred mice (Tac:Icr:Ha(ICR), Taconic, Germantown, NY) were housed in cages with access to food and water ad libitum on a 12 h light/dark cycle. Seven- to ten-week-old male mice weighing 20– 25 g were used for this study. All experiments were done in accordance with ethical guidelines for investigation of pain (Zimmermann, 1983) as well as the guidelines of the National Institutes of Health and The International Association for the Study of Pain and were approved by the Animal Care and Use Committee of Baylor College of Medicine. Mice were allowed to acclimate for at least three days before any behavioral tests. 2.2. Drug administration To induce persistent inflammation, we injected 10 ml of Complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO) into the plantar surface of a hind paw of the mouse. CFA is a commonly used inflammatory agent containing a suspension of heat killed and dried Mycobacterium tuberculosis in paraffin oil. The group I metabotropic Glutamate receptor (mGluR) agonist, (RS)-3,5-Dihydroxyphenylglycine (DHPG; Tocris Cookson Inc, Ellisville, MO) was dissolved in sterile phosphate buffered saline. The MEK inhibitor 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene (U0126; Calbiochem, San Diego, CA) and its inactive analog 1,4-Diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0124; Calbiochem, San Diego, CA) were dissolved in 10% dimethyl sulphoxide (DMSO; Sigma) at 1 mg/ml and then diluted with phosphate buffered saline (PBS) to the appropriate concentrations. The final concentration of DMSO was 0.8% for 0.5 nmol U0126 or U0124.
To test for effects of U0126 and U0124 on motor function, an accelerating rotarod treadmill (Ugo Basile, Comerio, Italy) was used to examine the motor ability of the animals. The mice were placed on the rotating rod at its slowest speed 15 min following intrathecal injection and the time for which the mice stayed on the rod was measured (Malmberg et al., 2003). The mice were not trained in this test prior to the experiment. Each trial was limited to 5 min. The mice were tested for 5 trials with an interval of 15 min between trials. 2.5. Sample preparation Spinal cords were isolated at the indicated time points and ipsilateral and contralateral lumbar spinal cord enlargements (L4 – S1) were homogenized using a Dounce homogenizer in ice-cold homogenization buffer (50 mM Tris– HCl (pH 7.5), 50 mM NaCl, 10 mM EGTA, 5 mM EDTA, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 200 mM paranitrophenylphosphate, 1 mM phenylmethylsulfonyl fluoride, 20 mg/ml leupeptin, and 4 mg/ml aprotinin; Sigma). Protein concentrations were determined using the DC protein assay kit (Bio-Rad, Hercules, CA). For membrane preparation, the section of the spinal cord was homogenized in a Dounce homogenizer 400 ml of 10% sucrose in 10 mM Tris –HCl (pH 7.4), with 1 mM dithiothreitol (DTT). The homogenate was centrifuged for 10 min at 1000 £ g: All centrifugation steps were done at 4 8C. The supernatant was saved, and the pellet re-homogenized in half the original volume of the same buffer. The homogenate was centrifuged for 10 min at 1000 £ g: The supernatants from both centrifugations were combined and centrifuged for 1 h at 60; 000 £ g: The pellets were re-suspended in 10 mM Tris – HCl (pH 7.4) buffer containing 1 mM DTT
H. Adwanikar et al. / Pain 111 (2004) 125–135
and 5 mM MgCl2. Samples were centrifuged for 30 min at 60; 000 £ g: The neuronal membrane pellets were resuspended in 150 ml of the same buffer and stored at 2 80 8C. 2.6. Immunoblotting for total and phospho-ERK Samples (10 mg protein) were electrophoresed in 10% SDS polyacrylamide gels, electrophoretically transferred onto protein-sensitive nitrocellulose membranes (Criterion blotter, Bio-Rad) and blocked in B-TTBS [3% bovine serum albumin (BSA), 50 mM Tris – HCl (pH 7.5), 150 mM NaCl, 0.02 mM Sodium orthovanadate, 0.05% Tween 20, 1 mM NaF, 1 mM Na4P2O7 and 0.01% thimerosal; Sigma] for 2 h at room temperature. Antibody applications were done in B-TTBS. An anti-phospho-ERK primary antibody that detects ERK1 (44 kDa) as well as ERK2 (42 kDa) phosphorylation at both Thr202 and Tyr204 (1:1000 dilution in B-TTBS; Cell Signaling Technology, Beverly, MA) was used for immunoblotting overnight at 4 8C. An anti-ERK primary antibody (1:1000 dilution in 3% BSA; Upstate Biotechnology, Lake Placid, NY) which detects total ERK1 and ERK2 (44/42 kDa) isoforms was used for immunoblotting for 1 h at room temperature. 2.7. Immunoblotting for mGlu1a and mGlu5 Proteins (10 mg) were electrophoresed in 10% SDS polyacrylamide gels. Proteins were transferred onto proteinsensitive nitrocellulose membranes and blocked in 4% milk in TTBS (50 mM Tris – HCl (pH 7.5), 150 mM NaCl and 0.05% Tween 20] for 1 h at room temperature. The antibody applications were done in 4% milk in TTBS. Anti-mGluRa antibody (1:1000, Upstate), anti-mGlu5 antiserum (1:1000) raised in our lab against a peptide corresponding to the mGlu5 C terminus (CSSPKYDTLIIRDYTNSSSSL) and Anti-Actin antibody (1:1000, Sigma) were used for immunoblotting overnight at 4 8C. The membranes were washed and incubated in HRP-conjugated secondary antibody for 1 h at room temperature. Blots were developed with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL). Densitometric quantification of immunopositive bands for total or phospho-p44/42 ERK was done using computerized image analysis software (Scion Corp., Frederick, MD). To ensure that the bands are in the linear range of the film, band densities were plotted against exposure times for a known amount of protein. Band densities in the linear range of the plot were used for quantification. 2.8. Immunocytochemistry Seven days following CFA injection into the hind paw, mice were anesthetized with Avertin (2,2,2 tribromoethanol, Aldrich, 0.8 –1.2 mg/g, i.p.) and perfused transcardially with warm saline (37 8C, 0.9% NaCl), followed by 250 ml of ice-cold 4% paraformaldehyde solution.
