Monash University Department of Anaesthesia, Monash Medical Centre, Level 5 Block E, Clayton, Victoria 3168, Australia*Corresponding author
Accepted for publication: December 21, 2001
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Abstract |
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Methods. Tail-flick latency and electric current threshold for nociception were measured in an acute pain model that allowed the study of the antinociceptive effects of intrathecally administered drugs that were due to actions of these drugs at spinal cord receptors. Experiments were performed in male Wistar rats with chronically implanted lumbar subarachnoid catheters. Doseresponse curves for spinally mediated antinociceptive effects of agonists selective for 5-HT receptor subtypes were constructed.
Results. The 5-HT1 agonist 1-(3-chlorophenyl)-piperazine dihydrochloride caused a dose-dependent antinociceptive effect, measured by both nociceptive tests. However, 8-hydroxy-DPAT (selective 5-HT1A agonist) produced antinociception assessed by electric current but not tail flick. A 5-HT1A-selective antagonist, 4-[3-(benzotriazol-1-yl)propyl]-1-(2-methoxyphenyl)-piperazine, reversed the antinociception in the electrical test produced by both of these agonists but the tail-flick latency effects after intrathecal 1-(3-chlorophenyl)-piperazine were not suppressed by this antagonist.
Conclusions. We conclude that 5-HT1A receptors in the spinal cord are involved in the nociceptive mechanisms assessed by noxious electrical stimuli. Other 5-HT1 receptors (non 5-HT1A receptors) are involved in the spinally mediated antinociception assessed by thermal noxious stimuli.
Br J Anaesth 2002; 88: 67984
Keywords: rat; serotonin (5-hydroxytryptamine); spinal cord; sensory block; pain
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Introduction |
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It has been shown that intrathecal injection of 5-HT in such a way that its actions are confined to the spinal cord causes antinociception, as revealed by the response to noxious electrical stimuli [electric current threshold (ECT) test] and by TFL.14 Methysergide reverses these antinociceptive effects, indicating the involvement of spinal cord 5-HT receptors. The investigators went on to show, in selective antagonist and cross-tolerance experiments, that the antinociception revealed by the noxious electrical stimulation paradigm (but not TFL) involved spinal cord µ opioid and GABAA receptors.15 16 Because different nociceptive testing paradigms were used, it is uncertain what 5-HT receptor subtypes were involved in the µ opioid and GABAA receptor interaction.
The purpose of the present study was to investigate the involvement of 5-HT1 receptor subtypes in the spinal cord control of nociception, assessed in the ECT and TFL paradigms. The response to noxious electrical stimulation and TFL were measured in a model that allowed the study of drug actions that were confined to the caudal segments of the spinal cord. Drugs selective for different 5-HT1 receptor subtypes were injected intrathecally into conscious rats with chronically implanted lumbar subarachnoid catheters. Antinociceptive effects were assessed with noxious heat and electric current. Antagonism of these effects was investigated by intrathecal injection of a selective 5-HT1A antagonist.
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Methods |
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Catheter implantation
Lumbar intrathecal catheters (dead space volume 35 µl) were implanted under halothane anaesthesia and aseptic surgical conditions as described previously.18 Portex catheters were introduced into the lumbar intrathecal space to lie next to the most caudal segments of the spinal cord, responsible for innervation of the tail and hind legs. The catheters were tunneled subcutaneously to an exit wound at the base of the neck. The exact volume of the catheter was measured before insertion, thus allowing accurate dosing of drugs injected through the catheter into the cerebrospinal fluid (CSF). Correct catheter placement was confirmed by injecting 2% lidocaine solution 10 µl down the catheter 10 min after recovery from general anaesthesia. The catheter was judged to be intrathecal if paralysis and dragging of the hind legs occurred within 30 s of this injection. All animals failing this test and those with neurological deficit after surgery were rejected from further study. The lidocaine test was also performed after each experiment to confirm that all drugs injected down the intrathecal catheters were injected into the lumbar intrathecal space. The rats were rested for 24 h before further experiments were performed. Only one experiment per day up to a maximum of five was performed on each animal.
