Damage to peripheral nerves often causes neuropathic pain associated with spontaneous pain, hyperalgesia, and allodynia (severe pain caused by nonpainful stimuli such as touching).1 These symptoms may not be relieved by either nonsteroidal antiinflammatory drugs or opioid analgesics, and supplemental analgesics are often tried for the treatment of neuropathic pain.2 Therefore, further research is required to develop effective neuropathic pain treatment.
Growing evidence indicates that the mitogen-activated protein kinase (MAPK) family plays an important role in the development and maintenance of neuropathic pain.3 MAPK family-mediated signal transduction is involved in diverse cellular events, such as proliferation, differentiation, survival, and activation after the regulation of gene expression.4 The MAPK family is activated by various physiological stimuli in vivo. The MAPK family includes three major molecules: extracellular signal-regulated kinase (ERK) 1/2 (p44/42), p38 MAPK, and c-Jun N-terminal kinase (JNK), and they each have different signal transduction mechanisms.5 Activation of ERK is required for neuronal plasticity, such as learning and memory,6 and it also plays an important role in the transmission of pain sensation. Indeed, phosphorylated ERK (pERK; the active form of ERK) was observed in the cell bodies of primary afferent neurons in the dorsal root ganglion (DRG) and in the spinal cord, after noxious stimulation.3,7 Inhibition of pERK by MAPK/ERK kinase (MEK) (an upstream kinase of ERK) attenuated these pain responses.8 Moreover, ERK was also activated in both DRG and spinal glial cells after peripheral nerve injury.3 There are some reports indicating that local injection of a MEK inhibitor could attenuate nerve injury-induced neuropathic pain.9,10 Not only ERK but also p38 MAPK and JNK may participate in inflammatory and neuropathic pain.11,12 Phosphorylation of both p38 MAPK and JNK were observed in DRG and glial cells (i.e., microglia and astrocytes) of the spinal cord after peripheral nerve injury associated with neuroinflammation.13,14 Treatment with each inhibitor of p38 MAPK and JNK as well as ERK attenuated the magnitude of neuropathic pain. The MAPK family including ERK is activated by major inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α,15 and it is thought that these cytokines play a key role in the development of neuropathic pain.16,17
Among these three major molecules in the MAPK family, pERK in the DRG and spinal cord has been well examined in several types of neuropathic pain.18,19 Previous reports showed an increase in pERK in injured sciatic nerve.20,21 However, the detailed role of pERK in the region of injured peripheral nerve is poorly understood. In this study, we investigated whether pERK in injured sciatic nerve contributes to neuropathic pain induced by partial sciatic nerve ligation (PSL) in mice, using intraneural and perineural injection of a MEK inhibitor, U0126, which is commonly used for the examination of neurological mechanism rather than therapeutic intervention.
Male Institute of Cancer Research (ICR) mice (SLC, Osaka, Japan) weighing 18-20 g were housed in plastic cages with water and food available ad libitum. Mice were maintained in an air-conditioned (23°C-24°C, 60%-70% relative humidity) vivarium with a 12-h dark/light cycle (light on from 8:00 am to 8:00 pm). Seven to eight mice per group were used in behavioral analysis and five to six mice per group were used in other experiments. All experimental procedures were approved by the Animal Research Committee of Wakayama Medical University and complied with the ethical guidelines of the International Association for the Study of Pain.22
Partial Sciatic Nerve Ligation
PSL of mice was performed according to the method described previously.23 Briefly, under sodium pentobarbital (75 mg/kg, i.p.) anesthesia, the common sciatic nerve at the high thigh level of the right hindlimb was exposed through a small incision. The position of sciatic nerve was identified using the femoral head as a landmark. Approximately one half of the sciatic nerve thickness was tightly ligated with a silk suture. The incision was closed with suture and washed with an antiseptic. Exposure of the sciatic nerve of the left hindlimb was performed as a sham control operation. In behavioral analysis, the response of the sham-operated left hindpaw was evaluated as a control for reducing the number of animals used in the experiment.
