In the von Frey test, milnacipran (60 mg/kg p.o.) significantly increased the withdrawal threshold of the ipsilateral paw between 40 and 160 min after p.o. administration compared with the preadministration baseline (0 min) (P < 0.05; Dunnett's test) although it did not change the withdrawal threshold of the contralateral paw (Fig. 2A). In the radiant heat test, milnacipran increased withdrawal latency of both ipsilateral and contralateral paws between 40 and 120 min and between 40 and 140 min, respectively compared with preadministration baseline (Fig. 2B).
To examine the involvement of spinal monoaminergic systems in the analgesic effect of milnacipran, spinal noradrenergic or serotonergic neurons were selectively denervated. The effects of chemical denervation on the contents of spinal NA, 5-HT and DA are shown in Table 1. 6-OHDA (i.t.) depleted the content of NA by more than 97% with only a slight (18%) but significant reduction of DA. 5,7-DHT reduced the content of 5-HT by more than 96% with no effect on NA or DA.
The peak effect of milnacipran via i.c.v. and i.t. routes was observed between 10 and 20 min after injection in both sham and nerve-ligated mice (data not shown). Either i.c.v. or i.t. milnacipran increased withdrawal threshold and withdrawal latency at the doses between 0.7 and 21 μg per mouse and between 7 and 21 μg per mouse, respectively, in nerve-ligated mice (Fig. 4). In sham-operated mice, milnacipran administered i.c.v. or i.t. did not change withdrawal threshold although withdrawal latency was increased at doses between 0.7 and 21 μg per mouse and between 2.1 and 21 μg per mouse, respectively. Local administration of milnacipran had no effect in either test on either nerve-ligated or sham-operated mice until 90 min after injection (Fig. 4).
The locomotor activity was tested in the open field apparatus in sham and nerve-ligated mice. No difference in the distance traveled during the 10 min test was observed between the groups after the administration of up to 120 mg/kg p.o. milnacipran (Fig. 5).
The oral route and the doses for examining systemic effect of milnacipran were based on previous studies,43,44 which showed analgesic, antidepressant, and antianxiety effects at these doses. Because paw withdrawal can be influenced by the sedative effect, the possible induction of sedative effects by milnacipran was evaluated in naïve mice in an open field test. No reduction of locomotor activity was observed even at the highest doses of milnacipran. This is consistent with results reported by Mochizuki et al.44 and suggests the behavioral change induced by systemic and i.c.v. milnacipran may not be the result of a sedative effect.
Milnacipran increased the withdrawal latency in the radiant heat test in the contralateral paw of nerve-ligated mice and in sham-operated mice, suggesting that it induced an antinociceptive effect. The increased withdrawal threshold in the von Frey test and withdrawal latency in the radiant heat test in the injured paw, suggests that milnacipran also has antiallodynic and antihyperalgesic properties. The antinociceptive, antiallodynic, and antihyperalgesic effects of milnacipran were dose-dependent up to 60 mg/kg p.o. but the effect of 120 mg/kg p.o. was not significantly greater than that of 60 mg/kg (Fig. 1). Dose-dependent effects have been observed in studies that examined the effect of other analgesics, such as opioids and TCAs, on neuropathic pain induced by SNL.6,16,19,45 The efficacy of TCAs against allodynia, however, is controversial. Reports using the rat SNL model indicate that TCAs are effective against hyperalgesia but not against allodynia.6,19 In the present study, milnacipran had an effect on both allodynia and hyperalgesia induced by SNL in mice (Figs. 1 and 2).
In the experiment using neurotoxin, both spinal NA and 5-HT depletion slightly but significantly reduced the withdrawal latency in the radiant heat test, but not the withdrawal threshold in the von Frey test in sham-operated mice, (Fig. 3B), suggesting that both spinal NA and 5-HT neurons are involved in nociceptive transmission. On the other hand, spinal monoamine depletion did not further reduce the withdrawal threshold and latency induced by SNL in the tests. In other words, there appeared no worsening of allodynia and hyperalgesia after monoamine denervation although withdrawal threshold and latency were already low in nerve-ligated mice.
