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Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: Relation to central neuropathic pain
Neuroscience Letters 384 (2005) 139–144
Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: Relation to central neuropathic pain E.D. Crown, Z. Ye, K.M. Johnson, G.-Y. Xu, D.J. McAdoo, K.N. Westlund, C.E. Hulsebosch ∗ Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Blvd, Route 1043, Galveston, TX 77555-1043, USA Received 28 February 2005; received in revised form 21 April 2005; accepted 22 April 2005
Abstract Spinal cord injury (SCI) often leads to the generation of chronic intractable neuropathic pain. The mechanisms that lead to chronic central neuropathic pain (CNP) following SCI are not well understood, resulting in ineffective treatments for pain relief. Studies have demonstrated persistent hyperexcitability of dorsal horn neurons which may provide a substrate for CNP. We propose a number of similarities between CNP mechanisms and mechanisms that occur in long-term potentiation, in which hippocampal neurons are hyperexcitable. One biochemical similarity may be activation of the transcription factor, cyclic AMP response element-binding protein (CREB), via phosphorylation (pCREB). The current study was designed to examine whether tactile allodynia that develops in segments rostral to SCI (at-level pain) correlates with an increase in CREB phosphorylation in specific neurons known to be involved in allodynia, the spinothalamic tract (STT) cells. This study determined that, in animals experiencing at-level allodynia 35 days after SCI, pCREB was upregulated in the spinal cord segment rostral to the injury. In addition, pCREB was found to be upregulated specifically in STT cells in the rostral segment 35 days after SCI. These findings suggest one mechanism of maintained central neuropathic pain following SCI involves persistent upregulation of pCREB expression within STT cells. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: FluoroGold; Immunocytochemistry; pCREB; Chronic pain
One of the most prevalent complaints among individuals suffering from spinal cord injury (SCI) is chronic intractable pain [1,3]. Siddall et al. [14] defined three categories of pain that result from SCI: (1) above-level pain which occurs at dermatomes rostral to the injury site in areas where normal sensation persists following injury, (2) at-level pain which occurs in dermatomes near the spinal injury, develops shortly after SCI, and is often characterized as either stabbing pain or a stimulus-independent type that is accompanied by allodynia (non-noxious stimuli become noxious), and (3) below-level pain, which is localized to dermatomes distal to the injury site, develops more gradually than does at-level pain, and is ∗
Corresponding author. Tel.: +1 409 772 2939; fax: +1 409 772 3222. E-mail address: [email protected] (C.E. Hulsebosch).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.066
often classified as a stimulus-independent continuous, burning pain. Multiple studies have indicated that the incidence of people with some form of chronic pain following SCI is up to 90%, with most studies reporting moderate to severe pain in the majority of cases [1]. Despite the prevalence of these complaints and our current understanding of the mechanisms underlying chronic neuropathic pain following SCI, there is a lack of clinically effective therapies, suggesting that these mechanisms are not entirely understood [1]. Research suggests that neuropathic pain which develops after peripheral injury or inflammation is a result of central sensitization [15,17]. Central sensitization refers to increased hyperexcitability observed in dorsal horn neurons following exposure to prolonged peripheral noxious stimulation. Recently, Ji and others (e.g., [6,12,16]) presented ev-
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idence linking the molecular mechanisms underlying central sensitization to the phenomenon of long-term potentiation (LTP). LTP is a phenomenon believed to underlie the synaptic changes in the hippocampus associated with learning and memory. The transcription factor cyclic AMP response elementbinding protein (CREB) has received a great deal of attention for its role in the formation of long-term memory, largely due to demonstrations of the importance of CREB activation in the late phases of both long-term facilitation and long-term potentiation. CREB is a 43 kDa nuclear transcription factor that is activated via phosphorylation at the serine 133 site. Once CREB is phosphorylated (pCREB), it can then bind to genes containing CRE promoters and begin transcription of a number of early immediate genes thought to play a role in learning and memory, including zif/268, arc, and homer [6]. Recently, CREB and the signaling cascades involved in CREB activation were examined in studies involving neuropathic pain. Evidence suggests that pCREB expression is upregulated by peripheral injections of chemical irritants (e.g., formalin, capsaicin, acidic saline) (for review see [6,18]). Upregulation of pCREB following sciatic nerve ligation has also been shown to mirror the time course of peripheral neuropathic pain, a period of 3 weeks [8]. While central sensitization appears to underlie the hyperreactivity of dorsal horn neurons following intense peripheral stimulation, it is unclear whether the same molecular mechanisms influence the development of chronic at-level pain following SCI. It is known from the work of McAdoo et al. [9] that contusive SCI causes a 37-fold increase in extracellular excitatory amino acid concentrations at the injury site and in the periimpact zone. Increases in excitatory amino acids like glutamate and aspartate could lead to the activation of NMDA receptors and intracellular signaling pathways that have been associated with central sensitization and neuropathic pain (e.g., extracellular signal related kinase [ERK], calmodulin kinase II [CaMK II], protein kinase A [PKA], protein kinase C [PKC], CREB) and cause the development of chronic at-level allodynia after SCI. Consistent with this, many laboratories present data that contusion SCI produces at-level allodynia that can be measured using a “girdling” procedure that tests vocalization responses to von Frey hairs applied to the trunk area surrounding the injury site [4,7,13]. To test the hypothesis that SCI leads to the activation of molecular pathways associated with long-term potentiation and neuropathic pain, rats given a contusion injury at spinal segment T10 were tested for the development of at-level allodynia by the “girdling” test and the spinal cords from the same rats were tested for pCREB expression by Western blot. To begin to determine the cellular populations that could be responsible for central neuropathic pain following SCI, colocalization studies using retrograde transport techniques to identify STT cells and immunocytochemistry to localize cells expressing pCREB were performed in some SCI rats. We report that SCI leads to increases in the average density
