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Anesth Analg 2008; 107:693-700
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e31817e7319
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ANALGESIA

Intrathecal Clonidine Suppresses Phosphorylation of the N-Methyl-D-Aspartate Receptor NR1 Subunit in Spinal Dorsal Horn Neurons of Rats with Neuropathic Pain

Dae-Hyun Roh, DVM, MS*, Hyun-Woo Kim, DVM, PhD{dagger}, Seo-Yeon Yoon, DVM, PhD*, Hyoung-Sig Seo, DVM, MS*, Young-Bae Kwon, DVM, PhD{ddagger}, Ho-Jae Han, DVM, PhD§, Alvin J. Beitz, PhD||, and Jang-Hern Lee, DVM, PhD*

From the *Department of Veterinary Physiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul, South Korea; {dagger}Department of Physiology, College of Medicine, Chungnam National University, Daejeon, South Korea; {ddagger}Department of Pharmacology, Institute for Medical Science, Chonbuk National University Medical School, Jeonju, South Korea; §Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwang-ju, South Korea; and ||Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St Paul, MN.

Address correspondence and reprint requests to Jang-Hern Lee, DVM, PhD, Department of Veterinary Physiology, College of Veterinary Medicine, Seoul National University, Seoul 151–742, South Korea. Address e-mail to JHL1101{at}snu.ac.kr.

Abstract

BACKGROUND: Intrathecal (IT) administration of the {alpha}-2 adrenoceptor agonist, clonidine, produces significant analgesic effects. Although several mechanisms underlying clonidine-induced analgesia have been proposed, the possible interaction with N-methyl-D-aspartate (NMDA) receptors as a major antinociceptive mechanism has not been addressed. We designed the present study to determine whether clonidine or other analgesics can affect spinal NMDA receptor activation in rats with chronic constriction injury (CCI)-induced neuropathy.

METHODS: Rats underwent unilateral CCI, and received IT clonidine (1, 5, 20 µg/rat), [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO, µ opioid receptor agonist, 1 µg/rat), gabapentin (anticonvulsant, 100 µg/rat) or vehicle 2 wks later. After drug injection, we measured the pain response to thermal or mechanical stimuli and used immunohistochemistry to evaluate spinal cord phosphorylated NMDA-receptor subunit 1 (pNR1) expression.

RESULTS: Two weeks after CCI surgery, rats displayed significant mechanical allodynia and thermal hyperalgesia, and the spinal cord dorsal horn showed a significant increase in the number of pNR1 immunoreactive neurons. IT injection of clonidine (20 µg/rat), DAMGO and gabapentin potently reduced mechanical allodynia and thermal hyperalgesia. Importantly, IT clonidine, but not IT DAMGO or gabapentin, dose-dependently reduced CCI-induced pNR1 expression in all lamina of the spinal cord dorsal horn by 30 min after injection. In addition, IT injection of the {alpha}-2 adrenoceptor antagonist, idazoxan (40 µg/rat) 10 min before clonidine injection completely reversed clonidine’s antihyperalgesic and antiallodynic effects, as well as clonidine’s suppressive effect on CCI-induced NR1 phosphorylation in the spinal cord dorsal horn.

CONCLUSIONS: Our data indicate that IT clonidine’s antihyperalgesic/antiallodynic effect on neuropathic pain is associated with a significant reduction in spinal NMDA receptor phosphorylation and suggests a potentially novel mechanism of clonidine’s action.

Intrathecal (IT) injection of the {alpha}-2 adrenoceptor agonist, clonidine significantly attenuates hyperalgesia and tactile allodynia associated with chronic nerve injury in the rat spinal nerve ligation model.1,2 Alpha-2 adrenoceptor agonists are thought to produce analgesia primarily by actions in the spinal cord, both by reducing the release of glutamate and substance P from central afferent terminals,3,4 and by hyperpolarizing dorsal horn neurons.5,6 These phenomena are related to the inhibition of N-type Ca2+ channels on the presynaptic membrane, and to an increase in the conductance of inwardly rectifying potassium channels in dorsal horn neurons, respectively. On the other hand, the {alpha}-2 adrenoceptor is coupled to a Gi protein, which reduces the activity of adenylyl cyclase, simultaneously suppressing both the production of cAMP and the activity of protein kinase A (PKA). The activation of {alpha}-2 adrenoceptor-associated Gz{alpha} and Go{alpha} proteins is necessary for the antinociception resulting from IT administered {alpha}-2 adrenergic agonists.7 It was reported that spinal nerve ligation-induced neuropathy increases {alpha}-2 adrenoceptor G-protein coupling in the spinal cord, and that this is associated with the enhanced potency of clonidine treatment in neuropathic pain.8