127
L4 – S1 lumbar spinal cord sections were dissected out and post-fixed at 4 8C for 4 h with paraformaldehyde, followed by overnight cryoprotection at 4 8C in 30% sucrose. After post-fixing, the ventral contralateral side of the spinal cord was marked by a small scalpel incision to identify ipsilateral and contralateral sides following immunocytochemistry. Tissue was embedded in OCT compound (Tissue-Tek, Miles Inc., Elkhart, IN) and stored at 2 80 8C. Coronal sections (30 mM) were cut using a freezing sliding microtome, and free floating sections were kept in PBS (pH 7.4). The sections were rinsed in 10% methanol and 0.3% H2O2 in 0.1 M PBS for 30 min and then blocked in 3% normal goat serum (NGS) with 0.2% Triton X-100 (NGST) two times for 10 min each. All antibodies were diluted in 1% NGST. Sections were then incubated at 4 8C for 36– 48 h in anti-phospho-p44/42 ERK primary antibody (1:1000 dilution in B-TTBS; Cell Signaling Technology. Following the antibody incubation, the sections were rinsed with 1% NGST two times for 10 min each, followed by incubation in a secondary biotinylated anti-rabbit IgG antibody for 90 min (1:200; ABC kit; Vector Laboratories, Burlingame, CA). The sections were again rinsed with 1% NGST two times for 10 min each and incubated in ExtraAvidin peroxidase (1:1000; Sigma) for 1 h at room temperature. They were then rinsed in 0.1 M PBS two times for 10 min each and then in phosphate buffer (two times for 10 min each) and stained with 3,30 -diaminobenzidine tetrahydrochloride (DAB) solution (0.025% DAB in phosphate buffer containing 0.0025% H2O2; Sigma) for 5 – 10 min. The sections were mounted onto gelatin-coated glass slides, air-dried, dehydrated, cleared with Hemo De, coverslipped with DPX mounting medium, and observed for phospho-ERK staining. For laminar distribution analysis, DAB-stained sections were compared to nissl stained sections (4.5 mg/ml Cresyl Violet, Sigma) from a comparable region of the lumbar spinal cord. Different treatments were processed in parallel using the same batch of solutions. The DAB positive cells were identified under a microscope. All cell counts were done while blind to treatment. 2.9. Data presentation and statistical analysis Behavioral responses to intrathecal DHPG are presented as the number of seconds spent in nocifensive behavior. Band densities of mGlu1a and mGlu5 in immunoblotting experiments were plotted relative to density of Actin band for each sample. Band density of phosphorylated ERK was plotted relative to density of total ERK band for each sample. Immunopositive profiles were plotted as average counts per section for each treatment group. Percent attenuation of the DHPG-induced nocifensive response is plotted as the percent decrease in the response of the CFA, saline or naı¨ve animals by U0126 pre-treatment compared to those with vehicle pre-treatment. Behavioral responses induced by intrathecal DHPG were analyzed using one way ANOVA and post-hoc analysis was
128
H. Adwanikar et al. / Pain 111 (2004) 125–135
carried out with Newman-Keuls Multiple Comparison test for pair-wise comparisons using GraphPad Prism 3 software (GraphPad Inc., San Diego, CA) when P , 0:05 for one way ANOVA. Dunnett’s Multiple Comparison test was used when comparing responses to vehicle controls for the dose response. For analysis of band density or ERK activation profiles, the unpaired t-test was used when comparisons were restricted to two means. One way ANOVA followed by post-hoc analysis using NewmanKeuls Multiple Comparison test was used for comparing more than two groups.