of pCREB expression and increased percentage of STT cells expressing pCREB. The increased pCREB expression correlated with the occurrence of trunk mechanical allodynia. Male Sprague–Dawley rats weighing between 225 and 250 g (Harlan Inc, Houston) received either a contusion injury at T10 using the Infinite Horizon device (150 kdyn, no dwell time) or were given a laminectomy to serve as sham controls. For the study involving measurement of tactile allodynia and Western immunoblotting, 15 injured rats and 15 age matched sham rats were used. For immunohistochemical studies, 5 injured rats and 5 age-matched sham rats were used. Following surgery, injured rats were given supplemental injections of Baytril twice daily for 7 days to prevent infection, and bladders were also expressed twice daily until the rats began to void on their own. Prior to sacrifice, sham and injured rats were examined for the development of tactile allodynia rostral to the injury using the procedure outlined in Hulsebosch et al. [4]. Briefly, a grid map of the girdle zone for allodynic responding was made on the rats using an indelible marker. A von Frey hair with bending force of 204.14 mN (26 g force) was applied to each point on the grid, and vocalization responses were recorded and mapped onto a grid map of that animal. Since animals do not normally vocalize with this stimulus, a vocalization response was interpreted to demonstrate that a noxious stimulus was experienced. In mapping the area of response, the number of vocalizations were recorded (Nv ) and normalized by the following formula: (Nv × 100)/total number of applications, indicating the percent vocalizations out of the total number of applications. For Western immunoblotting techniques to examine changes in pCREB expression following spinal cord injury, all subjects were overdosed with pentobarbital (100 mg/kg) and perfused intracardially with 250 ml cold heparinized (1 ml/l) saline (0.9%) and the spinal cord tissue immediately rostral to the injury site (T9) was removed and dissected while on dry ice. The dorsal aspect of the spinal cord was dissected from the ventral portion of the cord and frozen in dry ice. The collected tissue was mechanically homogenized in ice-cold tris-buffered saline containing 40 mM Tris–HCl (pH 7.5), 2% SDS, 2 mg/ml aprotinin, 2 mg/ml antipain, 2 mg/ml chymostatin, 2 mg/ml bestatin, 2 mg/ml pepstatinA, 2 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM EDTA. Homogenates were centrifuged at 10,000 × g for 10 min. The supernatant was collected and centrifuged again at 10,000 × g for 10 min and then stored at −80 ◦ C. Protein concentrations of the homogenate were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). To ensure that the maximum yield of nuclear protein was achieved by our extraction method, the cellular pellet from the first extraction was subsequently treated with a high salt buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 mg/ml antipain, 2 mg/ml chymostatin, 2 mg/ml pepstatin-A, and 2 mg/ml leupeptin) to maximize extraction of any nuclear transcription factors bound to DNA.
E.D. Crown et al. / Neuroscience Letters 384 (2005) 139–144
Briefly, the pellet was shaken for 30 min on a shaker/vortexer and then centrifuged at 14,000 × g for 5 min and the supernatant was collected for BCA assay. This assay determined that less than 1/800th of the total protein extracted remained in the pellet following the initial extraction. Importantly, an ANOVA performed on the data from the BCA assay on the high salt buffer extraction yielded no significant differences between the groups, F(1,8) < 1.0, p > 0.05, suggesting that protein extraction was comparable between the groups. In order to achieve an equal protein load of 10 ug per sample, the tissues from three subjects were homogenized together, heated for 4 min at 95 ◦ C in an equal volume of sample buffer (100 mM Tris, pH 6.8, and 2% SDS, 2% 2mercaptoethanol, 0.001% bromophenol blue, 20% glycerol) and then loaded onto a polyacrylamide gel in equal protein amounts (10 ug per lane). The stacking gel was 4% acrylamide, prepared in 0.13 M Tris, pH 6.8, and 0.1% SDS, and the separating gel was 10% acrylamide, prepared in 0.38 M Tris, pH 8.8, and 0.1% SDS. Samples were separated by electrophoresis in Tris–glycine buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) at 300 V for approximately 30 min. Proteins were transferred overnight (12–14 h) to a PVDF membrane at 30 V in transfer buffer containing 20% MeOH, 20 mM Tris, 150 mM glycine, pH 8.0. Membranes were incubated for 1 h at room temperature in blocking buffer containing 5% non-fat powdered milk in tris-buffered saline (TBS)–Tween (20 mM Tris, 137 mM NaCl, 0.1% Tween20), then washed for 10 min in TBS–Tween. Membranes were incubated overnight with primary antibodies to pCREB (1:2500). To control for equal protein loading, beta actin (1:5000) immunoreactivity was used to verify equal loading of proteins on the PVDF membrane and this method found no significant differences between the sham and SCI rats (F(1,8) < 1.0, p > 0.05; see Fig. 1C). After washing off primary antibody, membranes were incubated in horseradish peroxidase-conjugated anti-rabbit IgG diluted 1:10,000 in blocking buffer for 2 h and washed three times in TBS for 30 min. Peroxidase activity was detected using the Pierce SuperSignal West Femto Maximum Sensitivity Substrate kit, images were collected by exposing the membranes (exposure time varied from 30 s to 5 min) on chemiluminescence film (Hyperfilm ECL, Amersham Pharmacia Biotech, England), and integrated density values were calculated using LabWorks software (UVP, Upland, CA, USA). Seven days prior to sacrifice, rats used for our immunocytochemical colocalization study were given injections of FluoroGold (2%) into the ventral posterior lateral (VPL) nucleus of the thalamus to co-localize retrogradely labeled spinothalamic (STT) cells with immunocytochemical markers for pCREB. Briefly, while rats were deeply anesthetized, 14, 0.1 ul injections of Fluorogold were delivered into the right hemisphere of the VPL using the following coordinates (distance from bregma, distance from the right of the midline, distance from the surface of the skull) based on Paxinos and Watson [11]: −3.0 mm, 2.8 mm, 5.7/5.4 mm; −3.5 mm, 2.2 mm, 6.0/5.6 mm; −3.5 mm, 2.6 mm, 6.0/5.6 mm;
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Fig. 1. (A) Tactile allodynia in the injury “girdle” in spinal cord injured rats. The percentage of vocalization responses (y-axis) made by sham (Sham) and spinal injured (SCI) rats tested 35 days post-injury is graphed as bar graphs. The SCI rats showed a significant increase in responses to von Frey stimulation (∗ p < 0.0001). (B) Western blot data plotted in arbitrary units from the same sham and SCI rats as shown in (A). Phosphorylated CREB (pCREB) expression, was significantly upregulated in the spinal cord tissue rostral to the site of injury in SCI rats compared to sham rats (∗ p < 0.05). (C) A representative Western blot for sham and spinal injured rats. All data are represented as mean ± S.E.M.