The phosphorylation of the N-methyl-D-aspartate (NMDA) receptor subunit one (pNR1) via a number of protein kinases (e.g., PKC, PKA, PKG, CaMKII) is recognized as a major mechanism underlying the regulation of NMDA receptor (NMDAR) function.9 An increase in the number of pNR1 immunoreactive (pNR1-ir) neurons in the dorsal horn was observed in the spinal cords of rats showing secondary hyperalgesia after intradermal capsaicin injection.10 Gao et al. also showed that the number of pNR1-ir neurons was significantly increased in the dorsal horn (laminae I-II and III-VI) of neuropathic rats that received an L5 spinal nerve ligation and that this increase is associated with the development of mechanical allodynia.11 These studies support the idea that the phosphorylation of NR1 subunit is associated with the enhancement of synaptic efficacy and thus the development of central sensitization. It has also been demonstrated that clonidine treatment (0.025 mg/kg, subcutaneously), which does not specifically relieve neuropathic pain, enhances the neuropathic pain-relieving action of the NMDA antagonist, MK-801.12 In addition, {alpha}-2 adrenoceptor agonists depress NMDAR-mediated synaptic transmission in a neonatal rat hemisected spinal cord preparation.13 These findings suggest that the clonidine-induced activation of {alpha}-2 adrenoceptors might be capable of modulating the abnormal excitability of spinal NMDAR in neuropathic rats. However, the possible effect of clonidine on spinal NMDAR has never been directly examined.

In this regard, the present study was designed to evaluate whether the antihyperalgesic or antiallodynic effects of IT clonidine, [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO, a µ opioid receptor agonist) or gabapentin (GBP, anticonvulsant) are associated with suppression of NMDAR excitability, as measured by a reduction in spinal cord NR1 phosphorylation in sciatic nerve chronic constriction injury (CCI)-induced neuropathic rats.

METHODS

Animals and Neuropathic Surgery
Experiments were performed on male Sprague-Dawley rats weighing 200–250g. All experimental animals were obtained from the Laboratory Animal Center of Seoul National University. They were housed in colony cages with free access to food and water and maintained in temperature and light-controlled rooms (24 ± 2°C, 12/12h light/dark cycle with lights on at 07:00). All of the methods used in the present study were approved by the Animal Care and Use Committee at Seoul National University and conform to National Institute of Health guidelines (NIH publication No. 86–23, revised 1985).

A CCI of the common sciatic nerve was performed according to the method described by Bennett and Xie.14 Briefly, rats were anesthetized with 3% isoflurane in a mixture of N2O/O2 gas. The right sciatic nerve was exposed at the mid-thigh level, and 4 loose ligatures of 4–0 chromic gut were placed around the dissected nerve with a 1.0- to 1.5-mm interval between each ligature. Sham surgery consisted of exposing the sciatic nerve in the same manner, but without ligating the nerve.