3. Results 3.1. Nocifensive behavior caused by activation of spinal group I mGluRs is ERK dependent Activation of Group I mGluRs in the spinal cord has been shown to induce spontaneous nocifensive behaviors and ERK phosphorylation in C57/BL6 mice (Karim et al., 2001). We first tested whether ICR mice used in this study show similar behaviors. Mice were given a single intrathecal injection of vehicle or various doses of DHPG and the time spent in nocifensive behavior was recorded for 5 min. We observed dose-dependent induction of nocifensive behaviors by intrathecal application of the group I mGluR agonist, DHPG, in ICR mice (Fig. 1A). To determine whether ERK activation was necessary for this DHPG-induced nocifensive behavior, we used an inhibitor of the ERK kinase, MEK, to block the activation of ERK. We measured nocifensive behaviors for 30 min following intrathecal injection of 10 nmol DHPG, to examine the acute as well as longer lasting effects of activation of the spinal group I mGluRs. Mice were pre-treated with an intrathecal injection of vehicle (0.8% DMSO), the MEK inhibitor U0126, or its inactive analog U0124, for 15 min before intrathecal injection of (RS)-DHPG (10 nmol), and the time spent in nocifensive behavior was recorded for 30 min. The vehicle DMSO does not itself cause nocifensive behavior (data not shown). Both the low (0.5 nmol) and high (2.6 nmol) doses of U0126 significantly attenuated the nocifensive behavior observed for a 30 min period following intrathecal injection of DHPG (10 nmol), as compared to vehicle-injected controls (Fig. 1B). U0124 (0.5 nmol), a structural analog of U0126 that does not inhibit MEK (Favata et al., 1998), did not affect the nocifensive behavior induced by intrathecal DHPG as compared to vehicle controls. Neither U0126 nor U0124 have any effect on baseline behavior (data not shown). To ensure that the observed reduction in DHPG-induced nocifensive behavior by U0126 was not caused by motor impairment, mice were tested on an accelerating rotarod for motor function. Mice were treated intrathecally with 0.5 or 2.6 nmol of the MEK inhibitor U0126, its inactive analog U0124, or vehicle (0.8% DMSO), 15 min before measuring
Fig. 1. Intrathecal application of the group I mGluR agonist DHPG causes ERK dependent nocifensive behavior. (A) Total time (seconds/5 min) spent in spontaneous nocifensive behaviors after intrathecal injection of (RS)-DHPG. Points represent the mean ^ SEM time spent in nocifensive behaviors. n ¼ 4 – 6 animals per dose. *, indicates a significant increase in nocifensive behaviors induced by intrathecal DHPG injection, relative to vehicle control (P , 0:01; ANOVA followed by Dunnett’s Multiple Comparison test). (B) Effect of the MEK inhibitor U0126 on DHPGinduced spontaneous nocifensive behavior. Points represent the mean ^ SEM time spent in nocifensive behaviors. n ¼ 4 – 6 animals per dose. *, indicates a significant decrease in response relative to the DMSO/DHPG injected group (P , 0:05; ANOVA followed by Newman-Keuls Multiple Comparison test). (C) The effect of U0126 on the DHPG behavioral response is not due to motor impairment. Points represent the mean ^ SEM latency to drop off the rotarod after beginning of test. n ¼ 8 – 9 animals per dose.
their motor ability on an accelerating rotarod. The performance of mice treated with U0126 or U0124 was not significantly different from that of vehicle-injected controls, suggesting that the effects of U0126 on the DHPG-induced
H. Adwanikar et al. / Pain 111 (2004) 125–135
behavioral response were specific to nociception (Fig. 1C). Although not reaching significance, the higher dose of U0126 showed a trend towards decreased locomotor ability at later time points. Therefore, in all further experiments, the 0.5 nmol dose of U0126 or U0124 was used. 3.2. CFA induced inflammation potentiates nocifensive behaviors induced by activation of spinal group I mGluRs The results described earlier suggest that nociception induced by intrathecal DHPG involves ERK activation. As aforementioned, many forms of inflammatory pain involve ERK activation and are associated with functional upregulation of group I mGluRs. However, it is not known whether chronic inflammatory states change the behavioral responses induced by activating group I mGluRs. Consistent with previous observations, injection of 10 ml CFA into the plantar surface of a hind paw produced an area of localized swelling and hypersensitivity to mechanical stimuli that lasted for at least seven days. Normal withdrawal threshold force is in the range of 2.44 –3.84 g. Following inflammation, thresholds are reduced to as little as 0.008 g in the ipsilateral paw. There was a significant (, 55%) decrease in the ipsilateral threshold of force required to elicit paw withdrawal as compared to salineinjected controls (data not shown). We measured the nocifensive response to intrathecal DHPG at seven days following intraplantar injection of 10 ml CFA. The intermediate dose of 10 nmol DHPG was used to allow measurement of increases or decreases in nocifensive responses. The behavioral response to intrathecal DHPG was dramatically potentiated following inflammation as compared to naı¨ve or saline-injected controls (Fig. 2).
