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© 1999 International Anesthesia Research Society
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Abstract |
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Antinociception can be produced at the spinal level by activation of opioidergic, noradrenergic, and serotonergic systems. We tested the antinociceptive effects of combined activation of all three systems. Antinociception was assessed in the rat tail-flick test, and drugs were administered via an intrathecal catheter. Morphine, the norepinephrine uptake inhibitor desipramine, and serotonin produced antinociception of their own. The combination of subthreshold doses of morphine 1 µg and of desipramine 3 µg produced pronounced antinociception that was antagonized by yohimbine. The combination of subthreshold morphine with serotonin 50 µg or desipramine with serotonin caused only small antinociceptive effects. When morphine combined with desipramine was decreased to a subthreshold dose, we observed pronounced antinociception when a subthreshold dose of serotonin was added. A complex interaction can be supposed by results obtained with antagonists. The activation of all three neurotransmitter systems with small doses of agonists may represent an effective principle for pain control at the spinal level.
Implications: Pain sensations are modulated at the spinal level by opioids, noradrenergic drugs, and serotonin. Using a rat model, we showed that the concurrent use of drugs from each of these classes produces good pain control at doses that should avoid the side effects associated with larger doses of each individual drug.
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Introduction |
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At the spinal level, nociception is attenuated by local opioids, and norepinephrine and serotonin, which are released from descending inhibitory noradrenergic and serotonergic pathways (1, 2). The spinal administration of opioids (3), norepinephrine (4, 5), and serotonin (6) induces antinociception in the rat. Therefore, an interaction of these systems at the spinal cord may affect antinociception. Spinal
2-adrenoceptor activation enhances antinociception produced by intrathecal (IT) morphine (7). Combined activation of the other aforementioned systems has not been consistently studied at the spinal level; specifically, there has been no study on the triple combination. We studied these interactions with regard to antinociception using the rat tail-flick model (8) and tried to ascertain the involvement of specific receptors by using antagonists. |
Methods |
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Male Sprague-Dawley rats weighing 190–290 g were anesthetized with ketamine/xylazine (100 mg/10 mg/kg intraperitoneally). After fixation of the head in a stereotaxic device, the IT space was punctured through the atlantooccipital membrane, and a polyethylene catheter (PE 10) was inserted and advanced to the rostral margin of the lumbar enlargement, as described by Yaksh and Rudy (9), and fixed by suture to the nuchal skin. Only animals without paralytic signs of the hind limbs and showing a tail-flick reaction to radiant heat within 15 s, 2 days after surgery, were used for the experiments. At the end of the test, each animal was killed by CO2 inhalation, and correct catheter tip placement was verified using Evans blue dye. All experiments were performed in accordance with the German legislation for the use of experimental animals and with the European Communities Council Directive.
IT injections of agonists were made in a volume of 5 µL with a Hamilton syringe, followed by a 15-µL flush with saline (control injections were 20 µL of saline). Antagonists or vehicle were injected intraperitoneally, yohimbine 10 min and naloxone 20 min after and ritanserin 30 min before IT injections.
Antinociception was assessed using radiant heat from a light source at an energy setting 25% of maximum and with exposure time limited to 30 s. Tail-flick latency was measured twice before and five or six times after intrathecal injection.
Antinociceptive effects were determined as percentage of the maximal possible effect (% MPE) according to the formula:
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The following drugs were used: desipramine hydrochloride, 5-hydroxytryptamine creatinine sulfate, morphine hydrochloride, naloxone hydrochloride, ritanserin, and yohimbine hydrochloride. All drugs were dissolved in sterile saline except for ritanserin, which was dissolved in 40% PEG 400.