The antinociceptive effect of milnacipran, as measured by increases in withdrawal latency in sham-operated mice, was essentially unchanged after denervation of either NA or 5-HT (Fig. 3B) suggesting that its antinociceptive effect involves both noradrenergic and serotonergic neurotransmission.
Contrary to the antinociceptive effect, the antiallodynic and antihyperalgesic effects of milnacipran were attenuated by spinal NA denervation but not by 5-HT denervation. This suggests that the antiallodynic and antihyperalgesic effects of milnacipran are mediated principally through the spinal NA, but not 5-HT neurons, in nerve-ligated mice. These results are consistent with other reports investigating the effect of other analgesics in neuropathic pain induced by nerve injury, where spinal NA neurons appear to be involved to a greater extent in the antiallodynic and antihyperalgesic effects than spinal 5-HT neurons. A more important contribution of noradrenergic neurotransmission than serotonergic neurotransmission in the SNL model is suggested by the fact that the selective NA uptake inhibitors exhibit analgesic properties in neuropathic pain models, whereas selective 5-HT uptake inhibitors do not.6,8 Clinical studies have also shown analgesic and antiallodynic effects of antidepressants with a major noradrenergic component.23,46–48 On the contrary, Obata et al.34 reported the involvement of both NA and 5-HT in the antiallodynic effect of milnacipran in a rat neuropathic pain model. Although the exact mechanism of action of these antidepressants is still unknown, these results are consistent with our findings, suggesting the importance of noradrenergic systems in the antiallodynic and antihyperalgesic effect of these drugs.
In sham-operated mice, i.c.v. or i.t. but not local administration of milnacipran increased withdrawal latency in the radiant heat test, but did not increase withdrawal threshold in the von Frey test. This suggests that milnacipran's antinociceptive effect has its origin in the central nervous system. In ligated mice, antiallodynia and antihyperalgesia were observed after either i.t. or i.c.v. administration of milnacipran. Both antiallodynia and antihyperalgesia were dose-dependent. The site of analgesic action of TCA has been reported to be supraspinal, spinal and peripheral. Spinally injected TCAs inhibited biting and licking behaviors in a rat formalin test10 and spinal amitriptyline showed an antihyperalgesic effect in a rat SNL model.19 Korzeniewska-Rybicka and Plaznik20 suggested, however, that the supraspinal site is probably more important for the antinociceptive effect of imipramine and amitriptyline. On the other hand, spinally injected TCAs inhibited biting and licking behaviors in a rat formalin test10 and spinal amitriptyline showed antihyperalgesic effect in a rat spinal nerve ligation model.19 The peripheral action of TCAs has been well examined and characteristics other than monoaminergic uptake have been suggested. Amitriptyline and desipramine show the peripheral antinociceptive and antiallodynic effect, possibly by antiinflammation,49 by blocking sodium channels at peripheral sites,50,51 and by the action of adenosine.52,53 The involvement of NMDA receptors in spinal analgesia induced by TCAs has also been suggested, because TCAs have been shown to block NMDA receptors in high μM concentrations.25 On the other hand, milnacipran has been shown to block NMDA receptors in high μM concentrations.3 In our previous study, milnacipran blocked other ligand-gated ion channels, such as 5-HT3 and nACh receptors, in high μM concentrations but did not affect γ-aminobutyric acid type A (GABAA) receptors, even in high concentration.3 There are no studies investigating the potency of sodium channel blockers as opioid receptor blockers. It cannot be denied that no reaction by local injection of milnacipran always indicates lack of actions other than monoaminergic reuptake inhibition, because serotonin has been known to produce pronociception at peripheral site.54,55 Although more specific actions of milnacipran than TCAs might have less nonspecific effect, actions other than monoamine uptake inhibition are yet to be investigated.
In the present study, milnacipran had antinociceptive, antiallodynic, and antihyperalgesic effects in mice with SNL. Milnacipran has fewer side effects compared with amitriptyline and other TCAs, and may be useful as treatment for the relief of chronic pain, including conditions such as fibromyalgia.56
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