Intrathecal Drugs Injection
Rats were transiently anesthesized for 30 s before IT drug injection using 3% isoflurane in a mixed N2O/O2 gas in order to prevent any handling-induced stress that might be associated with the IT injection procedure. Animals awoke immediately after the IT injection procedure and were freely moving within 45 s after injection. Clonidine (1, 5, 20 µg; Sigma, St. Louis, MO; n = 7, respectively), the µ opioid receptor agonist, DAMGO (1 µg, Tocris, Avonmouth, UK; n = 6) or the anticonvulsant/antihyperalgesic drug, GBP (100 µg, Tocris; n = 6) was dissolved in 10 µL phosphate buffer saline (PBS) and injected IT in CCI-induced neuropathic rats. In sham rats, a maximal dose of clonidine (20 µg) was also injected (n = 6). Control animals were injected with 10 µL PBS (vehicle, n = 7). The {alpha}-2 adrenoceptor antagonist, idazoxan (IDA, 40 µg, Sigma; n = 6) was injected IT 10 min before IT injection of clonidine (20 µg). The IT dose of each drug was determined based on established doses reported in the literature.15–17 IT injections were made using a modification of the Mestre technique18 according to the method described by Fairbanks.19 Briefly, a 26-gauge needle (one inch in length) connected to a 50 µL Hamilton syringe was inserted into the skin and then through the L5–6 intervertebral space directly into the subarachnoid space. A flick of the rat’s tail provided a reliable indicator that the needle had penetrated the dura and at this point 10 µL of the drug was subsequently injected into the subarachnoid space. Behavior tests were performed before and at 10, 30, 60, and 120 min after injection of each dose of clonidine or PBS. In a separate experiment, rats were immediately euthanized 30 min after each drug injection (n = 5, respectively) that showed the maximal effect of clonidine, DAMGO or GBP, and the L4–5 spinal cord was removed for pNR1 immunohistochemistry.

Behavior Assessments
To assess nociceptive responses to heat stimuli, we measured paw withdrawal response latency (PWL) by using the procedure described by Hargreaves et al.20 A radiant heat source was positioned under the glass floor beneath the hindpaw to be tested, and PWL was measured using a plantar analgesia meter (IITC Inc., Woodland Hills, CA). The test was duplicated in each hindpaw, and the mean PWL was calculated. Cutoff time in the absence of a response was 20 s.

The number of paw withdrawals to normally innocuous mechanical stimuli was measured by using a von Frey filament with a force of 2.0 g (North Coast Medical, Morgan Hill, CA). Rats were placed on a metal mesh grid under a plastic chamber, and the von Frey filament was applied from underneath the metal mesh flooring to each hindpaw.21 The number of paw withdrawals after each stimulus was then counted. The results of mechanical behavioral testing in each experimental animal were expressed as a percent withdrawal response frequency (PWF, %), which represented the percentage of paw withdrawals out of 10 maximum.

pNR1 Immunohistochemistry and Imaging Analysis
Animals were deeply anesthetized with 5% isoflurane, perfused transcardially with calcium-free Tyrode’s solution, followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 6.9). The spinal cord was then removed immediately, postfixed at 4°C overnight in the same fixative and then cryoprotected in 30% sucrose in PBS (pH 7.4) for 48 hrs. Frozen serial frontal sections (40 µm) were cut through the lumbar L4–5 spinal cord using a cryostat (Microm, Walldorf, Germany). After elimination of endogenous peroxidase activity with 0.3% hydrogen peroxide and preblocking with 3% normal goat serum in PBS, the free-floating sections were incubated in rabbit anti-pNR1 antibody (1:1,000; Upstate/Chemicon Biotechnology, Temecula, CA) at 4°C for 48 hrs. To confirm the specificity of immunoreactivity, some sections were processed in an identical fashion, but without primary antibodies. The sections were subsequently processed using the avidin-biotin-peroxidase procedure previously described.22 Finally, visualization was performed using 3,3'-diaminobenzidine (DAB; Sigma) with 0.2% nickel chloride intensification.

The sections were examined under a brightfield microscope (Zeiss Axioscope, Hallbergmoos, Germany) at x100 to localize pNR1-ir neurons. For quantitative analysis of pNR1-ir neurons, five spinal cord sections from the L4–5 lumbar spinal cord segments were randomly selected from each animal and subsequently scanned. Individual sections were digitized with 4096 gray levels using a cooled CCD camera (Micromax Kodak 1317, Princeton Instruments, Tucson, AZ) connected to a computer-assisted image analysis system (Metamorph, Universal Imaging CO, West Chester, PA). To maintain a constant threshold for each image and to compensate for subtle variability of the immunostaining, we only counted neurons that were at least 70% darker than the average gray level of each image after background subtraction and shading correction. The average number of pNR1-ir neurons per section from each animal was obtained, and these values were averaged across each group and presented as group data. The expression of pNR1 was quantified in the following three dorsal horn regions: 1) the superficial dorsal horn (SDH, laminae I and II); 2) the nucleus proprius (NP, laminae III and IV); and 3) the neck region (NECK, laminae V and VI). These regions were identified based on cytoarchitectonic criteria as defined by Abbadie and Besson.23 All pNR1 quantitation procedures described above were performed blindly with regard to the experimental condition of each animal.

