DYNORPHIN A is an endogenous opioid peptide with a high degree of selectivity for κ-opioid receptors. It has been reported that high dosages or sustained exposure of dynorphin A may cause neurologic dysfunction including hyperalgesia, allodynia,1–3and paralysis.4There is also considerable evidence that levels of dynorphin A increase significantly at the sites of spinal cord injuries5and in the spinal cord after nerve injury,6,7and the increase of dynorphin levels in the spinal cord was associated with neurologic dysfunction.8As neuropathic pain remains a devastating sequela after spinal cord trauma and nerve injury, further understanding regarding pathophysiological mechanisms and treatment of dynorphin-induced behavioral dysfunction may become a key for strategies of pain managements in such patients.
Recent evidence suggested the antinociceptive effects of δ-opioid agonists in a variety of pain models.9,10SNC80, (+)-4-[(αR)-α((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3 -methoxybenzyl-N,N -diethylbenzamide, is a highly selective nonpeptidic delta opioid agonist. Although peptidic opioids may rapidly degrade, nonpeptidic opioids are proteolytically stable and have enhanced bioavailability relative to the peptidic opioids.11,12Although several investigators have demonstrated the antinociceptive effects of SNC80 in chronic pain models,12,13there has been no data on its antiallodynic effect. We hypothesized that δ-opioid agonist SNC80 can attenuate dynorphin A-induced tactile allodynia. First, to determine the dosage of dynorphin and time course of dynorphin-induced allodynia, dose–dependent effects of intrathecally administered dynorphin on the tactile allodynia were evaluated. Second, the effect of N -methyl-d-aspartate receptor antagonist MK801 on dynorphin-induced tactile allodynia was assessed. Finally, the effects of SNC80 on dynorphin A-induced tactile allodynia were investigated.
Materials and Methods
The Animal Experiment Committee of Nara Medical University approved this study. All experimental procedures were performed in accordance with the guidelines established in the Guide for the Care and Use of Laboratory Animals available from the National Academy of Science (Washington, DC).
Male Sprague-Dawley rats (300–350 g; Japan SLC, Shizuoka, Japan) were housed in cages with 12–24h light-dark cycle and were allowed free access to food and water.
Dynorphin A(1-13) (Biogenesis Ltd, Poole, England) and MK801 (EMD Biosciences Inc. San Diego, CA) were dissolved in saline, 0.9%. SNC80 (ALEXIS Biochemicals, Lausanne, Switzerland) was prepared in its vehicle (saline, 0.9%, and 100 mm HCl).
Intrathecal Catheter Implantation
Under isoflurane anesthesia (2% in oxygen-air), the rats were implanted catheters according to the method described by Yaksh and Rudy.14A PE-10 polyethylene tube (8.5 cm) was inserted through the atlanto-occipital membrane and to the lumbar enlargement.
Nociceptive Behavioral Testing
Mechanical allodynia was determined by measuring the paw withdrawal in response to probing with von Frey filaments. A 50% withdrawal threshold was determined by increasing and decreasing stimulus strength eliciting paw withdrawal and estimating the paw withdrawal threshold by a Dixon nonparametric test.15Rats with a baseline threshold less than 10 g were excluded from this study.
Rats were allowed 7 days to recover from the intrathecal implantation, and any rats exhibiting motor deficiency were discarded from testing. We measured the baseline paw withdrawal threshold (control value). In our preliminary study, we demonstrated that even 5 nmol of intrathecal dynorphin A (1-13) produced transient hind limb paralysis in some animals, resulting in a failure of behavioral assessment. Therefore, we used lower doses of dynorphin less than 5 nmol. In the first study, the rats were randomly allocated to one of four groups. Animals received saline (group S; n = 8) or dynorphin A (1-13) 0.25 nmol (group D0.25; n = 7), 1 nmol (group D1; n = 9), or 2 nmol (group D2; n = 7). All drugs were injected intrathecally in a volume of 5 μl followed by a 9-μl flush.14In a single blind manner, that is, the observer had no information as to the group designation, we measured paw withdrawal threshold at 10 min, 30 min, 1 h, 2 h, 4 h, 8 h, 1 day, 3 days, and 7 days after intrathecal injection. In the second study, rats were randomly allocated to one of three groups (n = 7 in each group). In the groups M5 and M10, 5 nmol or 10 nmol of MK801, respectively, was intrathecally administered 20 min before intrathecal injection of 2 nmol of dynorphin A (1-13). In the group S, saline was intrathecally administered. In study 3, the rats were randomly allocated to one of three groups (n = 9 in each group). In the groups S25 and S100, 25 nmol or 100 nmol of SNC80, respectively, was intrathecally administered 20 min before intrathecal injection of 2 nmol of dynorphin A (1-13). In the group S, saline with 100 mm HCl was intrathecally administered. In studies 2 and 3, an observer blinded to the drug applications measured paw withdrawal threshold at 10 min, 30 min, 1 h, 2 h, 4 h, and 8 h after intrathecal injection of dynorphin.