Mice were killed by decapitation and the sciatic nerve was collected. Tissue was sonicated in lysis buffer (20 mM HEPES, 100 mM NaCl, 5 mM KCl, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, pH 7.5) and the supernatant was collected after centrifugation at 15,000g for 10 min. A protein concentration of the prepared extract was evaluated by the Bradford method and sodium dodecyl sulfate (SDS) sample buffer (50 mM Tris, 10% glycerol, 2% SDS, pH 7.4) was added. Samples and molecular weight markers (Invitrogen, Carlsbad, CA) were electrophoresed in 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membrane (Millipore Co., Billerica, MA). The membrane was blocked with blocking buffer (5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween-20) at room temperature for 2 h and incubated with specific antibodies against ERK1/2 (p44/42; rabbit polyclonal antibody, 1:2000, Cell Signaling, Danvers, MA) or pERK1/2 (rabbit polyclonal antibody, 1:1000, Cell Signaling) diluted in blocking buffer at room temperature for 2 h or at 4°C overnight. The membrane was washed with Tris-buffered saline containing 0.1% Tween-20 and incubated in specific secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, 1:5000, Zymed Laboratories, San Francisco, CA) diluted in blocking buffer at room temperature for 2 h. Immunoreactive bands were detected using a chemiluminator with an enhanced chemiluminescent substrate for the detection of horseradish peroxidase (Millipore). The intensity of gray scale image was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). Data are presented as the intensity of ERK or the ratio of pERK/ERK.
Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 20 mL of phosphated buffered saline (PBS), pH 7.4, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Collected sciatic nerve (length of approximately 10 mm) was then postfixed in 4% paraformaldehyde for 3 h and was dehydrated overnight in 25% sucrose at 4°C. Tissue was frozen in optimal cutting temperature compound (SAKURA Finetechnical Co., Tokyo, Japan) and was cut longitudinally at 10 μm, using a cryostat. The sliced tissue was mounted on a silane-coated glass slide and air-dried at 37°C overnight. The section was washed with PBS containing 0.1% Triton X-100 (PBST) and incubated with 3% bovine serum albumin in PBST at room temperature for 2 h. The section was incubated with specific antibodies against pERK1/2 (rabbit polyclonal IgG, 1:100, Cell Signaling), glial fibrillary acidic protein (GFAP) (mouse monoclonal IgG, 1:100, Chemicon, Temecula, CA), and F4/80 (rat monoclonal IgG, 1:100, Cedarlane Laboratories, Burlington, Ontario, Canada) diluted in reaction buffer (1% bovine serum albumin, 0.01% Triton X-100 in PBS) at room temperature for 2 h or at 4°C overnight. The section was washed with PBST and incubated in specific secondary antibodies conjugated with fluorescent markers (Alexa-488-conjugated chicken anti-rabbit IgG, Alexa-594-conjugated donkey anti-mouse or anti-rat IgG, 1:200, Invitrogen) at room temperature for 2 h. The section was washed with PBST, followed by nuclear staining using Hoechst 33342 (Invitrogen), and a cover slip with Perma Fluor (Thermo Fisher Scientific, Pittsburgh, PA) was placed over it, and immunoreactivity was detected with a fluorescence microscope.
Intraneural and Perineural Injection
U0126 (an inhibitor of MEK, Merck, Tokyo, Japan) was dissolved in dimethylsulfoxide and was diluted in sterile PBS at a concentration of 10 nmol/μL. Thirty minutes before PSL and on Day 1 after PSL, intraneural and perineural injections were performed, respectively.24 Briefly, under pentobarbital (75 mg/kg, i.p.) anesthesia, sciatic nerve was exposed according to the methods mentioned above. U0126 (20 nmol) or vehicle (20% dimethylsulfoxide) was injected into the sciatic nerve in a volume of 2 μL using a microsyringe fitted with a 32-gauge needle. On Day 1, under pentobarbital (75 mg/kg, i.p.) anesthesia,25 U0126 (20 nmol) (in a volume of 10 μL using a microsyringe fitted with a 30-gauge needle) was injected into the area surrounding the sciatic nerve at a high thigh level of both hindlimbs without a skin incision. When the Evans blue dye was injected according to this procedure, complete deposition of the dye to the area surrounding the sciatic nerve was observed.