129
The response to intrathecal DHPG in saline-injected animals was similar to that in naı¨ve controls. It may be noted that in the naı¨ve group which received no intraplantar injection, the behavioral response to DHPG was greater in animals when pre-injected intrathecally with vehicle (DMSO, Fig. 1B) compared to animals with no pretreatments (Fig. 2). However, DMSO itself did not induce nocifensive behaviors (not shown). Therefore, this increase could be an effect of the presence of DMSO or of the act of giving two intrathecal injections (Fig. 1B). Increased responses to intrathecal DHPG following inflammation compared to naı¨ve or saline-injected controls were observed in both injection protocols. To ensure that any differences observed in the experiments looking at effects of intraplantar CFA are due to inflammation and not due to an intrathecal pre-injection effect similar to that described earlier, every experiment included a group of naı¨ve animals with no intraplantar injections and the same intrathecal manipulations. Thus, every drug effect is compared to a control with identical intrathecal treatments other than the application of vehicle instead of the drug being tested. These results indicate that inflammation potentiates the effect of group I mGluR activation in the spinal cord. To test whether up-regulation of mGlu1/5 accounts for the increased response to DHPG following inflammation, we analyzed expression of mGlu1/5 in the spinal cord. Mice were given a single intraplantar injection of CFA or saline in the hind paw. Seven days after the intraplantar injection, membrane preparations were made from ipsilateral and contralateral segments of the lumbar spinal cord, and these samples were immunoblotted with mGlu1a and mGlu5 antibodies. Actin blots from the same samples were used as loading controls. Consistent with previous reports, band intensities of mGlu5 were much higher than the mGlu1a bands. However, there was no significant change in the expression levels of mGlu1a or mGlu5 at 7 days following inflammation (Fig. 3). 3.3. CFA-induced inflammation causes persistent activation of ERK in the dorsal horn
Fig. 2. CFA induced inflammation potentiates responses to intrathecal DHPG injection. Points represent the mean ^ SEM time spent in nocifensive behaviors. n ¼ 9 – 10 animals. *, indicates a significant increase in response compared to naı¨ve or saline-injected controls (P , 0:01; ANOVA followed by Newman-Keuls Multiple Comparison test).
The ERK signaling cascade is important in generating acute pain hypersensitivity as well as the altered pain sensitivity following inflammation. Since Group I mGluR expression was not significantly increased following CFAinduced inflammation, we wanted to identify downstream components of the group I mGluR signaling cascade which may be influenced by inflammation. We tested the longterm effects of peripheral inflammation on ERK signaling in the dorsal horn. Mice were injected with intraplantar CFA and homogenates of the lumbar spinal cord were collected 7 days later. These homogenates were probed for phosphorylated ERK as well as total ERK. Analysis of band intensities from immunoblotting of the homogenates indicated that there was a significant increase in both ERK1 and ERK2 phosphorylation in the spinal cord 7 days following CFA
130
H. Adwanikar et al. / Pain 111 (2004) 125–135
Fig. 4. Persistent increases in phosphorylation of ERK1 and ERK2 in the dorsal horn following CFA induced inflammation. (A) Representative Western blot of phosphorylated ERK1 and ERK2 bands in mouse spinal cord homogenates seven days following the injection of CFA in one hind paw, using a phospho-ERK1/2 antibody (top) or total ERK1/2 antibody (bottom). The arrows indicate position of the 44 kDa (ERK1) and 42 kDa (ERK2) isoforms. (B) Densitometric analysis of phospho-ERK1 and phospho-ERK2 at seven days following CFA induced inflammation. Points represent the mean ^ SEM densities of phospho-ERK1 and phosphoERK2 bands normalized to total ERK for each sample. n ¼ 6 animals. *, indicates a significant increase in density as compared to saline-injected controls (P , 0:05; unpaired t-test). Fig. 3. Expression of group I mGluRs in the spinal cord dorsal horn 7 days following CFA induced inflammation. (A) Representative Western blot and densitometric analysis of mGluR1a expression in spinal cord membrane preparations. Points represent the mean ^ SEM densities of mGluR1a bands normalized to actin for each sample. n ¼ 5 – 6 animals. (B) Representative blot and densitometric analysis of mGluR5 expression in spinal cord membrane preparations. Points represent the mean ^ SEM densities of mGluR5 bands normalized to actin for each sample. n ¼ 5 – 6 animals.
