Since the first description in rats of powerful antinociception following small doses of intrathecal morphine , many opioids have been given spinally to human beings for pain relief with good effect, although there is much debate about dose and drug disposition [2-9]. It is often assumed that the analgesic effects in human beings and antinociception in animals following intrathecal or epidural injections of opioids are due to the spinal cord actions of the drugs. Many studies have shown antinociception after spinal morphine in animals and these are the basis of clinical practice [1,10-14]. The model of intrathecal cannulation, used commonly in those experiments, was shown to deliver drugs selectively to the spinal cord when it was first described. However, it is still possible in individual experiments, or in a series of experiments, that the catheters could be misplaced or the drugs injected through them spread rostrally to the brain . Small concentrations of morphine reaching sites in the brain can cause antinociception [16,17]. It is therefore possible that even an intrathecally administered drug - given at the lumbosacral level - can cause antinociceptive effects by actions at a brain site if it spreads away from its spinal site of injection towards the brain. Unless suitable experimental controls are performed to assure the experimenter that drug action is confined to the spinal cord, erroneous conclusions may be drawn about the site of action of a drug and thus the involvement of spinal cord receptors. This can greatly influence the side-effect profile of a drug and also the conclusion on its suitability for spinal administration to human beings. Such controls for confinement of drug action to the spinal cord have to be in place in every experiment. This has not been the case hitherto, and thus in all of the above-cited work it is uncertain how much of the antinociceptive effects observed was due to actions of the drugs at the spinal cord level; the spinal cord is assumed to be the site of action because of the presumed location of the tip of the catheter and the known presence of opioid receptors in the spinal cord. This criticism of the animal work seems to be supported by clinical observation; it is well known that morphine frequently reaches the brain after lumbar intrathecal injection in human beings leading to sedation, respiratory depression, nausea and vomiting [18-23]. These side-effects cause clinicians to limit the dose administered and these dose limitations restrict the analgesia that can be achieved.
Although this is one of the most researched and published areas in pain medicine, one can conclude that there is still great uncertainty about the relative contribution of spinal and supraspinal sites to antinociception following spinal morphine injections, and this uncertainty has major clinical implications. This study investigated the spinal cord actions of morphine given intrathecally to rats in a model that allows definition of drug action at the spinal cord level. This model has been used previously to show that fentanyl, a selective μ-opioid agonist, can cause antinociception after intrathecal injection by actions at spinal cord μ-opioid receptors . The opioid receptors involved with intrathecal morphine antinociception assessed with the model were investigated by giving intrathecal antagonists selective for μ, κ and δ sub-types at doses that produced 100% suppression of spinally mediated antinociception caused by drug actions at those receptors.
This work was carried out with permission of the Monash University Standing Committee on Ethics in Animal Experimentation (SCEAE Project No. 93017). In all experiments, attention was paid to ethical guidelines for the investigation of experimental pain in conscious animals . Experiments were performed on male Wistar rats (weight: 180-200 g).
Seventy-six rats were anaesthetized with halothane in oxygen-enriched air (FiO2, 0.4). A Portex catheter (ID 0.28 mm, OD 0.61 mm; Portex Ltd, Hythe, Kent, UK) was implanted under aseptic surgical conditions into the lumbar subarachnoid space to lie adjacent to lower lumbar and sacral segments of the spinal cord as described previously .
After recovery from general anaesthesia, the animals were observed for normal movement and behaviour. If there was any evidence of neurological damage, they were killed immediately by an overdose of halothane. If there was no neurological damage, lidocaine solution (2% 10 μL) was injected through the catheter to test for correct intrathecal position; correct placement in the intrathecal space was assumed if the animal became paralysed in the hind limbs within 30 s of this local anaesthetic injection. This test was performed immediately after intrathecal cannulation and recovery from general anaesthesia, and also after each experiment. Thus, we were confident that all drugs injected through the catheter were introduced into the lumbar subarachnoid space. A minimum period of 12 h elapsed between catheter implantation and nociceptive testing.
Experiments were performed on 76 rats with chronically implanted lumbar subarachnoid catheters. The animals were placed in a restrainer that was covered to exclude distracting sights and sounds. Nociceptive thresholds were measured by electrical current (electrical current test (ECT) and the tail flick latency (TFL) test) as described previously [26,27].