Thermal Paw Withdrawal Test
Thermal hyperalgesia was evaluated by withdrawal latency using the IITC 390 Plantar Test Analgesia Meter (Neuroscience, Tokyo, Japan). Mice were placed under plexiglas cages on top of a glass sheet and covered with a clear cage. Mice were allowed to adapt for 2-3 h. The radiant heat source was positioned under the glass sheet and applied to the plantar surface of hindpaw. Withdrawal latencies were measured three times for hindpaws. Data are presented as the mean latency of the three stimulations. The heat intensity was calibrated to give a control latency of approximately 8-10 s. A cutoff latency of 15 s was set for each measurement to avoid tissue damage and unnecessary suffering to mice.
von Frey test
Tactile allodynia was evaluated by withdrawal responses, using von Frey filaments (Neuroscience). Mice were placed on a 5 × 5-mm wire mesh grid floor and covered with an opaque cup to avoid visual stimulation. Mice were allowed to adapt for 2-3 h. The von Frey filaments were inserted through the mesh floor bottom and were applied to the middle of the plantar surface of the hindpaw with a weight of 0.16 g. Withdrawal responses were measured 10 times for each hindpaw. Tactile allodynia was considered the number of withdrawal responses to stimulation.
Data are presented as the mean ± sem. In Western blotting (Fig. 1) and behavioral analysis (Fig. 4), the statistical significances were determined by a two-way analysis of variance followed by Bonferroni multiple comparisons test and was established at P < 0.05.
PSL-Induced pERK in Injured Sciatic Nerve
We evaluated pERK1/2 in injured sciatic nerve on Days 1-14 after PSL using Western blotting. In sham-operated mice, the levels of pERK1/2 in sciatic nerve were constant and the same as that in naive mice during Days 1-14. In PSL-operated mice, a significant increase in pERK1/2 was observed on Day 1 after PSL and persisted until Day 3. After that, the level of pERK1/2 in PSL-operated mice was restored to that in sham-operated mice (Fig. 1A). The expression levels of ERK itself in PSL and sham-operated mice were similar to naive mice (Fig. 1B). The immunoreactivity of pERK1/2 in PSL-operated sciatic nerve was markedly increased in comparison with that in the sham-operated sciatic nerve on Day 1 after PSL (Fig. 2). By double immunostaining, the increased immunoreactivity of pERK1/2 was found to be colocalized with GFAP, a marker of Schwann cells, but not with F4/80, a marker of macrophages, in the sciatic nerve on Day 1 after PSL (Fig. 3).
Attenuation of PSL-Induced Neuropathic Pain by a MEK Inhibitor
To determine the involvement of pERK1/2 in PSL-induced neuropathic pain, U0126, a MEK inhibitor, was injected into the area of the sciatic nerve twice, that is, an intraneural injection at 30 min before PSL and a perineural injection on Day 1 after PSL. Thermal withdrawal latency in mice treated with PSL + vehicle was significantly less than that in mice given sham + vehicle on Days 3, 7, and 14 after PSL, demonstrating thermal hyperalgesia. The PSL-induced thermal hyperalgesia was significantly attenuated by treatment with U0126 (Fig. 4A). Using the von Frey test, the number of responses in mice treated with PSL + vehicle was significantly increased on Days 3, 7, and 14 in comparison with that in mice given sham + vehicle, showing tactile allodynia. The PSL-induced tactile allodynia was significantly attenuated by administration of U0126 on Days 7 and 14 after PSL (Fig. 4B). No effect of U0126 was observed in sham-operated mice on these days in either test. Moreover, we also tested the responses to mechanical stimulation using 0.4 g of filament, and then similar results were found (data not shown).