All data are expressed as mean ± SEM. Statistical analysis was performed using two-way analysis of variance with repeated measurements followed by Student-Newman-Keuls test for multiple comparisons. A P value of <0.05 was considered statistically significant.
Dynprphin-Induced Tactile Allodynia
As the withdrawal thresholds of left and right hind paws were similar in each group, mean values of the bilateral thresholds were used for further analysis. As shown in figure 1, the intrathecal administration of 2 nmol of dynorphin A (1-13) produced a significant reduction of the withdrawal threshold at 10 min, 30 min, and 1 h after injection compared with the control values and paw withdrawal threshold at 30 min and 1 h after injection was significantly lower than in the group S. The intrathecal administration of 1 nmol of dynorphin A (1-13) also produced a significant reduction in the withdrawal threshold at 30 min, 1 h, and 2 h after injection compared with the control values and paw withdrawal threshold at 30 min, 1 h, 2 h, and 4 h after injection was significantly lower than in the group S.
Effect of MK801 on Dynorphin-Induced Tactile Allodynia
Changes in withdrawal thresholds after 2 nmol of dynorphin intrathecal injection with or without MK-801 pretreatment are shown in figure 2. Rats pretreated with saline elicited a significant reduction in the withdrawal threshold at 10 min and 30 min after injection. Pretreatment with 5 or 10 nmol of MK801 failed to elicit a significant reduction in the withdrawal threshold compared with the control values. Furthermore, withdrawal thresholds at 10 min and 30 min after injection in the group M10 and at 30 min in the group M5 were significantly higher than in the group S.
Effect of SNC80 on Dynorphin-Induced Tactile Allodynia
Changes in withdrawal thresholds after 2 nmol of dynorphin intrathecal injection with or without SNC80 pretreatment are shown in figure 3. Rats pretreated with saline with 100 mm HCl elicited a significant reduction in the withdrawal threshold at 10 min, 30 min, and 1 h after injection. In the group S25, withdrawal thresholds were significantly reduced at 30 min after injection. In the group S100, withdrawal thresholds remained unchanged during the observation period. Withdrawal thresholds at 10 min, 30 min and 1 h after dynorphin injection were significantly higher in the group S100 compared with those in the group S.
This study shows that intrathecally administered dynorphin A (1-13) produced transient tactile allodynia and pretreatment with MK-801 or SNC80 dose-dependently attenuated the development of tactile allodynia induced by dynorphin A.
As reported previously,1–3intrathecal administration of dynorphin A produced tactile allodynia. Dynorphin-induced allodynia peaked at 30 min after injection, but lasted for only a short period. These findings are in contrast to the results in the previous study.1,2Vandrah et al. 1reported that intrathecal administration of dynorphin A produced a significant long-lasting tactile allodynia up to 60 days after injection. The reasons of these contradictory results are unknown. However, the dosage of dynorphin might have affected the results.
The mechanisms by which intrathecal administration of dynorphin induces tactile allodynia are unknown. Several possible explanations are as follows. First, dynorphin-induced allodynia may be attributable to ischemic injury after the reduction of spinal cord blood flow.16Second, N -methyl-d-aspartate receptors may be involved in the development of tactile allodynia after intrathecal administration of dynorphin A.1–3In fact, we confirmed that dynorphin-induced allodynia could be reversed by pretreatment with N -methyl-d-aspartate antagonist MK-801 in the present study.
Although the classic view has been that neuropathic pain is resistant to opioid therapy, recent evidence suggested an important role of delta opioid receptor agonists in antinociception at the level of the spinal cord.9,10These findings were compatible with the results in the present study, in which SNC80 attenuated tactile allodynia. The mechanisms of antiallodynic effect of intrathecally administered SNC80 observed in the present study are unknown. However, glutamate- and glutamate receptor-mediated responses might be at least involved in antiallodynic effect of SNC80. Zhang et al. 17demonstrated that delta opioid receptor agonist, [D-Ala2, D-Leu5]-enkephalin (DADLE), reduced glutamate-induced excitotoxic injury. Wang et al. 18reported that δ-opioid agonist, [D-Phe2, D-Phe5]-enkephalin (DPDPE) attenuated N -methyl-d-aspartate-evoked responses of nociceptive neurons. In fact, N -methyl-d-aspartate receptor antagonists have been shown to attenuate antinociception induced by δ-opioid receptor agonists.19,20Considering that dynorphin-induced allodynia was at least in part mediated through the N -methyl-d-aspartate receptor activation, SNC80 might exert antiallodynic effects by inhibiting glutamate- and N -methyl-d-aspartate-mediated responses in this model. Further investigations would be required to clarify the exact mechanisms of antiallodynic effect of SNC80.
The authors thank Osamu Kakinohana, Ph.D. (Assistant Project Scientist, Department of Anesthesiology, University of California, San Diego, San Diego, California), Joho Tokumine, M.D. (Assistant Professor, Department of Anesthesiology, University of the Ryukyus, Okinawa, Japan), and Tomoki Nishiyama, M.D. (Associate Professor, Department of Anesthesiology, The University of Tokyo, Tokyo, Japan) for their support in preparing the animal model.