Nociceptive tests
Two nociceptive tests were used in all experiments, the ECT test and the TFL test, as described previously.18 The electrical test allows demonstration of drug action at the level of the spinal cord after lumbar intrathecal injection, i.e. the measurement of an increase in nociceptive threshold in the tail with no change in the neck threshold measurements. Such a differential block proves that the effects of the drug are confined to the caudal segments of the spinal cord responsible for tail and hind limb innervation. A rise in TFL in the same experiment in which such a differential effect is demonstrated must also therefore be due to action of the drug on the caudal segments of the spinal cord. Both of these tests were applied in sequence to each rat every 5 min.
Electric current threshold test
Each rat was placed in a darkened restrainer and electrical stimulating electrodes were applied to the skin of the tail and neck. Brief bursts of electric current (0.5 s train, 2 ms pulse width, 50 Hz, 010 mA) were delivered in turn to each set of electrodes (tail first, 15 s before the neck) every 5 min. The minimum current necessary to evoke an obvious nociceptive response (a squeak or sharp withdrawal), defined by the updown method, was measured at each skin site every 5 min until three stable consecutive readings had been obtained. The intrathecal drug was then given and measurements continued at each skin site every 5 min for a further 4550 min. In order to standardize results obtained at each skin site, the response to intrathecal drug was calculated as a ratio with respect to the mean of the three stable readings taken before intrathecal drug injection, as described previously.18 These values for responses to each dose of drug were combined and expressed as the mean (SEM) on agonist doseresponse curves.
Tail-flick latency test
This was performed every 5 min immediately before the electrical measurement in the tail. The heat from a 150 watt projector bulb was directed onto the blackened tip of the tail and the power adjusted so that the rat flicked its tail away from the noxious stimulus in approximately 3 s. At this intensity of stimulation, three stable control (pre-drug injection) readings were obtained and then the test drug was injected. TFL measurement continued every 5 min thereafter for 3045 min. The response to each dose of drug was calculated as a percentage of maximum possible effect (MPE):
A cut-off time of 10 s was used. Values were combined for each drug dose, expressed as mean (SEM) and used to produce doseresponse curves.
Agonist doseresponse relationships
A range of doses (0.0055.0 µmol) of 1-(3-chlorophenyl)-piperazine dihydrochloride (non-selective agonist of 5-HT1)19 20 was given intrathecally to six rats with chronically implanted subarachnoid catheters. Sixteen experiments were performed (n=4 per dose of agonist). Similarly, a range of doses (0.010.5 µmol) of 8-hydroxy-2-dipropylaminotetralin hydrobromide (a 5-HT1A-selective agonist)21 22 was given intrathecally to 15 rats with chronically implanted subarachnoid catheters. Twenty-three experiments were performed (n=46 per dose of agonist). The drugs were dissolved in 6% dextrose solution to make the injectate hyperbaric compared with CSF. Doses were delivered into the lumbosacral CSF in 5 µl volumes while the rat was held at a 15° inclined plane, head up. This was done to restrict the spread of drug to the caudal segments of the spinal cord, as described previously.23 The dextrose solution has been shown not to affect nociceptive thresholds when given alone.18 Once-daily injections of agonist drugs were given on up to five occasions to individual rats. This has been shown in previous work on intrathecal 5-HT not to cause tolerance or tachyphylaxis.15 16
Antagonist doseresponse relationships
Doses of agonist drugs were chosen from their doseresponse curves for the experiments with antagonists. These were the roughly equieffective doses that produced a near-maximal response for tail ECT. This dose was 0.1 µmol for 8-hydroxy-DPAT hydrobromide and 0.5 µmol for 1-(3-chlorophenyl)-piperazine dihydrochloride. The agonist was given intrathecally alone at the beginning and at the end of a series of once-daily experiments in each rat. In the intervening days the same dose of the same agonist was given intrathecally combined with a range of doses (0.0010.05 µmol) of a 5-HT1A-selective antagonist [4-[3-(benzotriazol-1-yl)propyl]-1-(2-methoxyphenyl)-piperazine] dissolved in 6% dextrose. Twenty experiments were performed in nine rats using 8-hydroxy-DPAT hydrobromide (n=45 observations per dose of antagonist). Eleven rats were used in 27 experiments with 1-(3-chlorophenyl)-piperazine dihydrochloride (n=45 observations per dose of antagonist). Antinociceptive effects were measured using ECT in the tail and neck and TFL. The pre-series and post-series measurements of the antinociceptive effect for agonist alone in each rat were then averaged (R). The percentage suppression of agonist effect by each dose of antagonist was calculated using the following equation, described previously:18
where r is the individual response to agonist mixed with a particular dose of antagonist and R the mean response to agonist given intrathecally alone. The values of percentage suppression were calculated for both ECT and TFL tests. Values were combined to produce means and SEM, which were then plotted as doseresponse relationships for suppression of the antinociceptive effects of each agonist.