Pairs of electrical stimulating electrodes were placed on the skin surface of the tail and as needles (25 G) in the skin at the base of the neck. Electrical current was passed through each pair in turn (50 Hz, 1 ms pulses and 0.5 s train). The minimum current necessary to cause the rat to squeak or make a strong aversive movement was defined by the up-down method for each skin site. The ECT was conducted at two skin sites to show spinal effects of an intrathecal drug, either agonist or antagonist (a change in the tail threshold with no change in the neck threshold) [26-28]. These changes were standardized as a ratio of control (pre-drug administration) as previously described (ECT response) [26-28]. The TFL test was expressed as a percentage of maximum possible effect (%MPE) with the cut-off time set at 15 s to avoid damage to the tail, with starting (pre-drug) thresholds set at approximately 3 s by adjustment of lamp intensity. Both ECT and TFL test were measured every 5 min and the order of tests was TFL test, tail ECT and then the neck ECT.
Forty rats were given morphine intrathecally (0.1-10 μg; David Bull Laboratories, Mulgrave, Victoria, Australia) to construct dose-response curves for the ECT and TFL test. Six to eight replicate experiments were performed at each dose. Thus each rat had one experiment performed per day for up to 4 days when morphine was given and nociceptive measurements made as above; each rat received up to four of the above doses of morphine on 4 successive days. As on previous occasions when using this model, there was concern that repeating the experiments and repetitive testing might alter the results obtained and thus their interpretation, e.g. by causing stress. Drawing pieces of paper, labelled with the dose to be given, out of a hat randomized the order of doses administered to individual rats. The responses to each dose measured in the experiments performed, as the first experiment in the series for each rat, were compared with the responses obtained for that dose in experiments performed on rats that had undergone previous experiments earlier in the week using the same dose. The morphine used for the intrathecal injection was dissolved in dextrose solution (6%) in order to make it hyperbaric compared with cerebrospinal fluid (CSF). The intrathecal injections were given with the restrained rat on an inclined plane (head up) at 45° to the horizontal plane. Thus the spread of morphine rostrally in the CSF was potentially limited by gravity. This solution of dextrose has been shown previously to have no effects on ECT or TFL test values when given alone .
Experiments using antagonists
The involvement of spinal cord μ-, κ- and δ-opioid receptors with the antinociceptive effects of intrathecal morphine was investigated in 36 rats with chronically implanted intrathecal catheters. The animals were placed in a restrainer and the protocol shown in Figure 1 followed. Nociceptive thresholds, using the ECT (neck and tail) and TFL test, were measured every 5 min until three consecutive stable readings had been obtained at each skin site. Morphine (1 μg intrathecally) was then given and the rats remained in the restrainer. This dose was chosen to be just at the top of the intrathecal dose-response curve. Antinociceptive effects were assessed in four groups of rats, before and after intraperitoneal naloxone (David Bull Laboratories, Mulgrave, Victoria, Australia; 2.0 mg kg−1, n = 9) or intrathecal injections of selective opioid receptor antagonists: β-funaltrexamine (β-FNA) 10 pmol (Sigma Chemical Company, St Louis, MO, USA; μ-selective antagonist, n = 9); naltrindole 10 nmol (Sigma Chemical Company, St Louis, MO, USA; δ-selective antagonist, n = 9); nor-binaltorphimine (nor-BNI) 0.062 pmol (nor-BNI, κ-selective antagonist, n = 9; Research Biochemicals International, Natick, MA, USA). These doses have been shown previously to antagonize completely and selectively the spinally mediated antinociceptive effects of intrathecal injections of μ-, κ- and δ-selective opioid agonist drugs and to have no effects on nociceptive thresholds when given alone; the volume of the antagonist that was injected intrathecally was 5 μL [24,29-31]. This was chosen so that spread of the drug in the CSF was minimized and its effect would therefore be restricted to the lumbosacral spinal cord; all antagonists were also dissolved in a slightly hyperbaric 6% dextrose solution for the same reason. To confirm this restriction of the drug effect, the ECT was measured at both skin sites every 5 min. A change in the tail ECT following intrathecal drug - without a change in the neck ECT - would indicate that the drug's action was confined to the caudal segments of the spinal cord responsible for tail innervation.
