Antidepressants have antinociceptive effects and are widely used for treatment of chronic pain. Tricyclic antidepressants (TCAs) are often administered systemically to patients with neuropathic pain (1). The mechanisms of antinociceptive effects of antidepressants are not fully understood. Antidepressants inhibit the reuptake of monoamines, including noradrenaline (NA) and serotonin (5-HT), at the neuronal terminal (2), and the neuronal terminal might mediate antinociception as well. Most antidepressants, however, have multiple sites of action and can produce antinociceptive effects at supraspinal (3), spinal (4), and peripheral sites (5). This makes it difficult to clarify the mechanism of antinociceptive effects of antidepressants when administered systemically.
The brainstem-spinal descending NA and 5-HT systems suppress nociceptive signals from primary afferent neurons to the spinal dorsal horn. Intrathecal administration of adrenoceptor agonists and 5-HT receptor agonists produce antinociceptive effects for acute pain in rodents (6,7) and suppress allodynia in a rat model of neuropathic pain (8,9). Antidepressants might inhibit neuropathic pain in the spinal cord by blockade of NA or 5-HT reuptake. Recently, more selective monoamine reuptake inhibitors, such as serotonin noradrenaline reuptake inhibitors, selective serotonin reuptake inhibitors, and selective NA reuptake inhibitors, have become available and are clinically used for depression. It is not known whether these drugs inhibit hypersensitivity in a neuropathic pain state at the spinal site.
Tight ligation of the L5 and L6 spinal nerves leads to neuropathic pain in rats (10). In the present study, we examined the antiallodynic effects of intrathecal administration of a serotonin noradrenaline reuptake inhibitor, a selective serotonin reuptake inhibitor, or a selective NA reuptake inhibitor in the rat model of neuropathic pain.
All experiments were approved by the Animal Care and Use Committee of our institution. Male Wistar rats weighing 200 to 220 g underwent spinal nerve ligation under isoflurane anesthesia as previously described (10). On the left side, spinal nerves L5 and L6 were isolated and ligated tightly with 6–0 silk sutures distal to the dorsal root ganglion. After surgery, animals were housed individually with free access to food and water and allowed to recover for at least 14 d. Left paw mechanical allodynia was confirmed at this time by measuring the hindpaw withdrawal threshold in response to the application of von Frey filaments using the previously described up-down method (11). Only animals with a withdrawal threshold of <4 g were used. Then, an intrathecal catheter for spinal injection of drugs was inserted under isoflurane anesthesia as previously described (12). Catheters were advanced 8.0 cm caudally through an incision in the cisternal membrane. Rats with evidence of neuromuscular dysfunction were killed immediately. The other animals were allowed to recover 7 days before drug testing.
Behavioral testing was performed during the daylight portion of a regulated circadian cycle (0600 to 1800). Rats were placed individually in cages having a wire mesh bottom that allowed full access to the paws. The withdrawal threshold was calculated and 15 g was selected as the cut-off value (11). The general behavior of the rats was carefully observed. Motor function was evaluated by righting reflex, stepping reflex, posture, and ambulation. Sedation was assessed in terms of spontaneous movement, such as grooming and chewing, and evoked movement (a startle reflex evoked by tapping on the cage).
The first series of experiments examined the time-course of the antiallodynic effects and the dose-response effects of intrathecally administered antidepressants. The serotonin noradrenaline reuptake inhibitor milnacipran (3, 10, 30, and 100 μg), the selective serotonin reuptake inhibitor paroxetine (10, 30, and 100 μg), and the selective NA reuptake inhibitor maprotiline (10, 30, and 100 μg) were used. If the antiallodynic effects were observed after intrathecal administration, antiallodynic effects by intraperitoneal injections were determined using another groups of rats. Antagonist studies were also performed. We used yohimbine (30 μg) as an α2-adrenoceptor antagonist or methysergide (30 μg) as a 5-HT receptor antagonist, respectively. We also used atropine (30 μg) as a muscarinic receptor antagonist because muscarinic receptors contribute to monoamine receptor-mediated antiallodynic effects (13,14) The dose of the antagonist was selected according to previous studies (14,15).