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Results |
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The IT injection of any drug in the dose range tested did not interfere with the gross behavior of the animals. Before the IT injection of these drugs, the mean latency to the tail-flick reaction varied from 5.1 to 9.6 s between groups. Injection of morphine prolonged the latency of the tail-flick reaction (Fig. 1) starting at a dose of 3 µg, whereas after a dose of 1 µg, latencies did not significantly differ from the respective controls (Table 1). Maximal effects were observed 30–90 min after injection, and a maximum of approximately 76% MPE was obtained after the largest tested dose of 12 µg after 60 min. The IT injection of 3 µg of desipramine did not prolong tail-flick latencies (Table 1), but 6 and 12 µg were effective (Fig. 2); however, 6 µg was more potent than 12 µg. Only marginal and nonsignificant antinociceptive effects were observed with 50 µg of serotonin (Table 1), whereas the larger doses induced some short-lasting antinociception (Fig. 3).
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) (n = 40), 3 µg of morphine (
) (n = 10), 6 µg of morphine (
) (n = 10), and 12 µg of morphine (•) (n = 10) were compared with the respective pretest values and expressed as a percentage of the maximal possible effect (% MPE). *P < 0.05 versus controls.
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Subthreshold doses that did not induce significant antinociceptive effects, compared with vehicle injections alone, were 1 µg of morphine, 3 µg of desipramine, and 50 µg of serotonin (Table 1). A combination of subthreshold morphine with subthreshold desipramine elicited pronounced antinociception (Table 1), which was significantly different from the treatment with either substance alone. The combinations of subthreshold morphine with serotonin or desipramine with serotonin produced only small antinociceptive effects. To examine the effects of a triple combination containing subthreshold serotonin, the dose of the combination of morphine and desipramine was reduced to obtain subthreshold effects. This effect was observed with a 10 times smaller dose of the combination (Table 1). When subthreshold serotonin was introduced under these conditions, pronounced antinociception again occurred.
The crucial involvement of specific receptors in the antinociception elicited by the combination of subthreshold doses of the substances was investigated with receptor antagonists, which were injected at different time points in respect to their different pharmacokinetics. The 2-receptor antagonist yohimbine counteracted the effects of the combination of morphine and desipramine (Table 2 ). When PEG 400 was used as a solvent instead of saline, the antinociceptive effects produced by the combination of morphine and desipramine increased; the 5-HT receptor antagonist ritanserin, which was dissolved in PEG 400, did not affect the antinociception compared with these respective controls (Table 2).
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Using the triple combination of morphine 0.1 µg, desipramine 0.3 µg, and serotonin 50 µg, antinociceptive effects similar to those shown in Table 1 were obtained when saline was injected intraperitoneally instead of an antagonist (Table 3). Naloxone, but not yohimbine, antagonized the antinociceptive effect. Again, the solvent PEG 400, injected instead of saline, enhanced the antinociception. Ritanserin reduced the antinociceptive effects when compared with these controls (Table 3).
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Discussion |
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The antinociceptive effects of IT injected drugs can be assessed by interference in mechanical and thermal algesic tests in the rat, whereby the response to norepinephrine was most pronounced in a mechanical test, the tail-pinch test, and that to morphine and serotonin was most pronounced in a thermal test, the tail-flick test (10). The present study in the thermal rat tail-flick model confirms the antinociceptive effects by activation of all three systems, either by direct receptor activation by morphine or serotonin or by the enhancement of endogenous extraneuronal norepinephrine levels by the specific reuptake inhibitor desipramine, herein demonstrated by direct injection at the spinal level. In a previous study, a dose of 30 µg of desipramine IT failed to evoke antinociception of its own in rats (11), whereas desipramine was fairly active in mice (12), possibly because of species differences. Our results with a nonlinear dose response suggest the existence of a bell-shaped dose-response curve, with the dose used previously being too large for efficacy. The doses used in the present study suggest a local action because desipramine does not evoke antinociception after 30 mg/kg administered subcutaneously in the rat tail-flick test (13) or 25 mg/kg intraperitoneally in the rat hot plate test (14); the doses of morphine used are orders of magnitude lower than those necessary for systemic administration, and serotonin, if absorbed into the systemic circulation, will not reenter the central nervous system.
