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. Author manuscript; available in PMC: 2012 Jan 1.
Published in final edited form as: J Pain. 2010 May 26;12(1):13–21. doi: 10.1016/j.jpain.2010.03.018

A Spatiotemporal Pattern of Concurrent Spinal and Supraspinal NF-κB Expression Following Peripheral Nerve Injury

Chiu-Wen Chou 1,2, Gordon TC Wong 2, Grewo Lim 1, Shuxing Wang 1, Michael G Irwin 2, Jianren Mao 1
PMCID: PMC2978259  NIHMSID: NIHMS208996  PMID: 20537956

Abstract

The expression of NF-κB in the spinal cord is associated with neuropathic pain. However, little is known about its expression beyond the spinal cord. Here we examined a spatial and temporal pattern of the NF-κB expression in both spinal and supraspinal regions. After chronic constriction injury (CCI) of the sciatic nerve, the NF-κB (p65) expression was significantly increased in the ipsilateral spinal cord. In contrast, the NF-κB expression in the contralateral primary somatosensory cortex was decreased with no significant differences seen in the thalamus. In the contralateral anterior cingulate cortex, the NF-κB expression was increased significantly on day 14 as compared to the sham group. In the contralateral amygdala, the NF-κB expression showed a time-dependent downregulation after CCI, which became significant on day 14. MK-801 reduced nociceptive behaviors and reversed the direction of NF-κB expression. These results indicate that the CCI-induced expression of p65 NF-κB is both time-dependent and region-specific, in areas that process both sensory-discriminative and motivational-affective dimensions of pain.

Perspective

This article presents a spatiotemporal mapping of the NF-κB expression in spinal and supraspinal regions following peripheral nerve injury. These findings point to an involvement of NF-κB beyond the spinal cord in both the sensory discriminative and emotional affective aspects of neuropathic pain processing.

Keywords: neuropathic pain, NF-κB, affective, somatosensory cortex, thalamus, anterior cingulate cortex, amygdala

Introduction

Nuclear factor kappa B (NF-κB) is the first transcription factor family that was discovered in synaptic regions of the brain in 198914. Initially studied for its part in the immune response, NF-κB has since been recognized to have a role in the neuronal and glial cell function 12,27,44, transducing synaptic events directly linked to the nucleus, and processes related to neuronal development, plasticity, and degeneration 19,33. Evidence points to the involvement of NF-κB in both inflammatory pain and neuropathic pain in the peripheral nerve system and spinal cord20. In animal models of neuropathic pain, allodynia and thermal hyperalgesia may be attenuated by treatment with NF-κB inhibitor, NF-κB decoy antisense oligodeoxynucleotides or a viral vector-targeted blockade of NF-κB restricted to spinal glial cells 18,26,41. However, the nociceptive behavior in these rodents could not be completely reversed by NF-κB inhibition 18,26,41.

Pain is a subjective experience that involves both sensory-discriminative and emotional dimensions 31,39 with its signals being processed at different regions of the brain as well as at the spinal cord level. Observations from human brain imaging studies and the therapeutic responses to antidepressant drugs in neuropathic pain patients suggest that the emotional dimension may also play an important role in the underlying mechanism of neuropathic pain. Although neuropathic pain may be related to plasticity within the spinal cord5, therapies targeting this central locus alone has not been as effective as expected. Given the association of NF-κB in brain synaptic functions as well as neuro-inflammatory involvement in neuropathic pain at the spinal cord level, we hypothesized that the expression of NF-κB will also change in the supraspinal regions following induction of neuropathic pain. Using a rat model of chronic constriction sciatic nerve injury (CCI), we examined the expression of p65, a subunit of the NF-κB family, in both the spinal cord and brain regions in order to provide a chronological mapping of the NF-κB expression within the central nervous system following peripheral nerve injury. We also examined NF-κB expression following treatment with MK-801, an N-methyl-D-aspartate receptor (NMDA) receptor antagonist that is known to alleviate hyperalgesia and allodynia in neuropathic pain.

