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From the *Department of Anesthesiology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of China; Department of Anesthesiology, Tong Ren Hospital, Capital University of Medical Science, Beijing, People’s Republic of China; and Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of China.
Address correspondence and reprint requests to Yuguang Huang, Department of Anesthesiology, Peking Union Medical College Hospital, Shuai Fu Yuan 1, Dong Dan district, Beijing, People’s Republic of China 100730. Address e-mail to pumchhyg{at}yahoo.com.cn.
Abstract
BACKGROUND: Tumor necrosis factor 
(TNF-) could trigger p38 mitogen-activated protein kinase (MAPK) activation. Conversely phosphorylated p38 (p-p38) could induce the upregulation of TNF-. In this study, we examined the hypothesis that chronic constrictive injury (CCI) of the sciatic nerve could promote spinal cord release of TNF- and produce allodynia via the p38 MAPK pathway.
METHODS: Sprague–Dawley rats were divided into five groups: 1) naïve control rats, 2) sham surgery rats, 3) CCI surgery rats without treatment, 4) CCI surgery rats with saline (0.9%) treatment, and 5) CCI surgery rats with the p38 MAPK inhibitor SB203580 treatment. In treatment groups, saline or SB203580 (2 µg, twice a day) was given intrathecally starting 1 day before or 1 day or 7 days after CCI. All rats were killed at different times after surgery to examine p38 MAPK activity and TNF- levels in the spinal cord by Western blot analysis or immunohistochemistry. Mechanical allodynia was tested by a series of von Frey hairs 3, 7, and 14 days after surgery.
RESULTS: p-p38 MAPK was significantly increased at 3, 7, and 14 days after CCI surgery compared with time-matched shams (P < 0.05). Peripheral nerve injury induced mechanical allodynia and enhanced spinal concentrations of TNF- (P < 0.05). Pretreatment or early treatment with SB203580 inhibited p38 MAPK activity, resulting in reduction of TNF- synthesis and attenuation of mechanical allodynia (P < 0.05).
CONCLUSION: p38 MAPK activation is one aspect of the signaling cascade that culminates in TNF- synthesis and contributes to mechanical allodynia after peripheral nerve injury.
Neuropathic pain occurs after nerve injury or as part of diseases that affect peripheral nerve function, such as diabetes, herpes zoster, acquired immune deficiency syndrome, and cancer. Such pain can be characterized by hyperalgesia (increased responsiveness to noxious stimuli) and allodynia (painful responses to normally innocuous stimuli) (1). Therapeutics currently available for neuropathic pain are not generally very effective (2,3). Numerous studies have found that proinflammatory cytokines such as tumor necrosis factor (TNF-) may play an important role in neuropathic pain. Thus, TNF-, when applied on the sciatic nerve, evokes spontaneous firing of nociceptive primary afferent fibers in a dose-dependent manner (4). After subcutaneous injection of TNF-, a decrease in mechanical threshold is observed (5). Several lines of evidence have suggested (6) that TNF- expression increases in the dorsal horn of the spinal cord after peripheral nerve injury. In addition, the inhibition of TNF- reduces the hyperalgesia induced by chronic constriction injury (CCI) (7,8). However, the precise details of the signaling cascade involved in TNF- synthesis in the spinal cord have not been established. In cultured microglia, lipopolysaccharide stimulated induction of TNF- by activation of p38 mitogen-activated protein kinase (p38 [MAPK]) (9). Microglia are often considered resident macrophages, which are the source of multiple cytokines, including TNF- (10–13). Previous studies (14,15) showed that p38 is activated after peripheral nerve injury in spinal cord microglia in several neuropathic pain models. Thus, there should be a linkage between TNF- and p38, which plays an important role in the generation of neuropathic pain after nerve injury.
In the present study, we investigated the activation of p38 in the spinal cord after CCI of the sciatic nerve. In addition we examined whether the inhibition of p38 could block neuropathic pain at different time points during surgery. Finally, by using the p38 inhibitor, we determined whether inhibition of p38 affects TNF- levels in the spinal cord after CCI.
METHODS
Animals
Male Sprague–Dawley rats (n = 118) weighing 160–180 g were used in procedures approved by the Animal Care and Use Committee of Peking Union Medical College, Beijing, China. Experimental groups included: 1) naïve control rats (n = 14), 2) sham surgery rats (n = 14), 3) CCI surgery rats without treatment (n = 42), 4) CCI surgery rats with saline (0.9%) treatment (n = 24), and 5) CCI surgery rats with the p38 MAPK inhibitor SB203580 treatment (n = 24). In Group 4 and Group 5, saline or SB203580 (2 µg, twice a day) was given intrathecally starting 1 day before or 1 day or 7 days after CCI. The series of injections lasted 7 days in each subgroup of Groups 4 and 5.