Statistical Analysis
All values are expressed as the mean ± sem. Repeated measures ANOVA was performed to determine overall differences at each time point in PWL (sec) and PWF (%). A one-way ANOVA was used to determine differences in the number of spinal pNR1-ir neurons across all experiment groups. Post hoc analysis was performed using the Bonferroni’s multiple comparison test in order to determine the P value among experiment groups. A P < 0.05 was considered statistically significant.

RESULTS

Effect of IT clonidine, DAMGO or Gabapentin on CCI-Induced Pain Behaviors
In the CCI surgery group, prominent thermal hyperalgesia and mechanical allodynia behaviors were established by 14 days after surgery (Fig. 1A–D). IT injection of clonidine dose-dependently reversed (sec) and suppressed PWF (%) as compared to the PWL and PWF values obtained from the vehicle-treated group (Fig. 1A and B; *P < 0.05 and **P < 0.01). A significant antihyperalgesic effect was observed on CCI-induced thermal hyperalgesia from 10 min to 60 min after injection (Fig. 1A) and a significant antiallodynic effect was observed for up to 120 min on CCI-induced mechanical allodynia (Fig. 1B) after an injection of a 20 µg dose of clonidine (n = 7). An intermediate dose of clonidine (5 µg; n = 7) also produced an antihyperalgesic and antiallodynic effect, but over a shorter timeframe (10–30 min) after injection. Finally IT injection of the minimum dose of clonidine used in this study (1 µg; n = 7) did not produced any effect. IT injection of the {alpha}-2 adrenoceptor antagonist, IDA (40 µg, n = 6) 10 min before clonidine injection completely blocked the antihyperalgesic and antiallodynic effect of clonidine (20 µg) throughout the entire behavioral testing period (Fig. 1A and B), while IT injection of IDA alone did not affect thermal hyperalgesia or mechanical alldoynia. IT injection of DAMGO (1 µg, n = 6) or GBP (100 µg, n = 6) also produced a significant decrease in thermal hyperalgesia and mechanical allodynia (Fig. 1C and D; *P < 0.05 and **P < 0.01 as compared to those of vehicle-treated animals) in neuropathic rats. The antihyperalgesic and antiallodynic effect of DAMGO and GBP were comparable to that produced by the high dose (20 µg) of clonidine in CCI-treated rats.


Figure 150
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  		Figure 1. Graphs illustrating the time course and effect of IT clonidine (CLO, 1, 5, 20 µg, n = 7, respectively), [d-Ala2, NMe-Phe4, Gly-ol5]-enkephalin DAMGO(1 µg, n = 6) or gabapentin (GBP, 1 µg, n = 6) treatment on thermal hyperalgesia (A, C) and mechanical allodynia (B, D) 14 days (14D) after chronic constriction injury (CCI). IT clonidine significantly reversed paw withdrawal latency (sec) and suppressed paw withdrawal frequency (%) in a dose-dependent manner. (Fig. 1A and B; *P < 0.05 and **P < 0.01 as compared to those of the vehicle-treated group) In addition, idazoxan alone (IDA, 40 µg, n = 6) did not modify CCI-induced thermal hyperalgesia and mechanical allodynia, whereas IDA pretreatment totally reversed both the antihyperalgesic and the antiallodynic effects of clonidine (IDA+CLO, n = 6). IT DAMGO and GBP also significantly reduced thermal hyperalgesia and mechanical allodynia (Fig. 1C and D; *P < 0.05 and **P < 0.01 as compared to those of vehicle treated animals). The error bars represent the SEM.

