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From the Department of Anesthesiology, The University of Tokyo, Tokyo, Japan.
Address correspondence to: Tomoki Nishiyama MD PhD, 3-2-6-603, Kawaguchi, Kawaguchi-shi, Saitama, 332-0015, Japan. Phone: 81-3-5800-8668; Fax: 81-3-5800-8938; E-mail: NISHIYAMAT-ANE{at}h.u-tokyo.ac.jp
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Abstract |
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Methods: Sprague-Dawley rats (250–300 g) were implanted with chronic lumbar intrathecal catheters and were tested for their thermal withdrawal response using the hot plate test after intrathecal administration of AP-5 (NMDA receptor antagonist), ACEA 1021 (NMDA glycine site antagonist), or ACEA 2085 (AMPA receptor antagonist). The combinations of these three agents were also tested.
Results: Intrathecal administration of ACEA 2085 had a dose dependent analgesic effect while intrathecal AP-5 or ACEA 1021 could not induce dose dependent effect. Co-administration of AP-5 10 µg and ACEA 2085 intrathecally showed no changes in the thermal response latency compared with ACEA 2085 alone. ACEA 1021, 12 µg, and AP-5 showed left-ward shift of the dose effect curve only with small doses of AP-5 (1 µg, 3 µg). Only the smallest dose of ACEA 2085 (0.1 ng) with ACEA 1021 12 µg induced antinociception compared with that of ACEA 2085 alone.
Conclusions: The combination of the NMDA glycine site antagonist and low doses of the NMDA receptor antagonist or the AMPA receptor antagonist increased the analgesic effect on acute thermal nociception with increased side effects, while the NMDA receptor antagonist and the AMPA receptor antagonist had no such interaction.
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Introduction |
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Correspondingly, NMDA antagonists have little effect on acute nociception, but are effective in facilitated states of nociceptive processing.4,5 The NMDA glycine site antagonist also blocks facilitation of nociceptive transmission.3 In contrast, AMPA receptor antagonists have analgesic effects on acute nociception.6,7
The NMDA receptors and AMPA receptors are often co-localized on the post-synaptic membrane at the same synapse in rat hippocampus.8 The NMDA receptor antagonists and AMPA receptor antagonists have a supra-additive neuroprotective effect against hypoxic damage in the brain.9 This observation suggests a functional interaction in the brain between the NMDA and the AMPA receptor systems. However, there have been few data on the anatomical or functional interaction between the NMDA and the AMPA receptor systems in the spinal cord as related to nociception.
The NMDA glycine site antagonists administered intrathecally attenuate NMDA produced facilitation of nociception.3 This suggests that the glycine site is necessary in conjunction with the NMDA ionophore for facilitation of nociception in the spinal cord. Yet, there are no studies of the interaction in antinociceptive effects between the NMDA receptor antagonist and the NMDA glycine site antagonist.
Considering this evidence, we would expect some interaction in the effects on facilitated nociceptive processing but not on acute nociception among NMDA and AMPA receptor antagonists and NMDA glycine site antagonists.
The purpose of this study was to investigate the spinally mediated antinociceptive interaction among NMDA receptor antagonists, NMDA glycine site antagonist, and AMPA receptor antagonists on acute noxious stimulation to elucidate functional coupling in the nociception of different glutamate receptors in the spinal cord, as well as to investigate the interaction with respect to side effects.
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Drugs and injection
Drugs for intrathecal injection were dissolved in solvent such that 10 µl contained the desired quantity of the agent. The ACEA 2085 (AMPA receptor antagonist; CoCensys, Inc., Irvine, CA) and AP-5 ((±)-2-amino-5-phosphonopentanoic acid, NMDA receptor antagonist; Research Biochemical International, Natick, MA) were dissolved in normal saline and ACEA 1021 (5-nitro-6,7-dichloro-2,3-quinoxaline dion, non-strychnine-sensitive NMDA glycine site antagonist; Eagle-Picher Industries, Lenexa, KS) was dissolved in vehicle (tris buffer). After intrathecal drug injection, the catheter was flushed by injection of 10 µl of normal saline (saline, ACEA 2085, and AP-5 groups) or vehicle (vehicle and ACEA 1021 groups). A micro injector syringe was used for all injections. In each dose group, 10 rats randomly received one of these doses of ACEA 2085 (0.1 ng, 10 ng, 500 ng, 750 ng), AP-5 (1 µg, 3 µg, 10 µg, 30 µg), ACEA 1021(2.4 µg, 8 µg, 12 µg, 24 µg), saline or vehicle. The saline and vehicle groups were the control groups.
Nociceptive test
All animals were tested for acute nociceptive response using a hot-plate test.11 The rats were placed on a surface maintained at 52.0 ± 0.5°C and enclosed by Plexiglas walls. The behavioural endpoint was taken as licking of one hind paw or, less frequently, the jumping off of the plate. The cut-off time in the absence of a response was 60 sec to prevent tissue injury.
Behavioural and motor function test12
The general behaviour (including agitation and allodynia), motor function, flaccidity, pinna reflex, and corneal reflex were examined. They were judged as present or absent. Agitation was judged as spontaneous irritable movement and/or vocalization. The presence of allodynia was examined by looking for agitation (escape and/or vocalization) evoked by lightly stroking the flank of the rat with a small probe. Motor function was evaluated by the placing/stepping reflex and the righting reflex. The former was evoked by drawing the dorsum of either hind paw across the edge of the table. Normally rats try to put the paw ahead to walk. The latter was assessed by placing the rat horizontally with its back on the table, which normally gives rise to an immediate, coordinated twisting of the body to an upright position. Disturbance of the righting reflex also implies central nervous system damage. Flaccidity was judged as muscle weakness. Pinna and corneal reflexes were examined with a paper string. When a paper string was put into the ear canal or touched the cornea rats normally shake their head or blink, respectively, to avoid the stimulation.
