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From the *Department of Anesthesiology, Chang Gung Memorial Hospital; Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan; and Suao and Yuanshan Veterans Hospital, Yilan, National Yang-Ming University, Taipei, Taiwan.
Address correspondence and reprint requests to Ping-Wing Lui, MD, PhD, Suao and Yuanshan Veterans Hospital, No. 386, Rongguang Rd., Yuanshan Township, Yilan County 264, Taiwan. Address e-mail to pwlui{at}mail.ysvh.gov.tw.
Abstract
BACKGROUND: Recent reports have identified a role for cyclic adenosine monophosphate (cAMP) transduction in nociceptive processing. Spinal activation of the cAMP induced gene transcription through the activation of protein kinase A and cAMP response element-binding protein (CREB). Intrathecal injection of protein kinase A inhibitor reversed the mechanical hyperalgesia, whereas injection of CREB antisense attenuated tactile allodynia caused by partial sciatic nerve ligation (PSNL) in rats. In the present study, we aimed to assess the effects of spinal cAMP transduction on the nociceptive processing in a chronic neuropathic pain model.
METHODS: PSNL was performed in male Sprague-Dawley rats 1 wk after intrathecal catheterization. Nociception to mechanical and thermal stimuli was assessed at the hindpaw 2 h, 3, 7, and 14 days after PSNL. The effects of adenylate cyclase inhibitor, SQ22536 (0.7 µmol, intrathecal) on these nociceptions were evaluated. Changes in the expression and immunoreactivity of CREB and its phosphorylated proteins (CREB-IR and pCREB-IR) in the dorsal horn of the spinal cord were also measured.
RESULTS: The expression of CREB-IR and pCREB-IR proteins was shown to increase for 2 wk after PSNL. The increase in pCREB was partially reversed by the blockade of the cAMP pathway in the early 3 days, with a parallel increase in mechanical and thermal withdrawal thresholds.
CONCLUSION: These results revealed the possible contribution of an increase in pCREB to the PSNL-induced tactile allodynia and thermal hyperalgesia. Modulation of the cAMP pathway may be clinically relevant if early intervention can be achieved in patients with chronic neuropathic pain.
Neuropathic pain resulting from nerve injury due to trauma or disease is one of the most difficult challenges in pain management because of its varied mechanisms. Several reports have identified the roles in the cascade of cyclic adenosine monophosphate (cAMP) transduction involving nociceptive processing. Gene transcription was induced through the activation of protein kinase A (PKA) with the subsequent phosphorylation of the transcription factor, i.e., cAMP response element-binding protein (CREB) (1). In addition, several lines of evidence showed an increase in the phosphorylation of CREB in the superficial dorsal horn of neurons after partial sciatic nerve ligation (PSNL) (2). Peripheral activation of the adenylate cyclase-cAMP pathway was involved in the mediation of hyperalgesia in rats (3). Although an increase in cAMP was crucial in producing mechanical hyperalgesia, the PKA and CREB activities were also important in the maintenance of inflammatory pain (4). Injection of PKA inhibitor into the rodent spinal cord reversed mechanical hyperalgesia in response to intradermal injection of capsaicin (5). Furthermore, phosphorylation of CREB secondary to mechanical hyperalgesia was time-dependently reversed by blockade of the cAMP pathway after repeated intramuscular (IM) injections of acid (3). There is also some evidence that intrathecal (i.t.) injection of CREB antisense oligonucleotide attenuated PSNL-induced tactile allodynia (6). Despite evidence of the cAMP cascade in various pain models, the results were not consistent, most notably in the time course of CREB activation. The present study assessed the involvement of spinal cAMP in nociceptive processing in a chronic neuropathic pain model.
METHODS
This study was approved by the Committee of Institutional Animal Care and Use. Experiments were performed on adult male Sprague-Dawley rats (weighting 250–300 g) housed in pairs before surgery. Food and water were unrestricted. After sciatic nerve ligation, the animals were housed individually in clean bedding of organic cellulose fiber. The wound was checked daily and animals were excluded if there was any sign of wound infection or dehiscence. At the end of the protocol, all animals were killed with an overdose of pentobarbital administered intraperitoneally.
Animals were anesthetized with 1.5% to 2% isoflurane delivered via a nose cone (OH Medical Anesthetics) and were placed in prone position. After skin sterilization, a small incision was made in the atlanto-occipital membrane and a polyethylene catheter (PE-5, Becton-Dickinson, Sparks, MD) was inserted 8.5 cm. The external portion of the catheter was secured to the muscle at the back of the neck, and the wound was closed with sutures. Before sciatic nerve ligation, the function of the catheter was verified by i.t. injection of 2% xylocaine (10 µL). The catheter was appropriately placed if loss of motor power in the lower extremities was observed. Animals with any neurological deficits were excluded from the study. One week after i.t. catheterization, all rats were pretested for nociceptive baseline in response to von Frey filaments as well as radiant heat stimulation at the proximal part of the hindpaw (7–10).