induced inflammation, whereas the total ERK levels were unchanged (Fig. 4). To study the laminar distribution of CFA-induced persistent ERK activation, we carried out immunostaining for phosphorylated ERK in the spinal cord 7 days following induction of inflammation. ERK activation was enhanced both ipsilaterally and contralaterally in the dorsal horn (Fig. 5A), although there was significantly more activation ipsilaterally. Nissl staining of similar sections was used to estimate the positions of different laminae in the dorsal horn. Naı¨ve animals had no intraplantar injection. The cells showing pERK activation appear to be morphologically consistent with neurons under high magnification. However, it is formally possible that some of these cells are of glial
origin. Laminar quantification of the phospho-ERK positive profiles indicated that there was a significant increase in the number of phospho-ERK positive profiles in the superficial laminae of the ipsilateral dorsal horn, whereas the significant increases in the contralateral dorsal horn were in the deep laminae (Fig. 5B). In spinal cords taken from mice seven days after saline injected into the paw, there was no change in ERK activation as compared to naı¨ve controls. 3.4. Laminar distribution of DHPG-induced activation of ERK in the dorsal horn following inflammation To determine whether enhanced behavioral responses to intrathecal DHPG injection following inflammation are associated with an altered pattern of ERK activation in the cord, we measured phospho-ERK immunostaining induced by intrathecal injection of 10 nmol DHPG following intraplantar injection of saline or CFA. Seven days following an intraplantar saline injection, animals were intrathecally injected with DHPG and then perfused to collect spinal cord samples for immunocytochemistry.
H. Adwanikar et al. / Pain 111 (2004) 125–135
131
Fig. 5. Laminar distribution of phospho-ERK positive neurons in the spinal cord following CFA induced inflammation. (A) Immunocytochemistry showing phospho-ERK immunoreactivity in the dorsal horn 7 days following intraplantar CFA or saline injection. (B) Analysis of laminar distribution of phospho-ERK positive profiles in the spinal cord. Data represent the mean ^ SEM ERK phosphorylation profiles of 9 sections taken from 3 animals. *, indicates a significant increase in intrathecal DHPG-induced ERK activation profiles in each lamina following CFA-induced inflammation compared to profiles from saline-injected animals (P , 0:05; ANOVA followed by Newman-Keuls Multiple Comparison test).
Intrathecal DHPG caused bilateral increases in phosphoERK positive profiles in the saline injected animals. In animals with CFA-induced inflammation, however, there was no significant difference in the number of phospho-ERK positive profiles on the side ipsilateral to the inflamed paw in animals injected with DHPG compared to animals injected with vehicle (Fig. 6C). This indicates that DHPG activation of ERK may be occurring in the same subset of neurons, which are already activated following sustained inflammation. In the dorsal horn on the side contralateral to the inflamed paw, however, DHPG caused an increase in profiles with ERK activation in the superficial laminae similar to controls, which received a saline injection in the hind paw (Fig. 6D). The deep laminae did not show such an increase. These data suggest that the subset
of neurons in the spinal cord which are involved in transmitting group I mGluR mediated responses show a persistent increase in ERK activation following inflammation. 3.5. U0126 is significantly more effective in inhibiting DHPG-induced nocifensive behaviors following inflammation We observed persistent ERK activation and an increased behavioral response to spinal group I mGluR activation following inflammation. However, group I mGluR-mediated activation of ERK was not significantly enhanced following inflammation compared to controls. To elucidate the role of persistent ERK activation following inflammation in
132
H. Adwanikar et al. / Pain 111 (2004) 125–135
Fig. 6. Laminar distribution of ERK activation due to DHPG following CFA induced inflammation. (A,B) Comparison of laminar distribution of phospho-ERK positive neuron profiles induced by DHPG in the ipsilateral (A) and contralateral (B) dorsal horn in mice without inflammation. (C,D) Comparison of laminar distribution of phospho-ERK positive neuron profiles induced by DHPG in the ipsilateral (C) and contralateral (D) dorsal horn, 7 days following CFA-induced inflammation. Data represent the mean ^ SEM of 12 sections taken from 4 animals. *, indicates a significant increase in ERK activation profiles in each lamina of intrathecal DHPG-injected animals compared to PBS-injected controls (P , 0:05; Unpaired t-test).
processing Group I mGluR-induced behavioral responses, we compared the effects of inhibiting ERK activation in animals with inflammation to control animals. Seven days following CFA or saline injection, mice were pre-treated with an intrathecal injection of the MEK inhibitor U0126 15 min prior to the intrathecal DHPG injection. MEK inhibition leads to an attenuation of the behavioral response to intrathecal DHPG even in naı¨ve animals (Time spent in nocifensive responses 499.5 ^ 95 s in U0126 injected animals as compared to 777.5 ^ 84 s in vehicle injected controls). We compared the percent attenuation of the DHPG-induced nocifensive response by intrathecal U0126 pre-treatment for each intraplantar treatment to a group of animals with the same intraplantar treatment but intrathecal vehicle pre-treatment. U0126 inhibited the DHPG response to a significantly greater extent in animals with CFA induced inflammation (Fig. 7).