CCI Surgery
All experimental procedures were performed on rats (n = 118) that were deeply anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneal; supplemented as necessary). Special care was taken to prevent infection and to minimize the influence of inflammation, including covering clean paper on the surgery area and using sterile operating instruments. To produce the CCI model, a 7-mm segment of the left common sciatic nerve was exposed at the mid-thigh level under a dissecting microscope (40x magnifications). Four ligatures of 4-0 chromic gut were tied loosely around the nerve with 1 mm spacing between knots. Great care was taken to tie the ligatures such that the diameter of the nerve was seen to be just barely constricted (16). The incision was closed in layers. Rats without surgery were used as naïve controls and sham procedures (sciatic exposure without ligation) were done among the sham group.
Drug Delivery
Intrathecal cannulation was performed before CCI surgery. Under sodium pentobarbital anesthesia, a laminectomy of the L6 vertebra was performed. The dura was cut and a polyethylene-10 catheter (total length is 12–15 cm) was inserted into the rat’s subarachnoid space about 1.5 cm to lie approximately at the L3–4 spinal segmental level. The incision was sutured carefully. Before CCI surgery, the animals were allowed to recover from intubation for about 24 h. The p38 MAPK inhibitor 4-(4-fluorophenyl)-2- (4-methylsulfonylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (Sigma, St. Louis, MO) or vehicle (0.9% saline) was injected intrathecally (10 µL) and flushed with 10 µL of saline. In SB203580 treatment group, SB203580 (2 µg/once, twice a day, at 10 am and 6 pm) was given intrathecally starting 1 day before, or 1 day and 7 days after CCI. The series of injections lasted 7 days in each subgroup of Groups 4 and 5. The injections were performed without anesthesia.
Western Blots
At different time points after CCI (3 days, 7 days, and 14 days) animals were killed by decapitation under deep anesthesia, the ipsilateral lumbar enlargement was quickly removed and placed in ice-cold homogenization buffer that contained a mixture of proteinase and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA). The protein concentration of tissue lysates was determined with a BCA protein Assay Kit (Pierce, Rockford, IL), and 30 µg of protein was loaded in each lane. Protein samples were separated on SDS-PAGE gel (4%–15% gradient gel; Bio-Rad, Hercules, CA) and electrophoretically transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% low-fat milk in tris buffered saline containing 0.1% Tween 20 (Sigma, St. Louis, MO,) for 1 h at room temperature and incubated overnight at 4°C with anti-phospho-p38 antibody (anti-rabbit, 1:1000, in 5% bovine serum albumin (BSA); Cell Signaling Technology, Beverly, MA), anti-total p38 antibody (anti-rabbit, 1:2000, in 5% BSA, Cell Signaling Technology), anti-TNF- antibody (anti-goat, 1:1000, in 5% BSA, Santa Cruz Biotechnology) and β-actin (1:5000, in 5% BSA, Sigma). The blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (1:2000–1:5000, Santa Cruz Biotechnology), developed in ECL kit (Santa Cruz Biotechnology) for 1 min, and exposed onto films for 1–10 min. Data for phospho-p38 and TNF- are given in relation to the individual β-actin loading control and normalized to naive and sham controls within the same blot.
Immunohistochemistry
After defined survival times, control and injured rats were killed and perfused through the ascending aorta with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2–7.4, 4°C. After perfusion, the lumbar enlargement was carefully removed and postfixed in the same fixative for 3–4 h and then replaced with 25% sucrose overnight. Transverse free-floating spinal sections (thickness of 30 µm) were cut in a cryostat and processed for immunostaining. The cryostat sections were exposed to anti-phospho-p38 antibody (anti-rabbit, 1:100, in 5% BSA; Cell Signaling Technology, Beverly, MA). Sections were stained using the avidin-biotin technique (SP kit, Zymed, South San Francisco, CA) and 3,3'-diamino benzidine containing nickel sulfate.
Behavioral Analysis
Animals were habituated to the testing environment daily for 2 days before baseline testing. All the animals were placed on an elevated wire grid. For mechanical paw withdrawal threshold, the plantar surface of the hindpaw was stimulated with a series of von Frey hairs with ascending bending forces of 0.2g to 26g. The threshold was taken as the lowest force that evoked a brisk withdrawal response (17). All rats were tested for mechanical allodynia 3 days before surgery and 0 (on the surgery day, immediately before surgery), 3, 7, and 14 days after surgery.