 

Effect of IT clonidine, DAMGO or Gabapentin on pNR1 Expression in Spinal Dorsal Horn
CCI rats or sham rats were euthanized 30 min after vehicle, clonidine, DAMGO or GBP injection (n = 5 per group). Immunohistochemistry was then performed to determine the level of pNR1 expression in the spinal cord dorsal horn (Fig. 2A–F and Fig. 3A–C). In the sham surgery group, the maximal dose of clonidine (20 µg) had no effect on the number of pNR1-ir neurons in any of the spinal cord dorsal horn regions when compared to that of the vehicle-treated sham animals (Fig. 3A–C). The number of pNR1-ir neurons in vehicle-treated CCI rats was significantly increased in the SDH, NP and NECK regions of the dorsal horn as compared with that of vehicle-treated sham animals (Fig. 2A, B and Fig. 3A–C; *P < 0.05 and **P < 0.01). The IT clonidine injection dose-dependently suppressed the CCI-induced increase in the number of pNR1-ir neurons in each spinal cord region (Fig. 2C and Fig. 3A–C; #P < 0.05 and ##P < 0.01 as compared to vehicle-treated CCI animals, respectively). In addition, this suppressive effect of clonidine (20 µg) on spinal cord pNR1 expression was totally reversed in all three regions of the spinal cord dorsal horn by IT IDA pretreatment (Fig. 2D and Fig. 3A–C). On the other hand, IT DAMGO or GBP had no significant effect on the number of CCI-induced pNR1-ir neurons in laminae I-IV of the dorsal horn (Fig. 2E, F and Fig. 3A, B). GBP, however, significantly reduced pNR1 expression in the NECK region (lamina V-VI) of the spinal cord dorsal horn (Fig. 2F and Fig. 3C), but this reduction was much less than that produced by 20 µg IT clonidine.


Figure 250
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  		Figure 2. Photomicrographs of representative L4–5 spinal cord sections illustrating phosphorylated NR1 (pNR1)-ir neurons (arrowheads) in the ipsilateral dorsal horn of sham (A) and chronic constriction injury (CCI) (B–F) rats after IT injection of vehicle (B), clonidine (20 µg, C), DAMGO (1 µg, E), gabapentin (GBP, 100 µg, F) 2 wks after surgery. The number of pNR1-ir neurons in vehicle-treated neuropathic rats (B) was significantly increased as compared to vehicle-treated sham rats (A). The number of pNR1-ir neurons was dramatically reduced in the intrathecal (IT) clonidine-treated neuropathic group (C). Idazoxan (IDA) pretreatment completely reversed this suppressive effect of clonidine (D). On the other hand, IT DAMGO (E) or GBP (F) had no significant effect on CCI-induced pNR1 expression in the superficial dorsal horn (SDH) and nucleus proprius (NP) regions. Both clonidine and gabapentin significantly reduced CCI-induced pNR1 expression in the neck of dorsal horn (NECK) region (laminae V and VI) of the dorsal horn, but the effect of GBP was less than that of 20 µg clonidine (Fig. 3). SDH, superficial dorsal horn; NP, nucleus proprius; NECK, neck of dorsal horn. White arrowhead, representative pNR1-ir neurons. Scale bar = 200 µm.

 

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  		Figure 3. Graphs illustrating the effect of IT clonidine (CLO, 1, 5, 20 µg, n = 5, respectively) DAMGO (1 µg, n = 5) or gabapentin (GBP, 1 µg, n = 5) on the number of spinal pNR1-ir neurons in sham (SHAM, n = 5) or chronic constriction injury (CCI) rats. The number of pNR1-ir neurons in vehicle-treated CCI rats was significantly increased in the superficial dorsal horn (SDH) (A), nucleus proprius (NP) (B) and NECK (C) regions of the dorsal horn as compared with that of vehicle-treated sham animals (*, **P < 0.05 and P < 0.01, respectively). IT clonidine treatment dose-dependently suppressed the CCI-induced increase in the number of pNR1-ir neurons in each of the three dorsal horn regions examined (A–C; #, ## P < 0.05 and P < 0.01 as compared to vehicle-treated CCI animals, respectively), whereas in the SHAM group, even the maximum dose of IT clonidine (20 µg) had no effect on the number of pNR1-ir neurons in these three dorsal horn regions (A–C). In contrast to clonidine, DAMGO had no effect on the number of pNR1-ir neurons in any of the three dorsal horn regions, whereas GBP only partially suppressed pNR1 expression in NECK region (C), but had no significant effect on pNR1 expression in the SDH (A) and NP region (B) of CCI rats. The error bars represent the SEM.