Inhibition of the DHPG-induced nocifensive behavior in animals with CFA induced inflammation (time spent in nocifensive responses 281.6 ^ 51.1 s in U0126 injected animals as compared to 685 ^ 70.5 s in vehicle injected controls) was greater than the inhibition in control animals that received an intraplantar saline injection (time spent in nocifensive responses 593.3 ^ 20 s in U0126 injected animals as compared to 813.3 ^ 69.6 s in vehicle injected controls) or naı¨ve animals. In each set of animals, the nocifensive response to intrathecal DHPG was increased due to the effect of vehicle pre-injection, as mentioned earlier. It is possible that a ceiling to the observed behavioral effect was reached. However, the percent attenuation of the DHPG-induced behavioral response by U0126 pre-treatment was significantly greater following inflammation (Fig. 7). The attenuation of the response in saline injected animals was the same as in naı¨ve animals. Thus, the contribution of
H. Adwanikar et al. / Pain 111 (2004) 125–135
Fig. 7. Increase in U0126 inhibition of intrathecal DHPG-induced nocifensive response following inflammation. Points represent the mean ^ SEM percent attenuation of the DHPG response by intrathecal pre-injection of U0126 compared to vehicle/DHPG injected controls. n ¼ 5 – 6 animals. *, indicates a significant increase in percent inhibition compared to naı¨ve or saline-injected animals (P , 0:001; ANOVA followed by Newman-Keuls Multiple Comparison test).
ERK activation to DHPG-induced behavioral responses is greater in animals with inflammation compared to naı¨ve and saline-injected groups.
4. Discussion In summary, the studies described here have resulted in three main findings. First, CFA-induced inflammation of the hind paw causes a sustained increase in ERK activation in dorsal horn neurons. Second, this sustained increase in ERK activation is associated with a persistent increase in the behavioral response to intrathecally applied DHPG, a group I mGluR agonist. This behavior was shown to be dependent on ERK signaling. Finally, the behavioral response to intrathecal DHPG shows a greater ERK dependence following inflammation. A number of mechanisms could account for the potentiation of the behavioral response to intrathecal DHPG following CFA-induced inflammation. Among the possible mechanisms are increases in group I mGluR expression, increases in coupling of the receptors to intracellular signaling cascades, and increased excitability of dorsal horn neurons. We tested whether a change in mGluR expression in the dorsal horn mediates the potentiation of mGlu1/5 agonistinduced responses after inflammation. Such increases have been shown in a clinical model of persistent inflammation (Dolan et al., 2003) and a recent report shows an increase in the group I mGluR modulation of afferent evoked neurotransmission in substantia gelatinosa neurons seven days following CFA-induced inflammation (Giles et al., 2003). However, our results indicate that there is no
133
measurable change in mGlu1a or mGlu5 protein expression in the dorsal horn following inflammation. Thus, the enhanced sensitivity to group I mGluR activation does not appear to be mediated by increased receptor expression in the dorsal horn. It is possible that small but biologically significant changes in group I mGluR expression may remain undetected due to dilution of signal. The agonist sensitivity could also be enhanced due to changes in expression of receptor splice variants not detected by the anti-mGlu1a antibody, such as mGlu1b or mGlu1d, which are also present in the dorsal horn (Karim et al., 2001). Changes in these or other splice variants could be responsible for the observed effects. Another possibility is that while there is no change in the total expression of the group I mGluRs, more receptors could be inserted into or retained in the cell surface membrane following inflammation. It is also possible that feedback regulation by ERK of group I mGluR coupling to other signal transduction pathways mediates this enhanced sensitivity. These mechanisms cannot be ruled out by our experiments. We also tested whether there is an increase in coupling of the spinal group I mGluRs to ERK activation following CFA-induced inflammation. Inflammation did not potentiate the DHPG-induced activation of ERK. However, we find that basal levels of ERK activation in the dorsal horn are increased following inflammation. It is possible that this increased basal ERK activation underlies the enhanced response of the dorsal horn neurons to group I mGluR activation. ERK activation in the dorsal horn has been shown to occur transiently following intraplantar capsaicin, formalin and CFA (Ji et al., 1999, 2002; Karim et al., 2001). This observation has also been extended to models of neuritis and neuropathic pain (Kominato et al., 2003; Ma and Quirion, 2002). We find that at seven days following CFA-induced inflammation, enhanced ERK phosphorylation is still present. This suggests a more persistent activation of ERK than has previously been reported. Previous