−3.5 mm, 3.3 mm, 5.5/5.2 mm; −4.0 mm, 2.2 mm, 6.0/5.6 mm; −4.0 mm, 2.6 mm, 6.0/5.6 mm; −4.0 mm, 3.3 mm, 5.5/5.2 mm. Thirty-five days after injury, rats in the immunocytochemical study were overdosed with pentobarbital (75 mg/kg) and perfused intracardially first with 200 ml of heparinized warm 0.9% saline followed by 250 ml of cold 4% paraformaldehyde. The spinal segment immediately rostral to the injury (T9) was removed and post-fixed for 4 h in 4% paraformaldehyde prior to protection for 2 days in 30% sucrose at 4 ◦ C. The tissue was then embedded in OCT compound, frozen, mounted, and sectioned with a sliding microtome (model HM 400, Microm International, Waldorf Germany). Thirty micron sections from the T9 tissue were blocked in 5% normal goat serum for 30 min and incubated overnight in rabbit polyclonal pCREB antibody (1:200, Upstate Biotechnologies). The sections were then rinsed in phosphate-buffered saline (PBS) and incubated in goat anti-rabbit antiserum conjugated to Alexa Fluor 568-Red (1:400, Molecular Probes). After rinsing, the floating sections were mounted on gelatincoated slides and coverslipped with non-fade media. FluoroGold-labeled STT cells that were positive for pCREB expression in sham and injured rats were examined with a fluorescence microscope (E1000, Nikon). Briefly, the
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dorsal horn sections were viewed at 400× magnification and the FluoroGold positive STT cells were identified using a 330–380 nm wave band filter cube. Pictures of spinothalamic tract cells were captured using MetaMorph software (Universal Imaging Corporation, Downingtown, PA, USA) on the CoolSnap digital camera, following which a 532–587 nm wave band filter cube was used to visualize the expression of pCREB-positive cells in the same visual field. All pictures for imaging pCREB expression were taken with the same exposure conditions. Images were then merged using MetaMorph software and the percentage of pCREB-positive STT cells were counted and the average intensity of pCREB expression was measured with MetaMorph for sham and injured rats (five sections were measured per rat). Increases in the staining intensity of pCREB in STT cells are interpreted as an increase in the expression of pCREB within the tissues. To compare the group means in terms of responses during the girdling test for tactile allodynia, a one-way analysis of variance (ANOVA) was performed using SPSS 11.5 for Windows (SPSS, Chicago, IL, USA). To compare average intensity and percentage of pCREB-positive STT cells, Student’s t-tests were performed using SigmaPlot 9 (Systat Software, Point Richmond, CA, USA). In all cases, the alpha level for statistical significance was set at p < 0.05. All procedures were reviewed by the UTMB Animal Care and Use Committee and are consistent with the guidelines of the International Association for the Study of Pain
and the NIH Guide for the Care and Use of Laboratory Animals. Thirty-five days post-SCI, sham and injured rats were tested for the development of tactile allodynia rostral to the site of contusion injury. A one-way ANOVA indicated that injured rats vocalized a significantly greater number of times during testing than did sham rats, F(1,28) = 22.403, p < 0.0001 (Fig. 1A). Western immunoblotting to determine whether pCREB expression was upregulated (as assayed by increases in intensity) in the tissue of the dermatomes corresponding to those tested for tactile allodynia determined that pCREB expression was significantly upregulated in SCI rats relative to sham rats, F(1,8) = 5.487, p < 0.05 (Fig. 1B and C). To test whether cellular populations known to be involved in cutaneous pain transmission upregulate pCREB following SCI, FluoroGold-labeled pCREB-positive STT cells were analyzed. Fig. 2A depicts examples of FluoroGold-labeled pCREB-positive cells in the left dorsal horn of both sham (bottom panel) and injured (top panel) rats. For sham rats, the percentage of STT cells that expressed pCREB was 64%. In contrast, all (100%) of the labeled STT cells in injured rats were also positive for pCREB expression (Fig. 2B). In addition to the significant increase in the percentage of STT cells expressing pCREB following injury, SCI also produced a significant increase in the average intensity of pCREB expression within the labeled STT cells, both Student t values >2.78, p < 0.05 (Fig. 2C).
Fig. 2. Effects of SCI on pCREB expression in STT cells. (A) Example of STT cells that express phosphorylated CREB (pCREB) in sham (bottom middle panel) and SCI (top middle panel) rats. (B) Quantification of the significant increase in the percentage of STT cells that express pCREB following SCI (p < 0.05). (C) Quantification of the significant increase in the average intensity of pCREB expression in STT cells of injured rats relative to sham rats (∗ p < 0.05). All data are represented as mean ± S.E.M.
E.D. Crown et al. / Neuroscience Letters 384 (2005) 139–144
The goal of the current study was to examine whether mechanical allodynia associated with dermatomes corresponding to segments just rostral to the site of injury (spinal segment T9) correlates with upregulation of pCREB expression in the same spinal cord segments. To evaluate this hypothesis, rats were tested for the development of at-level mechanical allodynia following SCI. Results indicated that 35 days post-SCI, rats with an injury displayed a significant increase in response to von Frey stimulation in the “girdle” region rostral to the site of injury compared to sham controls; we interpret this as the development and maintenance of mechanical allodynia as a result of SCI. Western blot analysis revealed that the same rats also displayed significant increases in pCREB expression in the spinal cord segment rostral to injury compared to sham controls. To determine whether cells known to be involved in pain transmission (STT cells) upregulated the expression of pCREB, a separate group of rats was sacrificed for immunocytochemistry in order to localize the observed increases in pCREB expression. Thirty-five days after injury, pCREB expression in SCI but not sham rats was found to be significantly upregulated in a cellular population known to play a critical role in pain transmission, the STT cells. Following injury, there was an increase in both the number of STT cells that express pCREB and in the intensity of pCREB expression within these STT cells. The increase in intensity of pCREB expression in STT cells following SCI is interpreted to mean more active pCREB is available to bind CRE sites in target genes, as phosphorylation of CREB is required for gene transcription. This increase in pCREB correlates with increases in pCREB expression seen in other models of neuropathic pain [8,18]. These data also indicate that a number of STT cells in sham rats (64%) express pCREB when not in a state of neuropathic pain. This is not necessarily surprising, given that the transcription factor, CREB, coordinates the activity of a wide variety of genes involved in control of diverse cellular processes such as control of the cell cycle and long-term potentiation [10]. The current study reports increases in both the distribution and density of pCREB-positive STT cells rostral to the site of injury. This finding could have important implications for the mechanistic basis of chronic central neuropathic pain at the level of SCI. Spinothalamic tract cells are responsible for the transmission of noxious stimuli (e.g., pain, pressure, temperature) to the brainstem and thalamus. These cells are active during both acute and chronic pain and have been shown to undergo long-term potentiation during repeated exposure to a noxious stimulus like capsaicin [12,16]. Phosphorylation of the transcription factor, CREB, is thought to be critical for the nociceptive hypersensitivity that underlies neuropathic pain following peripheral insult [6]. The current finding is the first demonstration that pCREB expressing STT cells increase in number and density of pCREB expression as a result of SCI. Secondly, this is the first study to demonstrate persistent expression of pCREB in a chronic central neuropathic pain model.