 

DISCUSSION

The present study demonstrates that the activation of spinal {alpha}-2 adrenoceptors by IT clonidine injection produces a rapid and potent reversal of CCI-induced thermal hyperalgesia and mechanical allodynia in neuropathic rats. We also showed that the number of pNR1-ir neurons in the spinal cord dorsal horn is increased in CCI animals. In this regard phosphorylation of NR1, an essential subunit of the NMDAR, modulates NMDAR activity and facilitates pain. Importantly, we provide novel data in this study, which demonstrate that IT clonidine treatment causes a significant reduction in the number of pNR1-ir neurons in the dorsal horn of CCI animals and further shows that this reduction is dose-dependent and correlates with clonidine’s antihyperalgesic and antiallodynic effects. In addition, IT administration of the {alpha}-2 adrenoceptor antagonist, IDA before clonidine injection completely reversed the antihyperalgesic and antiallodynic effect of clonidine, as well as the suppressive effect of clonidine on CCI-induced spinal NR1 phosphorylation in all three regions of the spinal cord dorsal horn. These results imply that the clonidine-induced reduction in CCI-induced spinal pNR1 expression is not a nonspecific or unique property of clonidine itself, but rather a general property of activation of {alpha}-2 adrenoceptors in the spinal cord dorsal horn.

IT clonidine produces a significant analgesic or reversal effect in both acute and chronic pain states via direct activation of spinal {alpha}-2 adrenoceptors.24 Two of the mechanisms by which the IT injection of {alpha}-2 adrenoceptor agonists are thought to produce their analgesic or relieving effects are: 1) by reducing glutamate and excitatory neuropeptide release from central afferent terminals3,4 and 2) by hyperpolarizing dorsal horn neurons.5 More recently, it has been suggested that IT clonidine suppresses mechanical allodynia associated with nerve injury via a novel mechanism involving spinal M4 muscarinic receptors.25,26 In addition, it has been proposed that the direct application of clonidine to the site of nerve injury reduces cytokine production and, consequently, the activation of p38 mitogen-activated protein kinase in sensory neurons.27,28 In the present study, we demonstrate that IT clonidine administration can also significantly reduce the CCI-induced increase in pNR1 expression in dorsal horn neurons. Enhanced phosphorylation of the NR1 subunit has been shown to increase the response of NMDAR to NMDA in central nervous system neurons, and it is likely that NMDAR activation (phosphorylation) is associated with sensitization in chronic pain states.29 It has also been shown that subcutaneous clonidine treatment (0.025 mg/kg) enhances the neuropathic pain-relieving action of the NMDAR antagonist, MK-801, while preventing its neurotoxic and hyperactivity side effects.12 In addition, the {alpha}-2 adrenoceptor agonists xylazine, detomidine and dexmedetomidine, each depressed NMDAR-mediated synaptic transmission in a neonatal rat hemisected spinal cord preparation.13 Collectively, these studies in combination with our current data would argue that the antihyperalgesic and antiallodynic effect of IT clonidine may also be linked to its ability to modulate the enhanced spinal NMDAR activity that occurs after peripheral nerve injury.