Withdrawal responses to mechanical stimulation were determined using a calibrated Electronic von Frey Anesthesiometer (Model 2290CE, IITC Inc., CA) applied from beneath the cage through openings (12 x 12 mm) in the plastic mesh floor to the distal portion of the plantar aspect of the hindpaw. The stimulation was applied starting from 0 g and continuing until a withdrawal response was observed or a cutoff value (70 g) was reached. This was repeated three times with at least a 5-min test-free period between withdrawal responses. The lowest forces from the three tests producing a response were considered the withdrawal threshold. Finally, the flexible von Frey filaments above and below were tested to confirm the withdrawal threshold. For the withdrawal latencies to heat stimulation, rats were placed individually on an elevated plastic mesh floor covered with a clear plastic cage top (21 x 27 x 15 cm) and were assessed using a focused radiant heat source (Model-33 Tail Flick Analgesia Meter, IITC Inc., CA). The heat stimulus, a 50-W projector lamp with an aperture diameter of 6 mm, was applied from beneath a heat-tempered glass floor (3-mm thick) on the distal portion of the plantar hindpaw. Paw withdrawal latencies (PWL) were measured to the nearest 0.1 s. Three trials, 5–10 min apart, were used to obtain the average PWL. All rats were reanesthetized with isoflurane after the average PWL was attained. The right sciatic nerve was exposed high on the thigh and half of the nerve was tightly ligated with 6–0 polydioxanone suture (Ethicon, Arista, NY, NY) as described by Seltzer et al. (7). In sham animals, the sciatic nerve was exposed without ligation. Muscle and skin layers were then closed. Immediately after surgery, rats were randomly assigned into two different groups (n = 8 per group), an adenylate cyclase inhibitor, SQ22536 (0.7 µmol, dissolved and diluted with distilled water, Biomal, Plymouth Meeting, PA) or the same volume of distilled water (vehicle) was administrated i.t. After a recovery time of 2 h, responses to the mechanical stimulus and radiant heat were retested. The responses to these stimuli were determined for the next 3, 7, and 14 days in all groups.
A pilot dose-response test was performed 24 h after sciatic nerve ligation. Rats were randomly assigned into different groups (n = 6–8 per group) and received i.t. SQ22536 (0.1, 0.35, 0.7, or 1 µmol). The withdrawal threshold including preligation threshold (before), 24 h after nerve ligation (baseline) and every 30-min interval for 3 h after drug injection (after) were determined using a calibrated Electronic von Frey Anesthesiometer. The percentages of maximum possible effect, where % = 100 x (After – Baseline)/(Before – Baseline) of each dose were calculated as 20.4%, 25.8%, 55.0%, 57.6%, and 16.2% for vehicle, respectively (Fig. 1C). This result was in accordance with that of a study in which 0.715 µmol of i.t. SQ22536 was shown to increase the mechanical withdrawal threshold induced by IM acid injection (3). The dose of 0.7 µmol was then chosen for the present study.
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To observe the parallel changes in immunoreactivity (IR) of CREB-IR and its phosphorylated proteins (pCREB-IR) in the dorsal horn of the spinal cord after drug injections, different groups of animals were killed for immunohistochemistry at 2 h, 3, 7, and 14 days after PSNL. The sham-operated animals were used as controls. Rats were deeply anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused intracardially with cold saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.4). The L3–6 spinal cord segments were removed and fixed in the above fixative for 3–6 h. All tissues were cryoprotected by transferring to 30% sucrose in 0.1 M PBS at 4°C for 24 h. The spinal cord segments were cut on a cryostat at a 20-µm thickness. The free-floating sections were collected in PBS and blocked with 3% normal goat serum (Vector Laboratories, Burlingame, CA) in 0.3% Triton X-100 and 0.3% hydrogen peroxide for 1 h at room temperature. Sections were incubated for 36 h at 4°C in a rabbit polyclonal anti-CREB antibody and anti-pCREB antibody (1:1000, New England Biolabs, Beverly, MA), and were processed in biotinylated goat antirabbit IgG (1:200) using Elite Vectastain ABC kit (Vector lab, Burlingame, CA). Finally, the immunoprecipitates were developed with 0.05% diaminobenzedine in PBS for 3–10 min.
Eight L4–L5 spinal cord sections were randomly selected from each rat. Images of both ipsilateral and contralateral dorsal horns were captured at x250 magnification using a digital camera (Nikon DXM 1200 with Eclipse E800). Anatomical landmarks were used to identify laminar borders in gray matter of the spinal cord in reference to the standard anatomical mapping. The number of digitized pixels overlaid CREB-IR and pCREB-IR cells in the superficial laminae of the dorsal horn were measured automatically using image analysis software (Image-Pro Plus, Media Cybernetics, Inc., GA Avenue, Silver Spring). The values from these sections were averaged for each animal. To examine possible changes in the expression of CREB-IR and pCREB-IR within cells, the mean optical density of all positive (supra-threshold) objects were compared.