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Ji and others [6,16] have noted a number of similarities between long-term potentiation and central sensitization. For example, both phenomena have an early activity-dependent phase that alters neuronal excitability through synaptic strengthening and a later transcription-dependent phase that relies on the formation of new proteins [6,10]. In addition, both the late phase of hippocampal LTP and transcriptiondependent central sensitization have been linked to activation of the NMDA receptor, followed by subsequent activation of downstream intracellular enzymatic cascades involving adenylyl cyclase, protein kinase A, protein kinase C, and/or calmodulin kinase (CaMK). During both phenomena, these cascades can also be induced by intracellular increases in calcium, cAMP, nerve growth factor, nitric oxide, CaMK II (as reviewed in [10]). Stimulation of these cascades leads to activation of a number of mitogen-activated protein kinases (MAPK), including ERK, c-Jun N terminal kinase/stressactivated protein kinase (JNK/SAPK), and p38 MAPK which then, in turn, can lead to phosphorylation of transcription factors and changes in gene transcription [6]. Late phase LTP depends not only on activation of signaling cascades involving PKA, PKC, nitric oxide (NO) and/or CaMK II, but on subsequent cyclic AMP responsive element-binding protein (CREB)-mediated synthesis of mRNA and protein [10]. Consistent with this, it has been demonstrated that blocking or overexpressing CREB can impair or facilitate long-term potentiation and long-term memory, respectively (for review, see [10]). As discussed earlier, recent studies have demonstrated that pCREB expression increases in dorsal horn neurons following injection of chemical irritants such as formalin or capsaicin into the hindpaw (for review, see [6]). In addition, Willis and co-workers (e.g., [18]) have shown that transient increases in pCREB seen after intradermal capsaicin injection into the paw can be blunted by interfering with nitric oxide signaling cascades that have also been implicated in long-term potentiation and central sensitization. The tactile allodynia that follows sciatic nerve ligation has also been shown to correlate with upregulation of pCREB expression in the superficial dorsal horn [8]. The current study indicates that pCREB expression in STT cells is correlated with the observance of at-level tactile allodynia in rats given a spinal injury. The current data suggest that pCREB expression is an important component in the development and maintenance of chronic central neuropathic pain following SCI. Given that activating CREB by phosphorylation is critical for phenomena such as long-term potentiation and central sensitization, it is tempting to speculate that chronic central neuropathic pain following SCI shares molecular similarities with these phenomena. For example, both central sensitization following noxious peripheral stimulation and chronic central neuropathic pain following spinal cord injury involve the persistent phosphorylation of the NR1 subunit of the NMDA receptor [2,19]. Phosphorylation of the NMDA receptor allows for prolonged calcium influx and could thus provide a mechanism for CNP following spinal cord injury. We are also currently examining whether intra-
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cellular kinases associated with CREB activation (e.g., ERK, p38 MAPK, JNK/SAPK, CaMK II) are upregulated following SCI in animals that develop chronic central neuropathic pain. Given that there are no specific inhibitors of CREB activation, we are taking advantage of pharmacological manipulations that inhibit CREB phosphorylation (e.g., inhibition of MAP kinases such as ERK or p38 MAPK) to determine whether these inhibitors will prevent the induction and expression of chronic neuropathic pain following SCI. Inhibition of these intracellular signaling cascades has had limited benefit in models of transient central sensitization following peripheral insult (for review, see [5]); but may be of therapeutic benefit in persistent central sensitization. Despite the current finding that suggests a similarity between the signaling cascades involved in chronic central neuropathic pain due to spinal cord injury and long-term potentiation, there appears to be at least one important difference between these phenomena. Long-term potentiation and central sensitization refer to hyperexcitability in hippocampal and dorsal horn neurons, respectively, that lasts on the order of hours to days. The hyperexcitability that underlies chronic central neuropathic pain induced by SCI, on the other hand, is a permanent condition that persists for months to years to a lifetime after injury. Thus, it is critical to understand the mechanisms involved in maintained CREB activation, as it appears to be a key transcription factor involved in injury induced chronic central neuropathic pain. Acknowledgements This work was funded by CRPF grant CB1-0404-2 to EDC, Mission Connect of TIRR-Houston, the Dunn Foundation, Mr. Frank Liddell and NIH grants NS11255 and NS39161 to CEH. References [1] A. Beric, Spinal cord injury pain, Eur. J. Pain 7 (2003) 335–338. [2] G.J. Brenner, R.R. Ji, S. Shaffer, C.J. Woolf, Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons, Eur. J. Neurosci. 20 (2004) 375–384.