Experimental paradigm
Dose effect relationship: The first series of experiments was performed to determine the dose-dependency and time course of the analgesic actions of intrathecally administered ACEA 2085, ACEA 1021, and AP-5 on acute thermal nociception. The hot-plate, behavioural and motor function tests were performed before and at intervals of 15, 30, 60, 90, and 120 min (until 180 min for ACEA 2085) after the injection. In each group, 10 rats were tested.
The ACEA 1021 and AP-5 could not produce a response sufficient to allow calculation of a 50% effective dose (ED50). Therefore, to investigate the interaction between the two agents, a fixed dose of ACEA 1021 (12 µg) was co-administered with various doses of ACEA 2085 or AP-5 and AP-5 (10 µg) with various doses of ACEA 2085 was also tested. The higher doses of ACEA 1021 and AP-5 were considered not to be usable because of behavioral side effects. In each group, eight rats were tested.
Data analysis and statistics
Response latency (sec.) data from hot plate measurements were converted to a percentage of the maximum possible effect (%MPE) according to the formula: %MPE = [(postdrug latency - base line latency) / (cutoff time - base line latency)] x100.11,12
Data were expressed as mean ± standard error (SEM) due to the small number of rats in each group. The statistical analysis was done only for comparable doses with two way analysis of variance (ANOVA) followed by a post-hoc Contrast analysis if appropriate. A P value less than 0.05 was considered statistically significant.
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Results |
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Intrathecal administration of AP-5 (NMDA receptor antagonist), ACEA 1021 (NMDA glycine site antagonist), and ACEA 2085 (competitive AMPA receptor antagonist) resulted in dose-dependent increases in the thermal response latency (Figure 1
). The maximum usable dose of AP-5 or ACEA 1021 did not induce an effect of more than 50% MPE (Figure 2). The rank order of potency was ACEA 2085 > ACEA 1021 = AP-5 (Figure 1).
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Discussion |
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The NMDA antagonists have been reported to have little effect on acute nociception,4,5 because acute nociception is considered to be mediated by monosynaptic transmission in the spinal cord.13 The NMDA glycine site has an important role in the activation of the NMDA channel by glutamate.3 Thus, the functional properties of the NMDA glycine site antagonist resemble those of NMDA antagonists. These are consistent with the present results in which AP-5 and ACEA 1021 had no analgesic effects on thermally evoked nociception. In contrast, the AMPA receptors mediate acute excitation from primary afferent fibers to dorsal horn neurons.6 The AMPA receptor antagonist has an antinociceptive effect on acute thermal stimulus14 as ACEA 2085 in the present study.
From these previous observations, the NMDA receptor antagonist or the NMDA glycine site antagonist seem to have no spinally mediated analgesic interaction with the AMPA receptor antagonist on acute nociception. While the NMDA receptor antagonist (AP-5) had no interaction with the AMPA receptor antagonist (ACEA 2085), the NMDA glycine site antagonist (ACEA 1021) intensified the antinociceptive effect of the AMPA receptor antagonist.
In the brain, AMPA receptors are co-localized with NMDA receptors. Synaptically released glutamate causes rapid activation of both AMPA and NMDA receptors, resulting in a dual synaptic response.15 The NMDA receptor antagonists inhibit calcium ion (Ca2+) entry through the receptor -operated Ca2+ ionophore.16 The AMPA receptor antagonists inhibit Ca2+ entry through the NMDA receptor/ion channel, the voltage-operated Ca2+ channel and the sodium ion (Na+) /Ca2+ exchanger. The AMPA receptor antagonists suppress Na+ - and potassium ion (K+) - mediated cell depolarization, and therefore prevent the opening of voltage-operated Ca2+ channels.17 Thus, NMDA receptor antagonists and the AMPA receptor antagonists can act synergistically by blocking the interacting mechanisms of Ca2+ entry in the brain. For focal or global brain ischemia, combined treatment of the NMDA receptor antagonist and the AMPA receptor antagonist could decrease the infarct size or the incidence of damaged cells more than a single treatment.9 When low doses of the AMPA receptor antagonist were co-administered with NMDA receptor antagonists systemically, an interaction in decreasing the minimum alveolar concentration of halothane between the two antagonists was evident.18 This interaction between the two glutamate receptor antagonists was obvious when subanesthetic doses of the antagonists were combined. However, there is little evidence to suggest the interaction between the AMPA receptor and the NMDA receptor in the spinal cord, especially regarding nociceptive mechanisms.
The present study, however, showed that a low dose of NMDA glycine site antagonist had an interaction with the AMPA receptor antagonist in the spinal cord. The reason why the low doses of NMDA glycine site antagonist had interactions with the NMDA or AMPA antagonists might be 1) The effects of high doses might be masked by side effects, or 2) The participation of each glutamate receptor on acute nociception is limited by a ceiling effect. Regarding side effects, motor disturbance and flaccidity were not so severe as to inhibit the paw movement. In addition, if the disturbance was severe enough to block the response, higher doses of the combination would be expected to have a greater improvement. For these reasons, side effects might not cause this discrepancy. Therefore a ceiling effect might be the reason why the interactions were not dose dependent.
In conclusion, the combination of the NMDA glycine site antagonist and low doses of the NMDA receptor antagonist or the AMPA receptor antagonist had significant interactions in terms of their analgesic effects on acute thermal nociception, while the NMDA receptor antagonist and the AMPA receptor antagonist showed no such interaction.
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Acknowledgments |
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Accepted for publication April 8, 2000.
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References |
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