Milnacipran was donated by the Asahi Kasei Corporation (Osaka, Japan). Paroxetine was donated by GlaxoSmithKline (Worthing, UK). Other drugs were purchased from Tocris Cookson Inc. (Ellisville, MO). Milnacipran and atropine were dissolved in saline. Paroxetine and maprotiline were dissolved in 50% dimethyl sulfoxide solution. Yohimbine and methysergide were dissolved in distilled water. For intrathecal administration, drugs were delivered in a 10-μL volume of solution followed by 10-μL of saline to flush the catheter. Drugs were dissolved in a 1-mL volume of solution for intraperitoneal injection. Throughout the study, after determining the baseline value, paw withdrawal thresholds were measured at 15, 30, 60, 90, and 120 min after injection and at 60-min intervals thereafter. In the antagonist studies, one of each antagonist was coadministered with an antidepressant.
The data are presented as withdrawal threshold or percentages of the maximum possible effect (%MPE), where %MPE = (postdrug threshold − predrug threshold) × 100/(15 g − predrug threshold). Data were analyzed using a two-way analysis of variance, followed by a Student-Newman-Keuls post hoc test. A P value of <0.05 was considered to indicate statistical significance.
Intrathecal administration of milnacipran produced antiallodynic effects in a dose-dependent manner (Fig. 1, P < 0.01, two-way analysis of variance). The threshold increased within 15 min and peak effect was attained at approximately 1 h after injection. The effect continued 7 h after administration of 100 μg and 4 h after administration of 30 μg compared with the saline-treated group (P < 0.05). No adverse effects, such as motor effects or sedation, were observed. Intraperitoneal injection of milnacipran (3, 10, and 30 mg/kg) did not inhibit allodynia (Table 1). Intrathecal administration of either paroxetine or maplotiline had no antiallodynic effect at doses of 10 to 100 μg (Table 1).
An antiallodynic effect of intrathecal administration of 30 μg of milnacipran was attenuated by 30 μg of yohimbine, methysergide, or atropine (Fig. 2; P < 0.01, respectively, two-way analysis of variance). Intrathecal administration of yohimbine, methysergide, or atropine alone at the same doses used above did not alter the withdrawal threshold. Intrathecally administered distilled water or 50% dimethyl sulfoxide solution had no effect on withdrawal threshold (data not shown).
The present study demonstrated that intrathecal administration of milnacipran, a serotonin noradrenaline reuptake inhibitor, mediated dose-dependent antiallodynic effects in the spinal nerve ligation model in rats. The effects were attenuated by intrathecal coadministration with an α2-adrenoceptor antagonist, a 5-HT receptor antagonist, or a muscarinic receptor antagonist. In contrast, intrathecally administered paroxetine, a selective serotonin reuptake inhibitor, and maprotiline, a selective NA reuptake inhibitor, did not inhibit allodynia.
Among antidepressants, TCAs such as amitriptyline or imipramine are often used in pain therapy for patients with neuropathic pain. The mechanisms of the antinociceptive effects of TCAs are complicated. TCAs inhibit the reuptake of NA and 5-HT at neuronal terminals (3). TCAs have antagonistic action at the N-methyl-d-aspartate receptors (16) and inhibit adenosine reuptake (17). Further, most TCAs have affinity for opioid (18), NA, 5-HT, histamine, and muscarincic acetylcholine receptors (19). These multiple sites of actions of TCAs seem to modulate nociceptive transmission at supraspinal, spinal, or peripheral sites when they are administered systemically.
The spinal cord is an important site of action of antidepressant-mediated antinociception (4). The neurotransmitters NA and 5-HT have important roles in suppression of nociceptive transmission in the spinal cord. However, there are no studies demonstrating that the antinociceptive effects of antidepressants are attributable to the inhibition of reuptake of NA or 5-HT in the spinal cord in chronic pain. Eisenach and Gebhart (20) reported that intrathecal administration of amitriptyline reversed thermal hyperalgesia via N-methyl-d-aspartate receptor antagonism in a rat model of inflammation induced by carrageenin injection. Kawamata et al. (21), also using the rat model of inflammation, reported that intrathecally injected desipramine, a tricyclic NA reuptake inhibitor, produced analgesia unrelated to NA reuptake inhibition. Wang et al. (22) reported that intracerebroventricular injection of fenfluramine, a 5-HT reuptake inhibitor, produced antiallodynic effects in the rat spinal nerve ligation model, but the drug was not effective after intrathecal injection.