[3] M.D. Christensen, C.E. Hulsebosch, Chronic central pain after spinal cord injury, J. Neurotrauma 14 (1997) 517–537. [4] C.E. Hulsebosch, G.Y. Xu, J.R. Perez-Polo, K.N. Westlund, C.P. Taylor, D.J. McAdoo, Rodent model of chronic central pain after spinal cord contusion injury and effects of gabapentin, J. Neurotrauma 17 (2000) 1205–1217. [5] R.R. Ji, Peripheral and central mechanisms of inflammatory pain, with emphasis on MAP kinases, Curr. Drug Targets Inflamm. Allergy 3 (2004) 299–303. [6] R.R. Ji, T. Kohno, K.A. Moore, C.J. Woolf, Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci. 26 (2003) 696–705. [7] A.E. Lindsey, R.L. LoVerso, C.A. Tovar, C.E. Hill, M.S. Beattie, J.C. Bresnahan, An analysis of changes in sensory thresholds to mild tactile and cold stimuli after experimental spinal cord injury in the rat, Neurorehabil. Neural Repair (2000) 287–300. [8] W. Ma, R. Quirion, Increased phosphorylation of cyclic AMP response element-binding protein (CREB) in the superficial dorsal horn neurons following partial sciatic nerve ligation, Pain 93 (2001) 295–301. [9] D.J. McAdoo, G.Y. Xu, G. Robak, M.G. Hughes, Changes in amino acid concentrations over time and space around an impact injury and their diffusion through the rat spinal cord, Exp. Neurol. 159 (1999) 538–544. [10] P.V. Nguyen, N.H. Woo, Regulation of hippocampal synaptic plasticity by cyclic AMP-dependent protein kinases, Prog. Neurobiol. 71 (2003) 401–437. [11] G. Paxinos, G. Watson, The Rat Brain in Stereotaxic Coordinates, 3rd ed., Academic Press, New York, 1997. [12] J. Sandkuhler, Learning and memory in pain pathways, Pain 88 (2000) 113–118. [13] P. Siddall, C.L. Xu, M. Cousins, Allodynia following traumatic spinal cord injury in the rat, NeuroReport 6 (1995) 1241–1244. [14] P. Siddall, R. Yezierski, J. Loeser, Pain following spinal cord injury: Clinical features, prevalence, and taxonomy, IASP Newslett. 3 (2000) 3–7. [15] WD. Willis Jr., Progress in Sensory Physiology, vol. 3, SpringerVerlag, New York, 1982, 159. [16] WD. Willis Jr., Long term potentiation in spinothalamic neurons, Brain Res. Brain Res. Rev. 40 (2002) 202–214. [17] C.J. Woolf, Evidence for a central component of post-injury pain hypersensitivity, Nature 306 (1983) 686–688. [18] J. Wu, L. Fang, Q. Lin, WD. Willis Jr., The role of nitric oxide in the phosphorylation of cyclic adenosine monophosphate-responsive element-binding protein in the spinal cord after intradermal injection of capsaicin, J. Pain 3 (2002) 190–198. [19] Z. Ye, C.E. Hulsebosch, D.J. McAdoo, G-Y. Xu, K.N. Westlund, Phospho-NR1 NMDA receptor subunit increases in white matter oligodendrocytes in contused rat spinal cord, Soc. Neurosci. Abs. 31 (2001) 2035.
Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: Relation to central neuropathic pain E.D. Crown, Z. Ye, K.M. Johnson, G.-Y. Xu, D.J. McAdoo, K.N. Westlund, C.E. Hulsebosch ∗ Department of Neuroscience and Cell Biology, University of Texas Medical Branch, 301 University Blvd, Route 1043, Galveston, TX 77555-1043, USA Received 28 February 2005; received in revised form 21 April 2005; accepted 22 April 2005
Abstract Spinal cord injury (SCI) often leads to the generation of chronic intractable neuropathic pain. The mechanisms that lead to chronic central neuropathic pain (CNP) following SCI are not well understood, resulting in ineffective treatments for pain relief. Studies have demonstrated persistent hyperexcitability of dorsal horn neurons which may provide a substrate for CNP. We propose a number of similarities between CNP mechanisms and mechanisms that occur in long-term potentiation, in which hippocampal neurons are hyperexcitable. One biochemical similarity may be activation of the transcription factor, cyclic AMP response element-binding protein (CREB), via phosphorylation (pCREB). The current study was designed to examine whether tactile allodynia that develops in segments rostral to SCI (at-level pain) correlates with an increase in CREB phosphorylation in specific neurons known to be involved in allodynia, the spinothalamic tract (STT) cells. This study determined that, in animals experiencing at-level allodynia 35 days after SCI, pCREB was upregulated in the spinal cord segment rostral to the injury. In addition, pCREB was found to be upregulated specifically in STT cells in the rostral segment 35 days after SCI. These findings suggest one mechanism of maintained central neuropathic pain following SCI involves persistent upregulation of pCREB expression within STT cells. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: FluoroGold; Immunocytochemistry; pCREB; Chronic pain
One of the most prevalent complaints among individuals suffering from spinal cord injury (SCI) is chronic intractable pain [1,3]. Siddall et al. [14] defined three categories of pain that result from SCI: (1) above-level pain which occurs at dermatomes rostral to the injury site in areas where normal sensation persists following injury, (2) at-level pain which occurs in dermatomes near the spinal injury, develops shortly after SCI, and is often characterized as either stabbing pain or a stimulus-independent type that is accompanied by allodynia (non-noxious stimuli become noxious), and (3) below-level pain, which is localized to dermatomes distal to the injury site, develops more gradually than does at-level pain, and is ∗
Corresponding author. Tel.: +1 409 772 2939; fax: +1 409 772 3222. E-mail address: [email protected] (C.E. Hulsebosch).
0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.04.066
often classified as a stimulus-independent continuous, burning pain. Multiple studies have indicated that the incidence of people with some form of chronic pain following SCI is up to 90%, with most studies reporting moderate to severe pain in the majority of cases [1]. Despite the prevalence of these complaints and our current understanding of the mechanisms underlying chronic neuropathic pain following SCI, there is a lack of clinically effective therapies, suggesting that these mechanisms are not entirely understood [1]. Research suggests that neuropathic pain which develops after peripheral injury or inflammation is a result of central sensitization [15,17]. Central sensitization refers to increased hyperexcitability observed in dorsal horn neurons following exposure to prolonged peripheral noxious stimulation. Recently, Ji and others (e.g., [6,12,16]) presented ev-
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idence linking the molecular mechanisms underlying central sensitization to the phenomenon of long-term potentiation (LTP). LTP is a phenomenon believed to underlie the synaptic changes in the hippocampus associated with learning and memory. The transcription factor cyclic AMP response elementbinding protein (CREB) has received a great deal of attention for its role in the formation of long-term memory, largely due to demonstrations of the importance of CREB activation in the late phases of both long-term facilitation and long-term potentiation. CREB is a 43 kDa nuclear transcription factor that is activated via phosphorylation at the serine 133 site. Once CREB is phosphorylated (pCREB), it can then bind to genes containing CRE promoters and begin transcription of a number of early immediate genes thought to play a role in learning and memory, including zif/268, arc, and homer [6]. Recently, CREB and the signaling cascades involved in CREB activation were examined in studies involving neuropathic pain. Evidence suggests that pCREB expression is upregulated by peripheral injections of chemical irritants (e.g., formalin, capsaicin, acidic saline) (for review see [6,18]). Upregulation of pCREB following sciatic nerve ligation has also been shown to mirror the time course of peripheral neuropathic pain, a period of 3 weeks [8]. While central sensitization appears to underlie the hyperreactivity of dorsal horn neurons following intense peripheral stimulation, it is unclear whether the same molecular mechanisms influence the development of chronic at-level pain following SCI. It is known from the work of McAdoo et al. [9] that contusive SCI causes a 37-fold increase in extracellular excitatory amino acid concentrations at the injury site and in the periimpact zone. Increases in excitatory amino acids like glutamate and aspartate could lead to the activation of NMDA receptors and intracellular signaling pathways that have been associated with central sensitization and neuropathic pain (e.g., extracellular signal related kinase [ERK], calmodulin kinase II [CaMK II], protein kinase A [PKA], protein kinase C [PKC], CREB) and cause the development of chronic at-level allodynia after SCI. Consistent with this, many laboratories present data that contusion SCI produces at-level allodynia that can be measured using a “girdling” procedure that tests vocalization responses to von Frey hairs applied to the trunk area surrounding the injury site [4,7,13]. To test the hypothesis that SCI leads to the activation of molecular pathways associated with long-term potentiation and neuropathic pain, rats given a contusion injury at spinal segment T10 were tested for the development of at-level allodynia by the “girdling” test and the spinal cords from the same rats were tested for pCREB expression by Western blot. To begin to determine the cellular populations that could be responsible for central neuropathic pain following SCI, colocalization studies using retrograde transport techniques to identify STT cells and immunocytochemistry to localize cells expressing pCREB were performed in some SCI rats. We report that SCI leads to increases in the average density