Quantification and Statistics
Data are expressed as mean ± sd. For Western blots, the density of specific p-p38 bands was measured with a computer-assisted image analysis system (SynGene, Cambridge, UK) and normalized against total p38 level. Four to six rats were included for each group for quantification of Western blot and immunohistochemistry results. Differences in changes of values between the treatment groups (saline or SB203580, 6–8 rats per group were included for behavioral studies) were compared by Student’s t-test or ANOVA followed by Fisher’s PLSD post hoc analysis (SPSS 11.5, Chicago, IL). A difference was accepted as significant if P < 0.05.
RESULTS
p38 Was Activated in the Spinal Cord After CCI
An anti-p-p38 antibody was used to study changes in p38 activation. CCI induced a significant increase in phospho-p38 levels in the dorsal horn of the spinal cord as detected by Western blots. This increase was detectable at Day 3 after surgery, and was maintained for 2 wks (Fig. 1). p38 levels did not change after CCI (Fig. 1). Immunohistochemistry confirmed that there were low levels of phospho-p38 in dorsal horn under basal conditions; more importantly, 3 days after CCI there was a clear increase in both staining density and the number of cells positive for activated p38 (Fig. 1). The most prominent p-p38 positive increase was found in the medial laminae of the dorsal horn.
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CCI Induced an Increase in TNF- Synthesis in the Spinal Cord
An anti-TNF- antibody was used to assess TNF- synthesis in the spinal cord. Western blots analysis showed that as compared with the control and sham group, TNF- levels in the dorsal horn of the spinal cord were significantly increased in the CCI 3 days, 7 days, and 14 days group (Fig. 2). These suggested that CCI induced an increase in TNF- synthesis in the spinal cord.
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Inhibition of p38 Activation Attenuated CCI-Induced Mechanical Allodynia
CCI induced a mechanical allodynia that was present on Day 3 after the surgery and was maintained for more than 2 wks. To investigate the contribution of p38 activation to this altered behavior, we intrathecally administered the p38 inhibitor SB203580. First, to observe the preventive effect of p38 inhibitor, we injected SB203580 1 day before CCI surgery. This pretreatment protocol significantly reduced CCI-induced mechanical allodynia from Day 7 (n = 8, P < 0.05) to Day 14 after CCI (Fig. 3).
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To obtain a sustained inhibition of p38 over the time course of early development of neuropathic pain, we injected SB203580 (2 µg) twice per day for 7 days, with the first injection administered 1 day after the CCI injury. The CCI-induced mechanical allodynia was also significantly attenuated 7 days after the surgery (n = 8, P < 0.05) (Fig. 3).
To test whether p38 inhibition has any role in established pain hypersensitivity after CCI, we applied SB203580 (2 µg) twice per day from the 7th day after CCI surgery to the 14th day. This postsurgical treatment did not significantly reduce mechanical allodynia (n = 8, P > 0.1) (Fig. 3).
The Inhibitor of p38 Reduced TNF- Synthesis in the Spinal Cord After CCI
To test whether p38MAPK plays a role in TNF- synthesis in the spinal cord after CCI, the p38 inhibitor SB203580 (2 µg, twice a day) was given intrathecally, staring 1 day after, or 1 day and 7 days after CCI surgery. The series of injections of SB203580 lasted 7 days in each group. The dorsal horn of the L4–6 spinal cord was harvested and analyzed for TNF- level using western blots (In the sham group, the tissue was harvested at 7 days after the sham operation. Control group rates were killed at the same time. For the intrathecal saline and SB203580 groups, the tissue was harvested at 14 days after CCI). The data showed that compared with the saline treatment group, the synthesis of TNF- in the spinal cord induced by CCI was significantly attenuated in the SB203580 pretreatment subgroup (1 day before the CCI surgery) and early posttreatment (1 day after CCI surgery) subgroup (Fig. 4). However, when the CCI-induced mechanical allodynia had become established, e.g., 7 days after CCI surgery, posttreatment with SB203580 starting on Day 7 had no effect on mechanical thresholds or TNF- levels in the spinal cord (Fig. 4).
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These data suggested that pretreatment and early posttreatment with SB203580, which inhibited p38 MAPK activity, resulted in reduction of TNF- synthesis and attenuation of mechanical allodynia.
DISCUSSION
In the present study, we showed that CCI induced an increase in the phosphorylation of p38 in the dorsal horn of the spinal cord. This change coincided with the increasing synthesis of TNF- in the spinal cord after peripheral nerve lesion. Repeated intrathecal injection of the p38MAPK inhibitor SB203580, starting 1 day before or 1 day after CCI surgery, reduced CCI-induced mechanical allodynia from Day 7 to Day 14. We also found that the synthesis of TNF- in the spinal cord induced by CCI was significantly attenuated by the inhibitor of p38MAPK. However, posttreatment with SB203580 starting on Day 7 had no effect on mechanical thresholds nor on TNF- level.