This raises the issue of how clonidine modulates NMDAR activity. In this regard, {alpha}-2 receptors are G-protein linked receptors which, when activated, seem to exert their effects partly via the inhibition of cAMP formation, ultimately leading to a reduction in PKA activity.7,24 It was recently reported that peripheral neuropathy increased {alpha}-2 adrenoceptor G-protein coupling and spinal noradrenergic fiber sprouting via an increase in brain-derived neurotrophic factor in the spinal cord. This would imply that under neuropathic pain conditions, clonidine would exhibit an enhanced potency compared to that observed under normal conditions.8,30 It has been shown that NMDA current itself is dependent on PKA activity31 and thus if {alpha}-2 adrenoceptors reduce PKA activity, this may translate into a reduction in NMDA current. These results are consistent with our data showing that IT clonidine injection suppresses NR1 subunit phosphorylation in CCI-induced neuropathic rats, but not in sham surgery rats. As NMDAR phosphorylation can accelerate the rise and decay of NMDA currents32 and/or modulate the trafficking and clustering of NMDARs,33 it is feasible that the clonidine-induced reduction of NR1 subunit phosphorylation in the dorsal horn of CCI rats is correlated with an alteration of NMDA currents or with NMDAR trafficking and that this is associated with a reduction in CCI-induced hyperalgesia and allodynia. This mechanism may be similar to that reported for the general anesthetics isoflurane and propofol, which both reduce NR1 phosphorylation to ultimately depress glutamatergic transmission.34

Using whole cell patch clamp techniques in lumbar spinal cord slices or in vivo patch-clamp recording, several studies have reported that clonidine and noradrenaline suppress the generation of action potentials in nociceptive-specific and wide dynamic range neurons in the dorsal horn.6,35 It was demonstrated that noradrenaline acts on presynaptic sites to reduce noxious stimuli-induced excitatory postsynaptic currents, and on postsynaptic neurons to induce an outward current by G-protein-mediated activation of K(+) channels through {alpha}-2 adrenoceptors.35 Recently, Wolff et al. also demonstrated that at clinical concentrations clonidine partially inhibits voltage-gated Na(+) and K(+) channels, further suggesting that clonidine’s action is due to an interaction with these voltage-gated channels.6 Based on these data, it is possible that the rapid antihypernociceptive effect of IT clonidine is mediated by inhibition of voltage-dependent Na(+) ion channels or by G-protein-mediated activation of K(+) channels, and that the resulting repressed firing ultimately leads to suppression of NR1 subunit phosphorylation in spinal dorsal horn neurons. Although the precise mechanism by which IT clonidine induces suppression of NMDAR activity was not fully elucidated in this study, we would argue that there is a direct or indirect relationship between the clonidine-induced antihyperalgesic/ antiallodynic effect and its ability to modulate NMDAR activity in this CCI model of neuropathic pain.

On the other hand, while the µ opioid receptor agonist, DAMGO and the anticonvulsant, GBP significantly relieved thermal hyperalgesia and mechanical allodynia in neuropathic rats with effects similar to those of IT clonidine, these analgesics failed to reduce the CCI-induced increase in the number of pNR1-ir neurons in laminae I-IV of the spinal cord dorsal horn. This lack of effect of DAMGO and GBP on CCI-induced pNR1 expression in the SDH and NP region of the dorsal horn would argue that the antihyperalgesic and antiallodynic effects of DAMGO or GBP are not associated with modulation of spinal cord NR1 phosphorylation in neuropathic rats. In addition, we would argue that clonidine’s ability to reduce CCI-induced spinal cord NR1 phosphorylation is a distinctive feature of IT clonidine injection and that this suppression may be a novel mechanism by which clonidine produces its antihyperalgesic and antiallodynic effects in this CCI model of neuropathic pain.

In conclusion, the current study demonstrated that IT clonidine produces a significant reversal effect of both thermal hyperalgesia and mechanical allodynia in neuropathic rats. Simultaneously, this effect of clonidine is associated with a dose-dependent reduction of phosphorylation of the NMDAR NR1 subunit in the spinal dorsal horn. Collectively these data suggest that one of the mechanisms for the enhanced potency of IT clonidine administration in a rat model of neuropathic pain is its ability to modulate spinal cord NMDAR activation via suppression of NR1 phosphorylation.

Footnotes

Accepted for publication April 23, 2008.

Supported by a grant (M103KV010015 06K2201 01510) from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of the Republic of Korea and a grant (R01–2005-000–10580–0) from the Basic Research Program of the Korea Science & Engineering Foundation.

The author(s) declare that no financial support or compensation has been received from any individual or corporate entity for this research.

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2008 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press