of pCREB expression and increased percentage of STT cells expressing pCREB. The increased pCREB expression correlated with the occurrence of trunk mechanical allodynia. Male Sprague–Dawley rats weighing between 225 and 250 g (Harlan Inc, Houston) received either a contusion injury at T10 using the Infinite Horizon device (150 kdyn, no dwell time) or were given a laminectomy to serve as sham controls. For the study involving measurement of tactile allodynia and Western immunoblotting, 15 injured rats and 15 age matched sham rats were used. For immunohistochemical studies, 5 injured rats and 5 age-matched sham rats were used. Following surgery, injured rats were given supplemental injections of Baytril twice daily for 7 days to prevent infection, and bladders were also expressed twice daily until the rats began to void on their own. Prior to sacrifice, sham and injured rats were examined for the development of tactile allodynia rostral to the injury using the procedure outlined in Hulsebosch et al. [4]. Briefly, a grid map of the girdle zone for allodynic responding was made on the rats using an indelible marker. A von Frey hair with bending force of 204.14 mN (26 g force) was applied to each point on the grid, and vocalization responses were recorded and mapped onto a grid map of that animal. Since animals do not normally vocalize with this stimulus, a vocalization response was interpreted to demonstrate that a noxious stimulus was experienced. In mapping the area of response, the number of vocalizations were recorded (Nv ) and normalized by the following formula: (Nv × 100)/total number of applications, indicating the percent vocalizations out of the total number of applications. For Western immunoblotting techniques to examine changes in pCREB expression following spinal cord injury, all subjects were overdosed with pentobarbital (100 mg/kg) and perfused intracardially with 250 ml cold heparinized (1 ml/l) saline (0.9%) and the spinal cord tissue immediately rostral to the injury site (T9) was removed and dissected while on dry ice. The dorsal aspect of the spinal cord was dissected from the ventral portion of the cord and frozen in dry ice. The collected tissue was mechanically homogenized in ice-cold tris-buffered saline containing 40 mM Tris–HCl (pH 7.5), 2% SDS, 2 mg/ml aprotinin, 2 mg/ml antipain, 2 mg/ml chymostatin, 2 mg/ml bestatin, 2 mg/ml pepstatinA, 2 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM EDTA. Homogenates were centrifuged at 10,000 × g for 10 min. The supernatant was collected and centrifuged again at 10,000 × g for 10 min and then stored at −80 ◦ C. Protein concentrations of the homogenate were determined using the BCA Protein Assay Kit (Pierce, Rockford, IL, USA). To ensure that the maximum yield of nuclear protein was achieved by our extraction method, the cellular pellet from the first extraction was subsequently treated with a high salt buffer (20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2 mg/ml antipain, 2 mg/ml chymostatin, 2 mg/ml pepstatin-A, and 2 mg/ml leupeptin) to maximize extraction of any nuclear transcription factors bound to DNA.
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Briefly, the pellet was shaken for 30 min on a shaker/vortexer and then centrifuged at 14,000 × g for 5 min and the supernatant was collected for BCA assay. This assay determined that less than 1/800th of the total protein extracted remained in the pellet following the initial extraction. Importantly, an ANOVA performed on the data from the BCA assay on the high salt buffer extraction yielded no significant differences between the groups, F(1,8) < 1.0, p > 0.05, suggesting that protein extraction was comparable between the groups. In order to achieve an equal protein load of 10 ug per sample, the tissues from three subjects were homogenized together, heated for 4 min at 95 ◦ C in an equal volume of sample buffer (100 mM Tris, pH 6.8, and 2% SDS, 2% 2mercaptoethanol, 0.001% bromophenol blue, 20% glycerol) and then loaded onto a polyacrylamide gel in equal protein amounts (10 ug per lane). The stacking gel was 4% acrylamide, prepared in 0.13 M Tris, pH 6.8, and 0.1% SDS, and the separating gel was 10% acrylamide, prepared in 0.38 M Tris, pH 8.8, and 0.1% SDS. Samples were separated by electrophoresis in Tris–glycine buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) at 300 V for approximately 30 min. Proteins were transferred overnight (12–14 h) to a PVDF membrane at 30 V in transfer buffer containing 20% MeOH, 20 mM Tris, 150 mM glycine, pH 8.0. Membranes were incubated for 1 h at room temperature in blocking buffer containing 5% non-fat powdered milk in tris-buffered saline (TBS)–Tween (20 mM Tris, 137 mM NaCl, 0.1% Tween20), then washed for 10 min in TBS–Tween. Membranes were incubated overnight with primary antibodies to pCREB (1:2500). To control for equal protein loading, beta actin (1:5000) immunoreactivity was used to verify equal loading of proteins on the PVDF membrane and this method found no significant differences between the sham and SCI rats (F(1,8) < 1.0, p > 0.05; see Fig. 1C). After washing off primary antibody, membranes were incubated in horseradish peroxidase-conjugated anti-rabbit IgG diluted 1:10,000 in blocking buffer for 2 h and washed three times in TBS for 30 min. Peroxidase activity was detected using the Pierce SuperSignal West Femto Maximum Sensitivity Substrate kit, images were collected by exposing the membranes (exposure time varied from 30 s to 5 min) on chemiluminescence film (Hyperfilm ECL, Amersham Pharmacia Biotech, England), and integrated density values were calculated using LabWorks software (UVP, Upland, CA, USA). Seven days prior to sacrifice, rats used for our immunocytochemical colocalization study were given injections of FluoroGold (2%) into the ventral posterior lateral (VPL) nucleus of the thalamus to co-localize retrogradely labeled spinothalamic (STT) cells with immunocytochemical markers for pCREB. Briefly, while rats were deeply anesthetized, 14, 0.1 ul injections of Fluorogold were delivered into the right hemisphere of the VPL using the following coordinates (distance from bregma, distance from the right of the midline, distance from the surface of the skull) based on Paxinos and Watson [11]: −3.0 mm, 2.8 mm, 5.7/5.4 mm; −3.5 mm, 2.2 mm, 6.0/5.6 mm; −3.5 mm, 2.6 mm, 6.0/5.6 mm;
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Fig. 1. (A) Tactile allodynia in the injury “girdle” in spinal cord injured rats. The percentage of vocalization responses (y-axis) made by sham (Sham) and spinal injured (SCI) rats tested 35 days post-injury is graphed as bar graphs. The SCI rats showed a significant increase in responses to von Frey stimulation (∗ p < 0.0001). (B) Western blot data plotted in arbitrary units from the same sham and SCI rats as shown in (A). Phosphorylated CREB (pCREB) expression, was significantly upregulated in the spinal cord tissue rostral to the site of injury in SCI rats compared to sham rats (∗ p < 0.05). (C) A representative Western blot for sham and spinal injured rats. All data are represented as mean ± S.E.M.