Milnacipran is a new class of clinically effective antidepressants with equivalent inhibitory action at NA and 5-HT neuronal reuptake systems. The relative ratio of reuptake inhibition of 5-HT against NA (5-HT/NA) is 0.95 (23). Milnacipran has no relevant affinity for any other receptors tested, including α-adrenergic, 5-HT, histamine, muscarinic acetylcholine, opioid, and N-methyl-d-aspartate receptors (23). Alpha-2 adrenergic receptors, but not α1- adrenergic receptors, are implicated in the antinociceptive effect of NA for neuropathic pain in the spinal cord (8). In the current study, intrathecal coadministration of yohimbine, an α2-adrenoceptor antagonist, attenuated the effect of milnacipran. Among 5-HT receptor subtypes, the 5-HT2 receptor is the only receptor subtype that inhibits neuropathic pain in the spinal cord (9). Although methysergide has affinity for both 5-HT1 and 5-HT2 receptor subtypes (24), 30 μg of this drug reverses the antiallodynic effects of intrathecally administered 5-HT2 receptor agonists (9). Intrathecal coadministration of methysergide also inhibited the effects of milnacipran. These findings suggest that increased levels of NA and 5-HT in the spinal cord contribute to the antiallodynic effects of intrathecally administered milnacipran. NA and 5-HT interact to produce antinociception in the spinal cord. For example, the antinociception from intrathecally administered 5-HT is blocked by α-adrenoceptor antagonists or depletion of endogenous NA by a neurotoxin (25). 5-HT may increase the release of NA in the spinal cord via 5-HT1 receptors, including receptors currently called 5-HT2C, and NA reuptake inhibition by desipramine potentiates the antinociceptive effect of intrathecally administered 5-HT or 5-HT1 receptor agonists (25). Inhibition by methysergide in the antiallodynic effects of milnacipran in the current study support these observations. The synergistic interaction between NA and 5-HT systems may contribute to the antiallodynic effects of milnacipran.
Conversely, intraperitoneal administration of milnacipran up to 30 mg/kg did not produce antiallodynic effects. The concentration of milnacipran might not reach effective levels at the site of action in the spinal cord after intraperitoneal administration. We could not administer larger doses of milnacipran intraperitoneally because of its severe adverse effect. These findings suggest that the spinal cord is the site of action of antiallodynic effects produced by milnacipran.
Paroxetine inhibits 5-HT reuptake selectively. Radioligand binding studies demonstrated that paroxetine has little affinity for other receptor sites except for muscarinic receptors, to which it has weak affinity (26). Maprotiline is a tetracyclic antidepressant that selectively inhibits NA reuptake. This drug has some affinity for various receptors (19), but does not have affinity for N-methyl-d-aspartate receptors, as desipramine does (23). In the present study, paroxetine and maprotiline did not have antiallodynic effects. The findings suggest that specific NA or 5-HT reuptake inhibition alone in the spinal cord is not adequate to produce antiallodynic effects. Activation of the spinal muscarinic receptors produces antinociceptive effects for acute pain and neuropathic pain (13). Intrathecally administered α2-adrenergic agonists or 5-HT2 receptor agonists inhibit neuropathic pain via muscarinic receptors (13,14). Our data from antagonist studies suggest that muscarinic receptors are involved in the antiallodynic effects of milnacipran because atropine, a muscarinic receptor antagonist, completely reversed the effects of milnacipran. Paroxetine and maprotiline have weak affinity for muscarinic receptors and are antagonistic when they do bind (23,26). This might be another reason why these drugs did not produce antiallodynic effects in the spinal cord.
In summary, simultaneous inhibition of NA and 5-HT reuptake in the spinal cord by milnacipran effectively suppressed mechanical allodynia after spinal nerve injury. Selective reuptake inhibition of NA or 5-HT alone in the spinal cord did not have an effect. Administration of milnacipran might be a promising treatment for neuropathic pain.
We thank the Asahi Kasei Corporation and GlaxoSmithKline for their donation of drugs. We also thank Dr. J.C. Eisenach for his suggestions in this field of research.
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