Previous reports demonstrated that p38 MAPK is activated after peripheral nerve injury in spinal cord microglia in several neuropathic pain models, such as CCI (14) and spinal nerve ligation (SNL) (15). In our study, similar results with activation of p38 were obtained, although in the CCI model, p-p38 increases still could be seen 2 wks later, whereas in the SNL model it was not elevated 7 days after the surgery (15).
The p38 inhibitor, SB203580, does not inhibit the phosphorylation of p38 MAPK but, rather, binds to the ATP pocket in the enzyme, thereby inhibiting its activity (18). Intrathecal injection of SB203580 does not affect basal pain sensitivity (15) but reduces the mechanical allodynia after CCI when applied before or 1 day after the nerve injury. However, when CCI-induced mechanical allodynia was established for several days, the p38 inhibitor had no effect on mechanical thresholds. Similarly, in the SNL model, continuous intrathecal infusion of SB203580 (4 µg/d) was effective only if it was started before, but not 7 days, after SNL (19).
TNF- is the proinflammatory cytokine which has been most strongly implicated in the generation of pathological pain states. When applied to a normal dorsal root ganglia (DRG), TNF- caused pain (20). When administered to a mechanically compressed DRG, TNF- enhanced the hypersensitivity caused by the compression (20,21). Application of TNF- to the DRG was found to evoke discharges in silent fibers, enhance continuing activity of spontaneously active fibers, and increase neuronal sensitivity to electrical stimulation of the peripheral nerves (22). It was also demonstrated (23) that IM injection of TNF- induces muscle hyperalgesia in rats. Many studies observed increased levels of TNF- protein and mRNA after peripheral and central nerve injury. In the SNL model of neuropathic pain, TNF- receptors 1 and 2 were transiently upregulated in injured and adjacent uninjured DRG (24). Covey et al. (25) found that during persistent pain the level of TNF- mRNA increased in regions of the brain and spinal cord and the time course and distribution of the increases in TNF- accumulation support a neuromodulatory role for TNF- within the central nervous system in the development and maintenance of neuropathic pain. Interestingly, transgenically modified mice in which the TNF- was overexpressed displayed significantly higher mechanical allodynia after a peripheral nerve transaction as compared with the wild-type mouse (26).
Clearly, both p38MAPK and TNF- play important roles in neuropathic pain. We examined the association of p38 with TNF- synthesis in the spinal cord after CCI. After peripheral nerve injury (14,15), p38 is activated in microglia of the spinal cord in several neuropathic pain models. Meanwhile the TNF- level is also increased in the spinal cord. Microglia have an important role in the spinal cord and are the source of multiple cytokines, including interleukin-1β (IL-1β), IL-6, and TNF- (13). In our study, the inhibitor of p38 reduced TNF- synthesis in the spinal cord after CCI. It is assumed that after peripheral nerve injury the p38MAPK pathway is activated in the microglia of the spinal cord. The activated microglia could release many cytokines, including TNF-, which induced the allodynia and hyperalgesia. A previous study investigated another side of the relationship of TNF- and p38. The authors administered TNF antagonist by intraperitoneal injection to the SNL rats and found that TNF antagonist inhibited p38 MAPK activity, resulting in attenuation of mechanical allodynia (19). We conclude that TNF- and p38 MAPK activation in the spinal cord could be linked at two points, of which activated p38MAPK could induce the upregulation of TNF- or TNF- could trigger p38 MAPK activation.
Given the therapeutic window for the p38 inhibitor, we assume that p38 is involved in the development, rather than in the maintenance, of neuropathic pain. Seven days after CCI, when neuropathic pain is already developed, abundant cytokines, including TNF-, (IL-1), IL-6 and multiple pain mediators, are synthesized in the spinal cord (27). As discussed above, those cytokines may contribute to the activation of p38MAPK and other MAPK pathways. Therefore, the p38 inhibitor SB203580, which specifically blocks the activation of p38, cannot effectively attenuate established pain hypersensitivity when the drug is delivered several days after CCI initiation.
In conclusion, we demonstrated that the inhibitor of p38MAPK reduced TNF- synthesis in the spinal cord after CCI. p38MAPK is crucially involved in the signaling pathway leading to the induction of TNF- in CCI-induced neuropathic pain.
Footnotes
Accepted for publication August 21, 2007.
Supported by Natural Science Foundation of China, grant No. 30371370.
REFERENCES
requirement in the activation of p38 mitogen-activated protein kinase, which is linked to the induction of tumor necrosis factor in lipopolysaccharide-stimulated microglia. Neurochem Int 2004;44:205–14[Web of Science][Medline]
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