−3.5 mm, 3.3 mm, 5.5/5.2 mm; −4.0 mm, 2.2 mm, 6.0/5.6 mm; −4.0 mm, 2.6 mm, 6.0/5.6 mm; −4.0 mm, 3.3 mm, 5.5/5.2 mm. Thirty-five days after injury, rats in the immunocytochemical study were overdosed with pentobarbital (75 mg/kg) and perfused intracardially first with 200 ml of heparinized warm 0.9% saline followed by 250 ml of cold 4% paraformaldehyde. The spinal segment immediately rostral to the injury (T9) was removed and post-fixed for 4 h in 4% paraformaldehyde prior to protection for 2 days in 30% sucrose at 4 ◦ C. The tissue was then embedded in OCT compound, frozen, mounted, and sectioned with a sliding microtome (model HM 400, Microm International, Waldorf Germany). Thirty micron sections from the T9 tissue were blocked in 5% normal goat serum for 30 min and incubated overnight in rabbit polyclonal pCREB antibody (1:200, Upstate Biotechnologies). The sections were then rinsed in phosphate-buffered saline (PBS) and incubated in goat anti-rabbit antiserum conjugated to Alexa Fluor 568-Red (1:400, Molecular Probes). After rinsing, the floating sections were mounted on gelatincoated slides and coverslipped with non-fade media. FluoroGold-labeled STT cells that were positive for pCREB expression in sham and injured rats were examined with a fluorescence microscope (E1000, Nikon). Briefly, the
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dorsal horn sections were viewed at 400× magnification and the FluoroGold positive STT cells were identified using a 330–380 nm wave band filter cube. Pictures of spinothalamic tract cells were captured using MetaMorph software (Universal Imaging Corporation, Downingtown, PA, USA) on the CoolSnap digital camera, following which a 532–587 nm wave band filter cube was used to visualize the expression of pCREB-positive cells in the same visual field. All pictures for imaging pCREB expression were taken with the same exposure conditions. Images were then merged using MetaMorph software and the percentage of pCREB-positive STT cells were counted and the average intensity of pCREB expression was measured with MetaMorph for sham and injured rats (five sections were measured per rat). Increases in the staining intensity of pCREB in STT cells are interpreted as an increase in the expression of pCREB within the tissues. To compare the group means in terms of responses during the girdling test for tactile allodynia, a one-way analysis of variance (ANOVA) was performed using SPSS 11.5 for Windows (SPSS, Chicago, IL, USA). To compare average intensity and percentage of pCREB-positive STT cells, Student’s t-tests were performed using SigmaPlot 9 (Systat Software, Point Richmond, CA, USA). In all cases, the alpha level for statistical significance was set at p < 0.05. All procedures were reviewed by the UTMB Animal Care and Use Committee and are consistent with the guidelines of the International Association for the Study of Pain
and the NIH Guide for the Care and Use of Laboratory Animals. Thirty-five days post-SCI, sham and injured rats were tested for the development of tactile allodynia rostral to the site of contusion injury. A one-way ANOVA indicated that injured rats vocalized a significantly greater number of times during testing than did sham rats, F(1,28) = 22.403, p < 0.0001 (Fig. 1A). Western immunoblotting to determine whether pCREB expression was upregulated (as assayed by increases in intensity) in the tissue of the dermatomes corresponding to those tested for tactile allodynia determined that pCREB expression was significantly upregulated in SCI rats relative to sham rats, F(1,8) = 5.487, p < 0.05 (Fig. 1B and C). To test whether cellular populations known to be involved in cutaneous pain transmission upregulate pCREB following SCI, FluoroGold-labeled pCREB-positive STT cells were analyzed. Fig. 2A depicts examples of FluoroGold-labeled pCREB-positive cells in the left dorsal horn of both sham (bottom panel) and injured (top panel) rats. For sham rats, the percentage of STT cells that expressed pCREB was 64%. In contrast, all (100%) of the labeled STT cells in injured rats were also positive for pCREB expression (Fig. 2B). In addition to the significant increase in the percentage of STT cells expressing pCREB following injury, SCI also produced a significant increase in the average intensity of pCREB expression within the labeled STT cells, both Student t values >2.78, p < 0.05 (Fig. 2C).
Fig. 2. Effects of SCI on pCREB expression in STT cells. (A) Example of STT cells that express phosphorylated CREB (pCREB) in sham (bottom middle panel) and SCI (top middle panel) rats. (B) Quantification of the significant increase in the percentage of STT cells that express pCREB following SCI (p < 0.05). (C) Quantification of the significant increase in the average intensity of pCREB expression in STT cells of injured rats relative to sham rats (∗ p < 0.05). All data are represented as mean ± S.E.M.