Different groups of animals were killed for Western immunoblots and the lumbar spinal cords (L4–5) were excised. The dorsal halves were weighed, homogenized and centrifuged at 7000g for 15 min. The 50 mM Tris–HCl (pH 7.4) homogenizing buffer contained with 150 mM NaCl, 50 mM NaF, 2 mM EDTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, and 5 µg/mL of a mixture of protease inhibitors included bestatin, 4-(2-aminoethyl) benzene-sulfonyl fluoride, pepstatin A, aprotinin, and leupeptin. Total protein content in the homogenates was determined and separated by SDS-PAGE electrophoresis (15 µg of total protein per lane), transferred onto nitrocellulose membranes, and blocked with PBS solution containing nonfat, dry milk (5%) for 1 h. The membranes were first incubated with rabbit polyclonal antibodies to CREB (1:1000) and pCREB (1:500) overnight at 4'C, and then in secondary antibody at 1:10000 (Jackson, PA) for 1 h at room temperature. The signals were visualized by chemiluminescence and quantified using PharosFXTM Plus Molecular Imager® System (BIO-RAD Laboratories Inc. Hercules, CA). Values are expressed as mean ± sem. Groups were compared using one-way analysis of variance with Student Newman–Keuls multiple comparisons. P < 0.05 was considered significant.
RESULTS
After PSNL, most rats developed tactile allodynia and thermal hyperalgesia in the ipsilateral hindpaw, compared with the control group for at least 14 days. The allodynia and hyperalgesia in the ipsilateral hindpaw was significantly attenuated 1–3 days after i.t. injection of SQ22536, when compared with vehicle (Fig. 1). Increases in CREB-IR and pCREB-IR cell profiles were also found in the dorsal horn of L4–5 spinal cord with predominance in the superficial layers (Fig. 2). The mean pixel number, optical density, and protein expression in both groups were significantly higher than that of the preinjured baseline. However, differences in the total CREB-IR cells were not observed between SQ22536 and vehicle control (Figs. 3 and 4A). After i.t. SQ22536, pCREB-IR cell profiles were partially reduced, but not by vehicle, in the ipsilateral dorsal horn. Quantitatively, the mean pixel number, optical density and protein expression after i.t. SQ22536 were significantly decreased in comparison with control, especially after the first 3 days (Figs. 4B and 5).
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The results of this study demonstrated that PSNL significantly induced an increase in the phosphorylation of CREB for 2 wk. Furthermore, the expression of pCREB in cells of the superficial dorsal horn was correlated to the reduction in the mechanical and thermal withdrawal thresholds, indicating that behavioral changes were associated with an increase in pCREB. This result also showed that the increased pCREB depended on the activation of the cAMP pathway, as it could be prevented for at least 3 days by blocking the adenylate cyclase activity.
According to previous reports, activation of the cAMP pathway in the spinal cord was implicated in the mediation of nociceptive processing. Mechanical hyperalgesia was produced by spinal activation of the cAMP pathway (4,5), and spinal activation of adenylate cyclase increased the activities of neurons in the spinothalamic tract in response to pinch stimulation, which was blunted by pretreatment with a PKA inhibitor (11). Mice lacking adenylate cyclases manifested a reduction in behavioral responses to formalin or complete Freund’s adjuvant stimuli (12). Additionally, blocking adenylate cyclase or PKA prevented the mechanical hyperalgesia and allodynia induced by capsaicin injection (4,5). In the present study, PSNL-induced tactile allodynia and thermal hyperalgesia were attenuated by blockade of the cAMP pathway in the early phase.
Our results showed that PSNL significantly induced the expression of CREB, a transcription factor, at least for 2 wk, whereas i.t. adenylate cyclase inhibitor had no significant effect. Increases in CREB resulted in a greater resource of protein for phosphorylation. Previous studies reported that CREB mRNA and CREB-IR caused an increase in hippocampus after chronic administration of antidepressants in rats (13). Little information is available on the effect of chronic noxious stimulation on the expression of CREB, which is still controversial. The results of Sluka’s study revealed an increase in CREB level 24 h, but not in 1 wk, after repeated IM injections of acid in rats (3). In contrast, Miletic et al. (14) reported that there were no differences in the content of CREB between the PSNL model and control. Much work has still to be accomplished in the elucidation of different mechanisms underlying the control of the CREB expression in chronic pain disorders.