Materials and Methods

CCI model

The experimental protocol was approved by Massachusetts General Hospital Institutional Animal Care and Use Committee and carried out in accordance with the National institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA, USA) weighing 250–280g were used. Animals were housed under controlled temperature (21 ±2°C), relative humidity (50±10%) and artificial lighting (lights on from 7am until 7pm) with distilled water and food available ad libitum. CCI was induced by the method of Bennett and Xie (1988). In brief, the right side sciatic nerve was exposed in the mid-thigh of anesthetized animals (intraperitoneal pentobarbital 50mg/kg). Using a 4-0 chromic gut, four ligatures were made loosely around the nerve with a 1.0mm interval between each ligature. The wound was closed with wound clips. For the sham-operation group, the same surgical procedure was followed except for nerve ligation.

Drug Treatment

MK-801 (Methyl-10, 11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine hydrogen maleate) (Sigma) was dissolved in normal saline. The CCI rats were received either saline (n=6) or MK-801 0.1 mg/kg (n=6) daily for 7 consecutive days after CCI surgery. All injections were performed intraperitoneally (i.p.).

Behavioral test

After the rats were habituated to the test environment, the measurements were made before surgery as baseline, and on days 1, 7 and 14. The threshold of paw withdrawal to mechanical stimuli was measured by a serious of von Frey filaments (0.01g–20g) with an up-and-down approach46. Each rat was placed in a Plexiglas cage on a metal-mesh floor and a single filament was probed to the plantar surface of the hind paw perpendicularly for five times with an interval of 5s. A positive response was defined as the filament that evoked at least 2 clear paw withdrawals out of 5 applications and the filament of the next lower force was used. In a negative response, the filament of the next greater force was used. The cut-off force was 20gm. The threshold was the lowest force that caused a positive response.

Thermal hyperalgesia was assessed according to a previously report method9 using a paw Analgesia Meter (Model 390, IITC Life Science, Inc.). The radiant heat source was focused on the plantar surface of the hind paw and light intensity was preset to obtain a baseline latency of approximately 12s. A cut-off time was set at 20 s to avoid tissue damage. Each rat underwent two trials with a 5-minute interval and the mean value of these trials was used as the withdrawal latency.

Immunofluorescence staining

At each time point, rats were anesthetized with sodium pentobarbital (50mg/Kg, i.p.) and perfused transcardially with saline followed by 4% paraformaldehyde in phosphate buffer (PB, 0.1M. pH 7.2–7.4, 4°C). Lumbar spinal cords and the whole brain were dissected, post-fixed overnight and changed to 30% sucrose solution until the sample block sank to the bottom. Tissues were mounted using Cry O-Z-T embedding medium, then the transverse spinal cord sections (25μm) and the coronal brain sections (35μm) were cut using a cryostat (Jencons Scientific, Inc). A free-floating method was used for the staining processes. After being washed three times (5 min each) with 1×PBS, the sections were blocked in 0.1 M PBS containing 8% goat serum, 1% bovine serum albumin (BSA) and 0.3% Triton X-100 for 1 hour at room temperature and incubated for 24 hours at 4°C with first primary antibody NF-κB (p65 active form, 1:200, from CHEMICON and Santa Cruz). The sections were rinsed three times and then incubated for 1 hour at room temperature with Cy™3-conjugated Goat Anti-mouse or Anti-rat secondary antibodies (1:400, Jackson ImmunoResearch). For control samples, the primary antibody was omitted. Sections were washed subsequently and mounted onto chrome alum-coated slides, and covered with Vectashield Mounting medium (Vector Laboratories). Brain and spinal sections were examined using an Olympus fluorescence microscope, recorded with its digital camera, processed with Adobe Photoshop 7.0 (©Adobe System Incorporated).

Nuclear Extracts

Rats were rapidly decapitated on days 1, 7, or 14 under pentobarbital anesthesia (50 mg/kg, i.p.). Samples from the lumbar spinal cord enlargement (L3–L5), anterior cingulate cortex (ACC), amygdala, thalamus (primarily in the ventral posterolateral nucleus), and primary somatosensory cortex (S1) were removed and immediately placed on dry ice and stored at −80°C until use. These brain regions were identified according to the rat atlas37. Each segment was separated into the ipsilateral and contralateral side and nuclear extracts were prepared for every sample using Nuclear Extract Kit (Active Motif). Briefly, tissue samples were homogenized with 150μl ice-cold 1X Hypotonic Buffer containing 1μM DTT and detergent, incubated on ice for 15 minutes and then centrifuged for 10 minutes at 850x g at 4°C. The pellets were re-suspended in 100μl 1X Hypotonic Buffer and incubated on ice for 10 minutes. Then 5μl detergent was added, and the pellets were vigorously mixed and re-centrifuged for 2 min at a maximal speed at 4°C. The nuclear pellet was re-suspended in 50μl Complete Lysis Buffer, incubated on ice for 30 minutes on a rocking platform set at 150rpm and then centrifuged for 10 minutes at maximal speed at 4°C. The supernatant containing nuclear proteins was processed for the protein concentration assay and then used for Western blot analysis.