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The goal of the current study was to examine whether mechanical allodynia associated with dermatomes corresponding to segments just rostral to the site of injury (spinal segment T9) correlates with upregulation of pCREB expression in the same spinal cord segments. To evaluate this hypothesis, rats were tested for the development of at-level mechanical allodynia following SCI. Results indicated that 35 days post-SCI, rats with an injury displayed a significant increase in response to von Frey stimulation in the “girdle” region rostral to the site of injury compared to sham controls; we interpret this as the development and maintenance of mechanical allodynia as a result of SCI. Western blot analysis revealed that the same rats also displayed significant increases in pCREB expression in the spinal cord segment rostral to injury compared to sham controls. To determine whether cells known to be involved in pain transmission (STT cells) upregulated the expression of pCREB, a separate group of rats was sacrificed for immunocytochemistry in order to localize the observed increases in pCREB expression. Thirty-five days after injury, pCREB expression in SCI but not sham rats was found to be significantly upregulated in a cellular population known to play a critical role in pain transmission, the STT cells. Following injury, there was an increase in both the number of STT cells that express pCREB and in the intensity of pCREB expression within these STT cells. The increase in intensity of pCREB expression in STT cells following SCI is interpreted to mean more active pCREB is available to bind CRE sites in target genes, as phosphorylation of CREB is required for gene transcription. This increase in pCREB correlates with increases in pCREB expression seen in other models of neuropathic pain [8,18]. These data also indicate that a number of STT cells in sham rats (64%) express pCREB when not in a state of neuropathic pain. This is not necessarily surprising, given that the transcription factor, CREB, coordinates the activity of a wide variety of genes involved in control of diverse cellular processes such as control of the cell cycle and long-term potentiation [10]. The current study reports increases in both the distribution and density of pCREB-positive STT cells rostral to the site of injury. This finding could have important implications for the mechanistic basis of chronic central neuropathic pain at the level of SCI. Spinothalamic tract cells are responsible for the transmission of noxious stimuli (e.g., pain, pressure, temperature) to the brainstem and thalamus. These cells are active during both acute and chronic pain and have been shown to undergo long-term potentiation during repeated exposure to a noxious stimulus like capsaicin [12,16]. Phosphorylation of the transcription factor, CREB, is thought to be critical for the nociceptive hypersensitivity that underlies neuropathic pain following peripheral insult [6]. The current finding is the first demonstration that pCREB expressing STT cells increase in number and density of pCREB expression as a result of SCI. Secondly, this is the first study to demonstrate persistent expression of pCREB in a chronic central neuropathic pain model.
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Ji and others [6,16] have noted a number of similarities between long-term potentiation and central sensitization. For example, both phenomena have an early activity-dependent phase that alters neuronal excitability through synaptic strengthening and a later transcription-dependent phase that relies on the formation of new proteins [6,10]. In addition, both the late phase of hippocampal LTP and transcriptiondependent central sensitization have been linked to activation of the NMDA receptor, followed by subsequent activation of downstream intracellular enzymatic cascades involving adenylyl cyclase, protein kinase A, protein kinase C, and/or calmodulin kinase (CaMK). During both phenomena, these cascades can also be induced by intracellular increases in calcium, cAMP, nerve growth factor, nitric oxide, CaMK II (as reviewed in [10]). Stimulation of these cascades leads to activation of a number of mitogen-activated protein kinases (MAPK), including ERK, c-Jun N terminal kinase/stressactivated protein kinase (JNK/SAPK), and p38 MAPK which then, in turn, can lead to phosphorylation of transcription factors and changes in gene transcription [6]. Late phase LTP depends not only on activation of signaling cascades involving PKA, PKC, nitric oxide (NO) and/or CaMK II, but on subsequent cyclic AMP responsive element-binding protein (CREB)-mediated synthesis of mRNA and protein [10]. Consistent with this, it has been demonstrated that blocking or overexpressing CREB can impair or facilitate long-term potentiation and long-term memory, respectively (for review, see [10]). As discussed earlier, recent studies have demonstrated that pCREB expression increases in dorsal horn neurons following injection of chemical irritants such as formalin or capsaicin into the hindpaw (for review, see [6]). In addition, Willis and co-workers (e.g., [18]) have shown that transient increases in pCREB seen after intradermal capsaicin injection into the paw can be blunted by interfering with nitric oxide signaling cascades that have also been implicated in long-term potentiation and central sensitization. The tactile allodynia that follows sciatic nerve ligation has also been shown to correlate with upregulation of pCREB expression in the superficial dorsal horn [8]. The current study indicates that pCREB expression in STT cells is correlated with the observance of at-level tactile allodynia in rats given a spinal injury. The current data suggest that pCREB expression is an important component in the development and maintenance of chronic central neuropathic pain following SCI. Given that activating CREB by phosphorylation is critical for phenomena such as long-term potentiation and central sensitization, it is tempting to speculate that chronic central neuropathic pain following SCI shares molecular similarities with these phenomena. For example, both central sensitization following noxious peripheral stimulation and chronic central neuropathic pain following spinal cord injury involve the persistent phosphorylation of the NR1 subunit of the NMDA receptor [2,19]. Phosphorylation of the NMDA receptor allows for prolonged calcium influx and could thus provide a mechanism for CNP following spinal cord injury. We are also currently examining whether intra-
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cellular kinases associated with CREB activation (e.g., ERK, p38 MAPK, JNK/SAPK, CaMK II) are upregulated following SCI in animals that develop chronic central neuropathic pain. Given that there are no specific inhibitors of CREB activation, we are taking advantage of pharmacological manipulations that inhibit CREB phosphorylation (e.g., inhibition of MAP kinases such as ERK or p38 MAPK) to determine whether these inhibitors will prevent the induction and expression of chronic neuropathic pain following SCI. Inhibition of these intracellular signaling cascades has had limited benefit in models of transient central sensitization following peripheral insult (for review, see [5]); but may be of therapeutic benefit in persistent central sensitization. Despite the current finding that suggests a similarity between the signaling cascades involved in chronic central neuropathic pain due to spinal cord injury and long-term potentiation, there appears to be at least one important difference between these phenomena. Long-term potentiation and central sensitization refer to hyperexcitability in hippocampal and dorsal horn neurons, respectively, that lasts on the order of hours to days. The hyperexcitability that underlies chronic central neuropathic pain induced by SCI, on the other hand, is a permanent condition that persists for months to years to a lifetime after injury. Thus, it is critical to understand the mechanisms involved in maintained CREB activation, as it appears to be a key transcription factor involved in injury induced chronic central neuropathic pain. Acknowledgements This work was funded by CRPF grant CB1-0404-2 to EDC, Mission Connect of TIRR-Houston, the Dunn Foundation, Mr. Frank Liddell and NIH grants NS11255 and NS39161 to CEH. References [1] A. Beric, Spinal cord injury pain, Eur. J. Pain 7 (2003) 335–338. [2] G.J. Brenner, R.R. Ji, S. Shaffer, C.J. Woolf, Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons, Eur. J. Neurosci. 20 (2004) 375–384.
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