When CREB is phosphorylated, it binds to specific DNA consensus sequences such as cAMP responsive element, and regulates immediate-early genes, including c-fos (15) and c-jun (16). Some late effector genes such as those that transcribed dynorphin (17), substance P receptor NK 1 (18), and other elements (19) were also involved. Different animal models showed an association between the induction of nociception and the expression of pCREB at various intervals. For instance, an increase in the expression of pCREB occurred at the hindpaw of rats after subcutaneous injection of carrageenan (12), formalin or complete Freund’s adjuvant (12,18,20). Animals with repeated IM injection of acid (3) and those that suffered from neuropathic pain demonstrated a similar phenomenon (2,14). The expression of phosphorylated CREB is involved in the temporal effects of hyperalgesia in neuropathic (14) and inflammatory pain (20). Furthermore, the amount of phosphorylated CREB seemed to be stimulus dependent. Increasing the volume of formalin injected into the hindpaw resulted in an increase in phosphorylated CREB (20). Phosphorylation of CREB was also increased in rats with morphine tolerance as well as in CREB mutant mice, whose main symptoms of morphine withdrawal were significantly attenuated (21). Taken together, these observations strongly suggested that increased phosphorylation of CREB was likely to be involved in the development of inflammatory pain and neuropathic pain.
The results of previous observations also demonstrated that different response-time courses in the phosphorylation of CREB were demonstrated in various pain models. For instance, Hoeger-Bement and Sluka (3) reported that the mechanical hyperalgesia and phosphorylation of CREB depended on the early activation of the cAMP pathway during the first 24 h, but are independent of the cAMP pathway 1 wk after IM injection of acid. The temporal changes in the phosphorylation of CREB was observed in the following chronic neuropathic pain models, i.e., 2 h after loose ligation of the sciatic nerve (22), 3 wk after PSNL (2), and 7 days, but not 28 days after a chronic nerve constriction model (14). The results of the present study gave credence to the hypothesis that activation of the cAMP pathway was likely to be involved in the early phase of maintenance, but not the later phase of induction, in rats with chronic neuropathic pain. In fact, the temporal effects of cAMP pathway activation were manifested in other models of neuroplasticity. For instance, the early phase of long-term enhancement depended on the activation of the cAMP pathway, but was decreased by the inhibition of this pathway (23,24). In the hippocampus, the activity of PKA was rapidly increased in the initial stage of spatial learning and started to decrease when the activity of protein kinase C had reached to the maximum at the later stage (25). Previous evidence indicates that protein kinase C activation seemed to be critical in the maintenance phase of long-term enhancement such as memory (26). Although the results of the present study were compatible with the above phenomenon, further verification is still needed.
Consistent with our results, a previous study demonstrated a reduction in hyperalgesia secondary to the inhibition of the cAMP pathway at 24 h, but not 1 wk, in a repeated IM acid injection pain model (3). Changes in pCREB at 24 h were also found to have strong correlation with an increase in the mechanical withdrawal threshold, suggesting a role for CREB phosphorylation in the early phase of maintenance in mechanical hyperalgesia induced by IM acid injection. As demonstrated in our study, CREB phosphorylation in the early phase of neuropathic pain was mediated by the activation of the cAMP pathway because of the reversal of PSNL-induced pCREB enhancement secondary to cAMP inhibition. Although the role of phosphorylated CREB in the maintenance of neuropathic pain has been confirmed in the present study, other confounding factors underlying its mechanisms still need to be considered. For instance, in addition to PKA, a number of intracellular messengers resulting to the phosphorylation of CREB, such as calmodulin-dependent protein kinase, nerve growth factor-mediated Ras/Raf mitogen-activated protein kinase kinase-1/2 and extracellular regulated kinase, mitogen-activated protein kinase, p38, and other kinases may contribute to the regulation of CREB phosphorylation (1,27,28). One limitation of our study was the role of the cAMP-CREB pathway because the inhibition of cAMP only attenuated the PSNL-induced allodynia or hyperalgesia, but not the paw withdrawal threshold in response to mechanical and heat stimulation. This observation suggested that pathways other than cAMP-CREB may be involved in the mechanisms underlying the early phase of PSNL-induced tactile allodynia and thermal hyperalgesia.
Our results demonstrated that CREB and the phosphorylation of CREB in the spinal cord significantly increased for 2 wk after PSNL in parallel with an increase in the mechanical and thermal withdrawal threshold. The increase in pCREB level was partially reversed by the blockade of the cAMP pathway early in the 3 days. Our results strongly imply that increases in pCREB contributed to PSNL-induced tactile allodynia and thermal hyperalgesia. Modulation of cAMP pathway may be of clinical importance if early interventions can be performed in patients with chronic neuropathic pain.
Footnotes
Accepted for publication August 20, 2007.
Supported by a research grant (CMRPG 33012 and partial of CMRPG 34028) to J.T. Liou provided by Chang Gung Memorial Hospital and Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taiwan.
REFERENCES
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