Values for ECT responses to intrathecal morphine were combined for each dose of drug, as were those for the TFL test at each dose. Mean ± SEM (n = 6-8) were calculated for responses to each dose and plotted as dose-response curves. For experiments involving antagonists (protocol Fig. 1), time-response relationships for the antinociceptive effects of intrathecal morphine 1 μg were plotted. This was achieved by standardizing each ECT or TFL test reading taken after morphine administration by dividing it by the mean of the three control readings (before intrathecal morphine injection) for that experiment. All readings from all the rats for each particular time point in the protocol were then combined and the mean ± SEM (n = 9) were calculated. A change in nociceptive thresholds after intrathecal antagonist was considered significant if P < 0.05 in a two-way ANOVA comparing values of nociceptive thresholds after antagonist with those before antagonist administration but after intrathecal morphine.
Positive lidocaine tests were obtained with each rat after intrathecal catheter implantation and after each experiment reported here. Thus all drugs injected through the catheters were delivered to the lumbosacral intrathecal space. There were no significant differences in either the baseline ECT values before drug administration or the responses to each dose of morphine. The latter were those measured in experiments performed as the first experiment in the series for each rat compared with the responses obtained for that dose in experiments performed on rats that had undergone previous experiments earlier in the week. Thus the experimental paradigm did not alter the drug responses.
Intrathecal morphine caused dose-related antinociceptive effects in ECT and TFL test (Fig. 2). The dose of intrathecal morphine 1 μg was just at the top of the dose-response relationship for that route of administration. However, there was no dose of intrathecal morphine that caused a rise in ECT in the tail without causing a concomitant rise in ECT at the neck.
Experiments using antagonists
Naloxone given intraperitoneally at high dose (2 mg kg−1) completely reversed the antinociceptive effects of intrathecal morphine (Fig. 3). The ECT rises in the neck and tail, as well as the TFL test effects, that occurred within 5 min of intrathecal morphine, were reversed to normal pre-morphine values by 10 min after naloxone.
In contrast, neither β-FNA 10 pmol (μ-selective opioid antagonist) nor naltrindole 10 nmol (δ-selective opioid antagonist) reversed the antinociception after intrathecal morphine assessed with ECT or TFL test (Figs. 4 and 5). However, the results with nor-BNI 0.062 pmol (nor-BNI, κ-selective opioid antagonist) were different. Intrathecal nor-BNI reversed completely the antinociception assessed by ECT in the tail (P < 0.05 two-way ANOVA; Fig. 6). This occurred without any change of the neck ECT, thus indicating that the nor-BNI was confined in its action to the caudal segments of spinal cord responsible for tail innervation. TFL values were unaffected by intrathecal nor-BNI.
While many reports may be found in the literature reporting spinally mediated antinociceptive effects of opioid agonists, few have demonstrated that the antinociception achieved was due to an action of the drug confined to the spinal cord and excluding supra-spinal effects. The measurement using the ECT allows demonstration of changes in the tail ECT with no change in the neck attributable to a spinal cord action of a drug [32-34]. Antinociception revealed by a rise in TFL test must be due to a spinal cord effect of a drug given intrathecally if the tail ECT rises with no change in the neck. Such a result has been achieved readily with intrathecal injections of the μ-receptor selective opioid agonist fentanyl [24,28]. The TFL test is the most common and well-established technique for revealing the antinociceptive effects of opioids, such as morphine. It is a spinal reflex, since it is preserved in animals with spinal cord transection . However, caution must be used when using this test in animals with an intact neuraxis, since it is known that drugs like morphine do reach brain structures after lumbar intrathecal injection [18-23]; furthermore, the TFL test may be increased by the action of intrathecal drugs on the brain . Thus it is important to include appropriate controls in behavioural experiments to show that a spinally administered drug is confined in its effects to the spinal cord site of injection.