Western Blot analysis

Western blot was used to quantify the expression of p65 in the spinal cord and each brain region. Nuclear protein extracts (3 μg) were separated by SDS-PAGE gels (4%–15% gradient gel; Invitrogen) and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% milk for 1hour at room temperature, and then incubated overnight at 4°C with primary p65 antibody (1:1000, Santa Cruz). This was followed by three washes in PBS and incubation for 1hour with HRP-conjugated secondary antibody (1:7000, Amersham Biosciences). The blots were visualized in ECL solution (Thermo Scientific) for 5 minutes and exposed onto X-ray films (Kodak) for 1–10 minutes. The membranes were then incubated in a stripping buffer (Thermo Scientific) for 15 minutes in room temperature and re-probed with β-actin antibody (1:12000, mouse monoclonal, Abcam) as a loading control. The density of each band was measured by Quantity One software (Bio-Rad) and normalized against a corresponding loading control band. All Western analysis was made in four times.

Data and statistical analysis

All results are expressed as mean± standard error (SEM). For Western blot analysis, the protein expression in the sham group was normalized as 1, and the relative volume of the other groups was calculated proportionately within the same side. There were no significant differences between the ipsilateral and contralateral side in the sham group before adjusting. Differences in the values were compared using one-way analysis of variance (ANOVA) followed by a post hoc test (Turkey). Differences with a probability (P) less than 0.05 were considered to be statistically significant.

Results

Thermal hyperalgesia and mechanical allodynia following CCI

The threshold for both thermal and mechanical paw withdrawal of the right hind limb (ipsilateral to the ligation of sciatic nerve) was significantly decreased on post-operative day 1 and remained decreased up to at least post-operative day 14 as compared with sham-operated rats (Fig 1A, 1C, n=6; P< 0.05). There were no differences in these values for the contralateral hind limb (Fig 1B, 1D, n=6; P> 0.05).

Figure 1. Behavioral changes following CCI.

Figure 1

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The hind paw withdrawal thresholds to von Frey filaments are shown for the ipsilateral (A) and contralateral (B) hind limb. The values for paw withdrawal latency (PWL) in response to thermal stimuli are shown for the ipsilateral (C) and contralateral (D) hind limb. The sham group underwent a similar operative procedure without nerve ligation. The data for days 0, 1, 7 and 14 are displayed as mean ± SEM. * P < 0.05 (n=6).

p65 expression in the spinal cord after CCI

Immunofluorescence staining of the lumbar spinal cord showed that p65 was expressed in both dorsal and ventral horn of the spinal cord ipsilateral to the CCI (Fig 2A). When compared to day 1, the intensity was increased on day 7 and again on day 14, especially in lamina I, II and III (Fig 2B). This pattern was not visualized on the contralateral side (Data not shown). In the Western blot analysis, the nuclear p65 expression from spinal cord nuclear extracts was increased on post-operative day 14 (P < 0.05, n= 4; Fig 2C) as compared to the sham group.

Figure 2. NF-κB expression in the spinal cord.

Figure 2

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(A) The fluorescence image shows the distribution of p65 in the L4 spinal cord segment of a CCI rat on day 14. Right side of the image represents the ipsilateral side. (B) The expression of p65 in the ipsilateral side of the spinal cord dorsal horn at different time points. (C) Nuclear p65 expression (Western bolt) was upregulated and was significantly different compared to sham group on post-operative day 14. The protein expression in the sham group was normalized as 1, and the relative volume of the other groups was calculated proportionately within the same side. There was no significant difference between the ipsilateral and contralateral side in the sham group before adjusting. Each bar represents the mean± SEM of 4 rats and asterisks indicate a significant difference (P< 0.05).

CCI-induced p65 expression in the thalamus and S1 cortex

Immunostaining showed no obvious changes in p65 expression in the thalamus between the CCI and sham group (Fig 3A). Western blot also revealed no significant differences in p65 expression between groups from different time points, although there was a trend towards a modest decrease for both sides (Fig 3B).