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Results |
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Discussion |
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The study reported in this paper concentrated on the restriction of drug actions to the spinal cord and the provision of support for such confinement. The experiments were performed in rats with positive lidocaine tests, and antinociceptive effects occurred only in the tail, not the neck. It has been shown previously in this model that intrathecally administered 5-HT caused its ECT antinociceptive effects by involvement of spinal cord GABA and µ opioid receptors but no such interactions occurred for the TFL effects.16 23 The results of the experiments reported in this paper extend these findings. Again, there was a difference in the mechanisms of antinociception revealed by the TFL and ECT tests. The ECT test reveals antinociception involving 5-HT1A receptors, whereas the TFL test involves other 5-HT1 (but not 5-HT1A) receptors.
One study in mice used the same 5-HT1A-selective agonist as that used in the present study (8-hydroxy-DPAT).11 The investigators concluded that 5-HT1A receptors in the spinal cord were responsible for TFL antinociception. This result is contrary to the findings of the experiments reported in this paper. This may represent a supraspinal effect of the drug. Many of these drugs are very soluble in water and thus spread easily throughout the CSF. The investigators made no measurements, such as a comparison of rostral and caudal dermatome nociceptive thresholds, that would indicate confinement of drug action to the caudal segments of the spinal cord.
The importance of distinguishing spinal and supraspinal effects was highlighted recently in a study of the involvement of 5-HT1A receptors in opioid-induced bulbospinal inhibition.13 It was shown that fentanyl injected into CSF in the brain inhibited suralgastrocnemius reflex responses. Intrathecal administration of the selective 5-HT1A receptor antagonist WAY-100635 significantly reduced this inhibition. In a separate group of experiments, i.v. fentanyl depressed the suralgastrocnemius reflex. This inhibition was not affected by intrathecal WAY-100635 (100 µg), but combined administration of the 5-HT1A antagonist with an 2 adrenoceptor antagonist (RX 821002) significantly reduced the effects of i.v. fentanyl. They concluded that the bulbospinal and direct spinal actions of fentanyl may occur together to produce overall inhibition of the reflex.13
Other studies have claimed interaction of serotonergic receptors with other neurotransmitter systems in the spinal cord. It was reported that a 5-HT3 receptor selective agonist (2-methyl serotonin) caused antinociceptive effects that involved spinal opioid and GABA receptor systems.27 When this drug was injected intracerebroventricularly no antinociception was observed. It was therefore concluded that the antinociception after intrathecal injection was a spinal cord effect. However, 2-methyl serotonin does have affinity for 5-HT1 receptors.28 Selectivity of drugs for 5-HT receptor subtypes is often not high. Frequently, pKi values for a drug binding to different receptor subtypes differ by one log unit or less.28 29 Thus, Giordano27 may have been describing the system described in this paperthe activation of 5-HT1A receptors in the spinal cord. However, more confidence may be placed in the findings of the present paper because the 5-HT1A-selective drugs that were used have pKi values for the 5-HT1A receptor subtype that are two or three log units greater than values for other receptors.28 29 Clearly, the involvement of 5-HT1 receptors other than 5-HT1A receptors and of 5-HT2 and 5-HT3 receptors needs to be investigated because of the many reports of antinociceptive effects after intrathecal drugs reported to be selective for these 5-HT receptor subtypes. Furthermore, more detailed doseresponse studies must await the arrival of more selective agonist and antagonist drugs that are also water-soluble, so that it will be possible to inject these drugs intrathecally and to study actions confined to the spinal cord. These issues are clearly important if there is to be development of a drug designed to target the spinal cord 5-HT receptors involved in antinociception. For example, an intrathecal drug selective for a 5-HT receptor subtype that is only involved in antinociception in the brain will work only if a high dose of the drug is given intrathecally and the drug subsequently spreads to the brain. Such an approach to therapy will inevitably lead to side-effects. We conclude that 5-HT1A receptors in the spinal cord are involved in nociceptive mechanisms that can be assessed by measuring responses to noxious electrical stimuli. Other 5-HT1 receptors (non-5-HT1A receptors) are involved in spinally mediated antinociception that can be assessed by measuring responses to thermal noxious stimuli.
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