If such controls are not performed, incorrect conclusions may be made about the spinal cord being the site of drug action and also about the types of spinal cord receptors involved. In the experiments reported here, there was no dose of intrathecal morphine that caused a rise in the ECT in the tail without causing a concomitant rise in the ECT at the neck. This occurred in spite of the small volume injected intrathecally and the use of a hyperbaric solution with an inclined plane to restrict the spread of the drug. Thus it was not possible to be sure that the antinociception caused by intrathecal morphine was due to a spinal cord action of the drug; it had clearly spread away from the lumbosacral site of drug delivery - at least to the cervical spinal cord and therefore possibly to centres in the brain. The first of the experiments with intrathecal morphine reported in this paper used the same rat preparation with an implanted intrathecal catheter that has been used before to show that intrathecal fentanyl, which is a selective μ-opioid agonist, does cause a rise in TFL test by an action at the level of the spinal cord . Smaller doses of intrathecal morphine did cause a rise in TFL test without a rise in the ECT in the neck. From that observation, one might think that rises in the TFL test at those doses might be due to a spinal cord effect; however, the ECT was less sensitive and could not show a rise at the tail or the neck. However, subsequent experiments with intrathecal antagonists did not reverse that rise even though those antagonists were used at doses that were shown previously to block their respective spinal cord receptors sufficiently to cause 100% reversal of spinally mediated antinociception by μ-, κ- and δ-selective opioid agonists [29-31].
It is clear that the effects of intrathecal morphine, judged from the ECT and TFL test, were mediated by opioid receptors, since all the effects were suppressed by intraperitoneal naloxone. The ECT effects were unaffected by intrathecal injections of β-FNA or naltrindole. However, nor-BNI, the κ-selective opioid antagonist, reversed the ECT antinociceptive effects of intrathecal morphine only in the tail. The neck ECT thresholds were unaffected by nor-BNI, indicating that this drug was combining with spinal cord κ-opioid receptors that were responsible for the ECT antinociceptive effects of intrathecal morphine.
More recent studies have examined antinociception caused by morphine when the concentrations of opioid receptors in the spinal cord have been reduced. For example, morphine antinociception was reduced by the induction of neuropathic pain by peripheral nerve or spinal cord damage [35,36]. A correlation was observed between decreased opioid antinociception and μ-opioid receptor concentration in the spinal cord. However, this cannot be used as an argument for causation, since other receptor sub-types are also reduced in these models. Gene knockout studies, in which the μ-opioid receptor 1 (MOR1) has been removed by genetic manipulation [37-42], have shown that the antinociceptive effects of μ-opioids were reduced. However, when the opioids were injected intrathecally in the knockout animals, no confirmation was given that the drug actions were confined to the spinal cord. The reduction in efficacy in TFL and hot plate nociception could have been due to lack of target μ-receptors in the brain.
Clearly opioids can target spinal cord μ-opioid receptors to cause TFL test antinociceptive effects; the experiments reported previously with fentanyl - using the same preparation, with the ECT in combination with TFL test measurements - attest to this fact and also to the fact that this preparation is capable of demonstrating such effects. However, fentanyl is much more lipid soluble than morphine; it is more likely to bind to spinal cord tissue and to remain there compared with morphine. Morphine is more water soluble and easily spreads to the brain, even after a low lumbar intrathecal injection. Thus, it is possible that morphine, which is known to bind with μ-opioid receptors, does so simultaneously in the spinal cord and the brain after the drug has been given intrathecally. This would explain why a selective antagonist for μ-opioid receptors, when confined to the spinal cord, could not reverse the TFL antinociceptive effect following intrathecal morphine.
We conclude that the antinociceptive effects following intrathecal and intraperitoneal morphine involve spinal and supraspinal opioid receptors. Antinociception revealed by the ECT is caused by morphine acting at spinal cord κ-opioid receptors. However, the tail flick effect frequently described in experiments with morphine administration to rats, with an intact neuraxis, involves actions at opioid receptors in the brain that override any action that may be caused by combination of morphine with μ-opioid receptors in the spinal cord. Thus, when using an opioid spinally in human beings on the basis of animal experimental work, one must be aware that resultant analgesic effects can be caused by drug action in the brain as well as the spinal cord. This rostral spread will be accompanied by side-effects also due to brain actions of the drug; side-effects that one was seeking to avoid by the use of a spinal route of administration.
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