Figure 3. NF-κB expression in thalamus.

Figure 3

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(A) The figure shows the distribution of p65 in contralateral sides of the thalamus in the sham-operated rat and the CCI rat on day 7. (B) The nuclear p65 expression in thalamus (Western bolt) has no significant difference between CCI groups and the sham group. Each bar represents the mean± SEM of 4 rats.

In the contralateral S1, a decreased p65 expression was visualized using immunostaining on day 7 (Fig 4A). The western blot data showed that the p65 expression on the contralateral S1 was decreased on both day 1 and day 7 following CCI (Fig. 4B). Although not statistically significant, the p65 expression was still decreased at day 14 (p=0.08). There were no differences in the ipsilateral S1 for all three time-points (Fig 4B).

Figure 4. NF-κB expression in S1 area.

Figure 4

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(A) The fluorescence image shows the expression of p65 on a sham rat and the CCI rat on day 7. (B) The nuclear p65 expression on the contralateral side of S1 area (Western bolt) was downregulated and had significant difference on post-operative day 1 and 7 compared to sham group. Each bar represents the mean± SEM of 4 rats and asterisks indicate a significant difference compared to sham group (P< 0.05)

CCI-induced p65 expression in the ACC and amygdala

Immunostaining of the contralateral ACC showed a small increase in p65 expression on day 1 and further increased by day 14 (Fig 5A). From the Western blot analysis, a difference was demonstrated on day 14, as compared to day 1 and day 7 on the same contralateral side (Fig 5C). There were no differences on the ipsilateral side at all three time points (Fig 5A, C).

Figure 5. NF-κB expression in ACC and amygdala.

Figure 5

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The expression of p65 is shown in ACC (A, C) and amygdala (B, D). (A) The fluorescence image shows the distribution of p65 in ACC. (B) The fluorescence image shows the distribution of p65 in the amygdala. Sh: sham rats. (C) The nuclear p65 expression in the contralateral ACC (Western bolt) was upregulated on day 14 as compared to day 1, day 7 and sham group. (D) The nuclear p65 expression on the contralateral side of the amygdala area (Western bolt) was downregulated after CCI and has a significant difference on post-operative day 14 as compared to the sham group. Each bar represents the mean± SEM of 4 rats and asterisks indicate a significant difference between different time points (P< 0.05).

In contrast, there was a small decrease in the p65 expression in the contralateral amygdala from day 1 that became significant by day 14 when compared with the sham group. This pattern of the p65 expression was not observed on the ipsilateral side (Fig 5B, D).

Effect of MK-801 treatment on CCI rats

On the ipsilateral side, mechanical allodynia was alleviated in MK-801-treated rats on post-operative day 7 but returned on day 14 after the cessation of drug treatment, while hyperalgesia was attenuated on both post-operative day 7 and 14. The MK-801 treatment had no effect on the contralateral paw, which indicated that the changes in pain behaviors were more likely a suppression of neuropathic activity (antihyperalgesic) rather than a non-specific analgesic effect (Fig 7).

Figure 7. Western blot analysis of p65 expression.

Figure 7

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Panels show the p65 expression in the ipsilateral side of the spinal cord dorsal horn and the contralateral side of the thalamus, S1, ACC and amygdala on day 14. * as compared with sham group (P < 0.05, n=3). # comparison between CCI+ saline and CCI+ MK-801 rats on postoperative day 14 (P < 0.05, n=3)

With MK-801 treatment, the p65 expression in the ipsilateral spinal cord dorsal horn and contralateral ACC was both decreased although statistically significant differences were absent on day 14, which is corresponded with the behavioral results. In the thalamus, there were no differences between CCI rats treated with saline or MK-801 on day 14. In the S1 area, the altered expression of p65 was prevented with the MK-801 treatment, whereas in the amygdala MK-801 reversed the downregulation of the p65 expression in CCI rats.