The potentiation of spinal morphine antinociception by desipramine has been observed previously (13); however, desipramine was administered systemically; therefore, the sites of interaction were not unequivocally defined. In a later study, direct local activation of 2-adrenoceptors by a subanalgesic dose of clonidine potentiated local morphine antinociception (7). The antinociceptive effects of IT clonidine were attenuated by IT yohimbine, which confirms the involvement of 2-adrenoceptors in this effect (7). The antagonism by systemically administered yohimbine in the present study to the antinociceptive effects of the combination of subthreshold morphine and subthreshold desipramine argues in favor of the participation of enhanced extraneuronal norepinephrine, although IT yohimbine may also attenuate the effects of IT morphine (7). The failure of ritanserin to interfere with the effect of the combination demonstrates that the respective 5-HT receptors are not involved in the cascade after activation of µ-opioid and 2-adrenoceptors by morphine combined with desipramine.
The other dual combinations of subthreshold doses of morphine, desipramine, and serotonin—namely morphine plus serotonin or desipramine plus serotonin—produced only moderate antinociceptive effects. It has been suggested that serotonin causes antinociception by subsequent activation of an opioidergic link (15); therefore, the low level of activation produced by the small doses of morphine and serotonin at the same final target may not result in a prominent potentiation of the total effect. Potentiation of spinal serotonin antinociception by systemic desipramine has been reported previously in the rat hot plate test (14). This effect was more pronounced with a large (150 µg) compared with a small (75 µg) dose of serotonin. In conjunction with a further study (16), it was supposed that serotonin acts via an enhanced release of norepinephrine at the spinal level; we also observed an enhanced release of norepinephrine from spinal cord slices by a serotonergic agonist (17). The small potentiation seen in the present study is therefore probably due to the small doses of serotonin used, which may also apply to the lack of effect of the combination of morphine and serotonin.
An unexpected and previously unreported finding is the strong effect of the triple combination. In this combination, the doses of morphine and desipramine are extremely small (0.1 and 0.3 µg, respectively). The short-lasting antagonism to the antinociceptive effects by naloxone coincides with its short-lasting biological half-life, and its potency provides evidence for the crucial role of opioid-receptor activation for the overall effect. Antagonism by the purported 5-HT2 antagonist ritanserin may also point to a crucial role of 5-HT2–like receptor activation. The lack of complete antagonism by ritanserin is not clear because 5-HT2–like receptors have been claimed to be responsible for spinal antinociception produced by serotonin (18). However, the effects of serotonin on spinal norepinephrine release were supposed to be due to activation of either the "5-HT1C" or "5-HT1S" receptor subtypes (16), and effects are therefore not antagonized by ritanserin. Whether ritanserin antagonizes the direct receptor activation by serotonin or whether it acts further downstream cannot be determined because the serotonin antagonists methysergide and ketanserin also attenuate the antinociception produced by IT morphine (15). Given the pronounced antagonistic effect of yohimbine to the antinociception produced by the combination of morphine and desipramine, the complete lack of antagonism against the triple combination remains obscure and can only be subject to speculation. For instance, activation of serotonin receptors could release norepinephrine at sites more specific for antinociception and less accessible for yohimbine; however, a more pronounced effect of the dual combination of serotonin and morphine would then be expected. Further, serotonin could release norepinephrine at sites at which it can act on sites other than 2-adrenoceptors; again, a strong effect of the dual combination would be expected. These results with the antagonists give rise to the assumption that the triple combination induces a more complex interaction than that observed with the combination of just two substances. The precise nature of this phenomenon cannot be elucidated by the present study.
In conclusion, the results of this study confirm the production of pronounced spinal antinociceptive effects by combined µ-opioid and 2-receptor activation compared with the combined activation of µ-opioid and serotonin-receptors or serotonin and 2-receptors. Serotonin receptor activation, however, produces profound antinociceptive effects when added to a subthreshold dose of the combination of morphine with desipramine. This combination offers efficacy and avoids the use of large doses of either drug. Because of different modes of action, a wider spectrum of pain may be controlled compared with the use of single drugs.
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