In this study, maximal increases in pERK were observed in injured sciatic nerve on Day 1 after PSL, and significant activation continued to Day 3, followed by a gradual decrease (Fig. 1). The increased pERK was localized in Schwann cells but was not observed in macrophages in the sciatic nerve (Figs. 2 and 3). Although the increased pERK was restored to the basal level on Day 7, PSL-induced thermal hyperalgesia and tactile allodynia persisted until Day 14. Local injection of U0126 suppressed PSL-induced thermal hyperalgesia and tactile allodynia. The suppressive effect of U0126 on neuropathic pain persisted until Day 14 after PSL (Fig. 4). These results indicate that the activation of ERK in damaged Schwann cells may play an important role in the early stage of peripheral nerve injury-induced neuropathic pain.
It was previously demonstrated that pERK was increased in primary sensory neurons after nerve inflammation and nerve injury, such as nerve transection, crush, or ligation.19–21 However, the detailed mechanism of ERK activation has been poorly understood. The increase of pERK in DRGs has been well examined and the consensus was that pERK was necessary for inflammatory and neuropathic pain.26,27 On the other hand, the activation of ERK in the injured region of the peripheral nerve, including Schwann cells, macrophages, and axons of sensory neuron, was not determined. In our study, pERK was localized in GFAP-positive Schwann cells after PSL. Although macrophages were recruited and activated in the injured region after peripheral nerve injury,28 pERK was not localized in F4/80-positive macrophages. Therefore, it is possible that the activation of ERK in Schwann cells, but not macrophages, is important for the development of neuropathic pain after PSL. Although the key activators of ERK in Schwann cells are still unclear, it was reported that damaged Schwann cells and infiltrating immune cells produced several inflammatory cytokines, such as interleukin-1β and tumor necrosis factor-α, after nerve injury.29–31 Therefore, we hypothesize that these inflammatory cytokines may participate in ERK activation after nerve injury.
In Schwann cells, ERK activation participates in various cellular events, such as cell proliferation, differentiation, survival, and activation after gene expression.32 Indeed, damaged Schwann cells showed a disorder of myelin conformation.33 In general, myelin conformation is essential for normal saltatory conduction in sensory neurons and demyelination of sciatic nerve fiber caused by nerve injury elicits neuropathic pain.34 Demyelination of sciatic nerve fibers may be involved in pERK-dependent neuropathic pain. Moreover, damaged Schwann cells produce several inflammatory cytokines, which are involved in inducing neuropathic pain, and it is thought that these components of Schwann cells are important for the development of neuropathic pain.31,35 It was previously shown that inhibition of these inflammatory cytokines in the peripheral nervous system (PNS) prevented neuropathic pain. These results suggest that the inhibition of ERK activation attenuates the development of neuropathic pain and is consistent with our results.36–38
In this study, we examined the relationship between pERK-dependent Schwann cell activation in the PNS and neuropathic pain. The increase in ERK plays a key role in nerve injury-induced neuropathic pain in the spinal cord and in the PNS.10 In addition, other molecules of the MAPK family, which include p38 MAPK and JNK, are important for the development of neuropathic pain.38 Therefore, not only ERK activation but also p38 MAPK and JNK activation may play a role in neuropathic pain through the regulation of Schwann cells.
In conclusion, we demonstrated that the increase in pERK in Schwann cells of the injured PNS played an important role in the development of neuropathic pain. The underlying mechanism of neuropathic pain, pERK, in Schwann cells may elicit several dramatic events leading to chronic pain. Our results suggest that pERK itself and ERK-related mediators are potential therapeutic targets for the treatment of neuropathic pain.
The authors gratefully acknowledge Dr. James H. Woods and Dr. Gail Winger (Department of Pharmacology, University of Michigan Medical School) for review of this manuscript, and also thank Mr. Takashi Iguchi and Ms. Sachiyo Kogure for technical assistance.
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© 2009 International Anesthesia Research Society
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