Discussion

In rats with ipsilateral thermal hyperalgesia and mechanical allodynia after CCI, we demonstrated a characteristic spatial and temporal mapping of NF-κB (p65 subunit) expression in the central nervous system. When compared with sham rats, the p65 expression within the ipsilateral spinal cord began to increase on day 1 and became significant by day 14. For the supraspinal regions, we grouped the results according to regions involved in the processing of the sensory discriminative [primary somatosensory cortex (S1), thalamus] or emotional and affective (amygdala and ACC) aspects of pain. Interestingly, the p65 expression was reduced in the contralateral S1 area on day 1 and day 7 and then returned to the baseline by day 14. No changes in the thalamus were seen on any tested postoperative days. In the contralateral amygdala a decrease was seen on day 14, whereas an increase was seen in the contralateral ACC for the same time point. In addition, both hyperalgesia and allodynia were reduced by a systematic MK-801 treatment, together with the reversal of the p65 expression in both spinal and supraspinal regions.

It is recognized that physiological pain processing involves a number of steps. For example, the nociceptive signal from somatic areas is transmitted from the spinal dorsal horn, through ascending pathways such as the spinothalamic tract, to the ventral posterolateral (VPL) nucleus of thalamus and then onto the somatosensory cortex 3,25. Synaptic plasticity of neurons is believed to be fundamental to pathological pain11 and the importance of spinal sensitization is well recognized. Although functional studies have demonstrated the increase in metabolic activity in most brain regions following the onset of neuropathic pain 21, insights into the cellular mechanisms are still lacking. NF-κB is an important transcriptional factor for studying the involvement of supraspinal regions in neuropathic pain because it is regulated by many of the same signaling pathways that are involved in the induction of synaptic plasticity in the brain 1,24. Neurobiological functions that are related to neuropathic pain, such as neurodegeneration and apoptosis5,42, have also been shown to be modulated by NF-κB23. Furthermore, NF-κB has a major role in inflammatory pain and the involvement of immune cells and glia in neuropathic pain.

The relationship between NF-κB and neuropathic pain has been studied in various forms over the past decade. Most studies have focused at the peripheral nerve and spinal cord level 18,34. This study aimed at providing information regarding NF-κB expression in various brain regions but was subjected to several methodological limitations. For example, behavioral tests were limited only to the sensory discriminative aspects of pain; spontaneous or other dimensions of pain-related behavioral changes were not examined. Western blot analysis was technically limited to detect regional changes rather than changes at the sub-nuclei level of a given brain region due to the technical difficulty of obtaining tissue samples from small brain areas. Therefore, the functional implication of this observational study needs to be made within these methodological limitations.

Our spinal cord data showed the NF-κB expression was significantly different ipsilaterally on day 14 when compared with the sham group. This is consistent with these previous findings that collectively suggest NF-κB could affect the development of neuropathic pain via influencing the expression of inflammatory cytokines, adhesion molecules, iNOS, PG and COX-2 in the spinal cord 26,34,41. However, it is unclear whether this up-regulation of p65 expression resulted from neuronal plasticity or inflammatory response from spinal glial or a combination of both. Meunier et al reported that selective inhibition of NF-κB activity targeting glial cells in the dorsal spinal cord could attenuate hyperalgesia and allodynia26, which suggested a role for NF-κB in the glial cell modulation after CCI. From our data this upregulation of NF-κB could be abated by the systemic treatment with MK-801, an NMDA receptor antagonist known to improve neuropathic pain behaviors in rats 4,22. NMDA receptor activation may activate NF-κB in neuron, suggesting some involvement of this transcriptional factor with synaptic plasticity33. Therefore NF-κB upregulation in the spinal cord following peripheral nerve injury may reflect changes in both glial cell and NMDA-related neuronal function.

Reorganization of S1 is correlated with ongoing pain intensity in neuropathic pain patients with phantom limb pain or spinal cord injury 7,49 and increased activation of contralateral S1 is a key feature of abnormally enhanced nociception in a rat model of neuropathic pain using fMRI analysis6. In contrast to this and the increased activities seen in 2-DG imaging studies21, our data for the contralateral S1 revealed an initial decrease in p65 expression that gradually returned toward normal on day 14. This early decrease of NF-κB might reflect an initial impact caused by the peripheral nerve injury, and the late recovery might be related to the reorganization or neural plasticity of S1 associated with persistent pain30. This suggests that levels of NF-κB expression may change in accordance to whether it is the early or late phase of neuropathic pain. Furthermore, the reversal of this pattern of NF-κB expression in SI by the MK-801 treatment suggests that part of its role in cortex might be highly NMDA receptor mediated.