studies have indicated genotypic variability of nocifensive behaviors (Mogil et al., 1999), and thus it is possible that there is a genetic component to these differences in ERK activation. Analysis of the laminar distribution of phospho-ERK immunopositive cells showed a sustained enhancement of ERK activation in both the ipsilateral and contralateral segments of the dorsal horn as compared to saline injected controls or naı¨ve mice, although the activation is significantly higher ipsilaterally than contralaterally. In contrast, previous studies show increases only ipsilaterally (Ji et al., 2002). It is possible that the longer lasting ERK activation reflects a central sensitization effect, which is bilaterally mediated rather than acute activation in ipsilateral neurons. We further find that the DHPG-induced increase in the number of pERK positive neurons in the ipsilateral dorsal horn is occluded by inflammation, compared to its effect on the contralateral dorsal horn. This suggests that the DHPGinduced nocifensive response could be mediated by the same
134
H. Adwanikar et al. / Pain 111 (2004) 125–135
subset of neurons which show an increase in ERK activation following inflammation. Dorsal horn neurons can operate in several functional states with different capabilities for information transfer that can be modulated by the group I metabotropic glutamate receptors (Derjean et al., 2003). These receptors act through ERK to modulate A-type potassium currents and neuronal excitability (Gereau and Hu, 2003; Hu and Gereau, 2003; Hu et al., 2003). Thus, regulation of the tonic level of ERK activation in dorsal horn neurons may be an important mechanism by which nociceptive plasticity is mediated. Inflammation leads to an enhancement of ERK activity in dorsal horn neurons, and it is plausible that this regulates plasticity in the spinal cord and underlies a component of central sensitization. The results from the present study indicate that there is indeed a change in the tonic level of ERK activation in dorsal horn neurons associated with inflammation. Moreover, this change correlates with a change in the behavioral response to group I mGluR activation. There is a significant body of evidence supporting increased involvement of the group I mGluRs in nociceptive processing following inflammation (Dolan et al., 2003; Neugebauer et al., 2003; Varney and Gereau, 2002). However, whether the observed change in ERK activity underlies enhanced excitability due to inflammation still remains to be tested. Noxious stimulation and inflammation can lead to activation of CREB and the induction of CRE-containing genes in dorsal horn neurons (Ji et al., 2002, 2004; Seybold et al., 2003). Blocking ERK activation reverses both the transcriptional changes and the development of a late-onset component of inflammatory pain hypersensitivity (Ji et al., 2002). Thus, it is believed that the maintenance of latephase central sensitization involves ERK mediated activation of CREB and subsequent transcriptional changes in dorsal horn neurons (Ji et al., 2004). Our data suggest that the persistence of ERK activity itself is also important for the enhanced group I mGluR mediated signaling in dorsal horn neurons following inflammation. Whether this sustained ERK activity serves to drive gene transcription, regulate ion channel function, or both, remains to be determined. In conclusion, our data show that inflammation induces a persistent ERK activation as well as ERK-dependent potentiation of group I mGluR-mediated nociceptive signaling. These results suggest that modulation of the level of ERK activity in dorsal horns neurons may be an important mechanism by which central sensitization is mediated.
Acknowledgements These studies were supported by National Institutes of Health Grant R01 MH-60230 to R.W.G., and by funds from the Arthritis Foundation to F.K.
References Alvarez FJ, Villalba RM, Carr PA, Grandes P, Somohano PM. Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord. J Comp Neurol 2000;422:464 –87. Berthele A, Boxall SJ, Urban A, Anneser JM, Zieglgansberger W, Urban L, Tolle TR. Distribution and developmental changes in metabotropic glutamate receptor messenger RNA expression in the rat lumbar spinal cord. Brain Res Dev Brain Res 1999;112:39–53. Derjean D, Bertrand S, Le Masson G, Landry M, Morisset V, Nagy F. Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states. Nat Neurosci 2003;6:274 –81. Dolan S, Kelly JG, Monteiro AM, Nolan AM. Up-regulation of metabotropic glutamate receptor subtypes 3 and 5 in spinal cord in a clinical model of persistent inflammation and hyperalgesia. Pain 2003; 106:501– 12. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM. Identification of a novel inhibitor of mitogenactivated protein kinase kinase. J Biol Chem 1998;273:18623–32. Fisher K, Coderre TJ. Comparison of nociceptive effects produced by intrathecal administration of mGluR agonists. Neuroreport 1996;7: 2743–7. Fundytus ME, Fisher K, Dray A, Henry JL, Coderre TJ. In vivo antinociceptive activity of anti-rat mGluR1 and mGluR5 antibodies in rats. Neuroreport 1998;9:731 –5. Galan A, Lopez-Garcia JA, Cervero F, Laird JM. Activation of spinal extracellular signaling-regulated kinase-1 and -2 by intraplantar carrageenan in rodents. Neurosci Lett 2002;322:37 –40. Galan A, Cervero F, Laird JM. 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 2003;116:126–34. Gereau RW, Hu HJ. Metabotropic glutamate receptor 5 modulates the potassium channel Kv4.2 subunit via the ERK signaling pathway in isolated spinal cord dorsal horn neurons. Society for Neuroscience, 33rd Annual Meeting; 2003. pp. 382– 16. Giles PA, Davies CH, King AE. Synaptic modulation by group I metabotropic gluramate receptor (mGluR) agonists in rat substantia gelatinos in vitro: a comparision between normal and arthritic rats. Society for Neuroscience, 33rd Annual Meeting; 2003. pp. 695–7. Hu HJ, Gereau RW. ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. II. Modulation of neuronal excitability. J Neurophysiol 2003;90:1680–8. Hu HJ, Glauner KS, Gereau RW. ERK integrates PKA and PKC signaling in superficial dorsal horn neurons. I. Modulation of A-Type K þ currents. J Neurophysiol 2003;90:1671–9. Ji RR, Baba H, Brenner GJ, Woolf CJ. Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 1999;2:1114–9. Ji RR, Befort K, Brenner GJ, Woolf CJ. ERK MAP kinase activation in superficial spinal cord neurons induces prodynorphin and NK-1 upregulation and contributes to persistent inflammatory pain hypersensitivity. J Neurosci 2002;22:478 –85. Ji RR, Kohno T, Moore KA, Woolf CJ. Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci 2003;26: 696 –705. Karim F, Wang CC, Gereau RW. Metabotropic glutamate receptor subtypes 1 and 5 are activators of extracellular signal-regulated kinase signaling required for inflammatory pain in mice. J Neurosci 2001;21:3771–9. Kominato Y, Tachibana T, Dai Y, Tsujino H, Maruo S, Noguchi K. Changes in phosphorylation of ERK and Fos expression in dorsal horn neurons following noxious stimulation in a rat model of neuritis of the nerve root. Brain Res 2003;967:89– 97. Lever IJ, Pezet S, McMahon SB, Malcangio M. The signaling components of sensory fiber transmission involved in the activation of ERK MAP kinase in the mouse dorsal horn. Mol Cell Neurosci 2003;24:259–70.
H. Adwanikar et al. / Pain 111 (2004) 125–135 Ma W, Quirion R. Partial sciatic nerve ligation induces increase in the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus. Pain 2002;99:175–84. Malmberg AB, Gilbert H, McCabe RT, Basbaum AI. Powerful antinociceptive effects of the cone snail venom-derived subtype-selective NMDA receptor antagonists conantokins G and T. Pain 2003;101: 109–16. Millan MJ. The induction of pain: an integrative review. Prog Neurobiol 1999;57:1– 164. Mills CD, Fullwood SD, Hulsebosch CE. Changes in metabotropic glutamate receptor expression following spinal cord injury. Exp Neurol 2001;170:244 –57. Mogil JS, Wilson SG, Bon K, Lee SE, Chung K, Raber P, Pieper JO, Hain HS, Belknap JK, Hubert L, Elmer GI, Chung JM, Devor M. Heritability of nociception I: responses of 11 inbred mouse strains on 12 measures of nociception. Pain 1999;80:67–82. Neugebauer V. Metabotropic glutamate receptors–important modulators of nociception and pain behavior. Pain 2002;98:1–8. Neugebauer V, Lucke T, Schaible HG. Requirement of metabotropic glutamate receptors for the generation of inflammation-evoked hyperexcitability in rat spinal cord neurons. Eur J Neurosci 1994;6: 1179–86. Neugebauer V, Chen PS, Willis WD. Role of metabotropic glutamate receptor subtype mGluR1 in brief nociception and central sensitization of primate STT cells. J Neurophysiol 1999;82:272–82.
135
Neugebauer V, Li W, Bird GC, Bhave G, Gereau RW. Synaptic plasticity in the amygdala in a model of arthritic pain: differential roles of metabotropic glutamate receptors 1 and 5. J Neurosci 2003;23:52–63. Seybold VS, McCarson KE, Mermelstein PG, Groth RD, Abrahams LG. Calcitonin gene-related peptide regulates expression of neurokinin1 receptors by rat spinal neurons. J Neurosci 2003;23:1816–24. Stanfa LC, Dickenson AH. Inflammation alters the effects of mGlu receptor agonists on spinal nociceptive neurones. Eur J Pharmacol 1998;347: 165– 72. Varney MA, Gereau RW. Metabotropic glutamate receptor involvement in models of acute and persistent pain: prospects for the development of novel analgesics. Curr Drug Target CNS Neurol Disord 2002;1: 283– 96. Yashpal K, Fisher K, Chabot JG, Coderre TJ. Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats. Pain 2001;94:17–29. Young MR, Fleetwood-Walker SM, Mitchell R, Munro FE. Evidence for a role of metabotropic glutamate receptors in sustained nociceptive inputs to rat dorsal horn neurons. Neuropharmacology 1994;33:141– 4. Young MR, Fleetwood-Walker SM, Dickinson T, Blackburn-Munro G, Sparrow H, Birch PJ, Bountra C. Behavioural and electrophysiological evidence supporting a role for group I metabotropic glutamate receptors in the mediation of nociceptive inputs to the rat spinal cord. Brain Res 1997;777:161–9. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 1983;16:109–10.