The thalamus is known to have a pivotal role in pain processing and has been shown to be involved in the neuropathic pain 2,10,40. Thalamic stimulation has been suggested to serve as a treatment modality for neuropathic pain patients 8,36. In our data, although the expression of p65 in the thalamus was slightly decreased after CCI, no significant differences between CCI and sham rats were observed. Of interest to note is that, although there is a lack of statistical differences in the thalamic expression of p65 NF-κB, the p65 expression was decreased at least initially after CCI, and this pattern was corresponded to the changes of p65 in the S1 area. Therefore, it is possible that the lack of enhanced NF-κB expression in the sensory-discriminative system in the early phase might be related to the mechanisms contributory to the initiation of neuropathic pain. 35,51

The amygdala and ACC are two main brain regions that have been recognized to be involved in the affective aspect of pain. Lesions of ACC could alleviate intractable pain in chronic pain patients and significantly decrease escape/avoidance behaviors, a paradigm reflecting affective responses, in rodent neuropathic pain models 16,17. Studies using brain imaging, electrophysiology, behavioral test and molecular analysis have also demonstrated an important role for the amygdala in the emotional dimension of pain 31,32. Our results showed that the NF-κB expression in the ACC was increased significantly on day 14 while the p65 expression in the amygdala was significantly decreased. Considering the time course, these results indicate a possible delayed modulation of the NF-κB expression within ACC and amygdala in this CCI model, which may be responsible for the maintenance of neuropathic pain.

Interestingly, the NMDA receptor antagonism has a significant effect on amygdala but not ACC. Noticeably, recent studies suggest that amygdala is not only involved with the affective component but also sensory discriminative component in neuropathic rats38 and that different nuclei within the amygdala might either facilitate or inhibit a painful condition 32. Therefore, our results suggest the role of NF-κB within the amygdala might be related to its sensory discriminative aspect and share functional similarities with S1 in the late phase of neuropathic pain. With regard to the ACC, the MK801 treatment did not affect the pattern of NF-κB expression despite its antihyperalgesic effect. The ACC has become a brain area of significant interest in the study of persistent pain43,53. Multiple studies have pointed to the importance of the NMDA receptor in the ACC because upregulation or overexpression of the NMDA receptor enhanced persistent pain behavior47,48,50,52. It also has been demonstrated that chronic pain induced astrocyte activation in the mouse ACC15,28. It is possible that the upregulation of NF-κB in this model may reflect an increased astrocyte activity in this region. Other studies have also pointed to contributions of immediate early genes expression in the ACC, such as CREB47, c-fos29,45 and Egr113, in different animal models of pain. Of interest is that the NF-κB change seems to become more prominent in the late stage of our neuropathic pain model. Therefore, it would be of significance to examine a possible relationship between NF-κB and immediate early genes expression in various brain regions.

It would be of considerable interest in future studies to determine whether the NF-κB involvement is related to modulation of immune responses and/or synaptic function in the brain. Nonetheless, our findings demonstrate significant changes of p65 expression in the ipsilateral spinal cord and several contralateral brain regions corresponding to the ascending pain pathways. These changes are reversed by the NMDA receptor antagonism in association with a reduction in pain behaviors. Changes in the NF-κB expression may therefore be involved in neuropathic pain processing not only in the spinal cord but also in supraspinal regions. This pattern of NF-κB expression is in concordance with previous suggestions that neuropathic pain is a multidimensional experience involving both sensory and affective dysfunction in a time-dependent manner. Therefore, examining spatial and temporal patterns of NF-κB expression may provide an alternative research approach to mapping the involvement of various CNS structures following peripheral nerve injury and response to therapy, which may complement other modalities such as neuroimaging and electrophysiological recording.

Figure 6. Effects of daily treatment for 7 days with saline or MK-801.

Figure 6

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The hind paw withdrawal thresholds to von Frey filaments are shown for the ipsilateral (A) and contralateral (B) hind limb. The values for paw withdrawal latency (PWL) in response to thermal stimuli are shown for the ipsilateral (C) and contralateral (D) hind limb. * Difference while compared with pre-test (D0) (P < 0.05, n=6). # Difference from MK-801 (0.1mg/kg) treatment group compared with saline treatment group (P < 0.05, n=6) on respective time points.

Acknowledgments

This study is supported by NIH RO1 grants DE18214, DE18538, and NS45681.

Footnotes

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The authors claim no conflict of interest.

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