Peripheral nerve injury may result in chronic neuropathic pain, which is characterized by spontaneous pain, hyperalgesia, and allodynia (1). Unilateral ligation of the L5 and L6 (fifth and sixth lumbar) spinal nerves produces signs that appear representative of neuropathic pain (2). Signs of mechanical allodynia were most evident in this model among several experimental animal models (3). Within a day or two, these animals displayed profound and long-lasting tactile allodynia (4). The spinal pharmacology of this nerve injury-induced allodynia is distinct from that associated with acute nociceptive input. Intrathecal administration of cholinesterase inhibitors has an antiallodynic effect in a dose-dependent manner, which is mediated by the spinal muscarinic receptor system (5). In addition, α2-adrenergic receptor agonists, γ aminobutyric acid (GABA) receptor agonists, and adenosine receptor agonists have been shown to have an antiallodynic effect at the level of the spinal cord (6–9). The efficacy of morphine in neuropathic states is controversial. Some authors have suggested that morphine is ineffective against neuropathic pain in both clinical (10) and animal studies (7,11–13), whereas others have found that opioids may alleviate neuropathic pain in humans (14,15). Although the synergistic antinociceptive interaction of spinal cholinesterase inhibitors with morphine has been demonstrated in normal rats (16), there are no studies about the antiallodynic interaction in this neuropathic model. The present study was therefore designed to investigate the drug interaction between intrathecal morphine and neostigmine in the rat model of neuropathic pain.
The following investigations were performed under a protocol approved by our animal care committee. Experiments were conducted in male Sprague Dawley rats (weight 160–180 g; Asan LSI, Seoul, Korea), which were housed individually in a temperature controlled (21 ± 1°C) vivarium and allowed to acclimate for 3 days in a 12/12-h light/dark cycle. For creating the neuropathic rat model, a surgical procedure was performed according to the method devised by Kim and Chung (2). Under halothane anesthesia (2% in 100% oxygen), a dorsal midline incision was made from L3 to S2 dermatomal area. By using a combination of sharp and blunt dissection, the left L6/S1 posterior interarticular process was exposed and resected to permit adequate visualization of the L6 transverse process. A partial excision of process was made, and the left L5 and L6 spinal nerves were gently isolated and ligated tightly with 6.0 black silk distal to the dorsal root ganglion and proximal to the formation of the sciatic nerve. Animals that could not flex the left hind limb postoperatively, indicating damage to the L4 nerve, were excluded. After a 7-day postoperative period, implantation of the intrathecal catheter was performed if the rat showed a withdrawal threshold of 4.0 g or less by postoperative Day 7. Such rats were defined as demonstrating the touch-evoked allodynia. For spinal drug administration, rats were chronically implanted with catheters as previously described (17). Under halothane anesthesia, the rats were placed in a stereotaxic head holder. The occipital muscles were separated from their attachment points and retracted caudally to expose the cisternal membrane at the base of the skull. Intrathecal catheters (PE-10 polyethylene tubing) were passed caudally from the cisterna magna to the T12-L1 spinal cord level of the lumbar enlargement (8.5 cm). The catheter was tunneled subcutaneously and externalized through the skin, and the end of the tip was plugged with a 28-gauge wire. Only animals with no evidence of neurologic deficit after catheter insertion were studied.
All the pharmacologic experiments were conducted 3 wk after spinal nerve ligation because tactile allodynia develops within 1 wk after surgery and lasts for 6–8 wk. At least 5–7 days of recovery were allowed before the animals were used in experiments. Rats that exhibited motor deficiency were not used. There was at least a 3-day interval between successive experiments with any rat after each intrathecal administration of drug. Each animal received a total of three injections.
For intrathecal administration, the drugs were given by using a microinjection syringe over a 60-s interval in a volume of 10 μL, followed by a 10-μL flush. When drug combinations were given, the intrathecal injections were concurrent, because the times of peak effect of intrathecal neostigmine and morphine coincided. For the evaluation of reversal of mechanical allodynia and the dose (ED50) estimated to produce 50% maximal possible effect (%MPE) of each drug, neostigmine bromide and morphine sulfate were administered intrathecally. The doses of 1, 3, 10, and 30 μg were injected for morphine and 0.3, 1, 3, and 10 μg for neostigmine. All drugs were dissolved in 0.9% sodium chloride solution. Animals were given a 5-day rest period between drug injections to minimize any possibility of tolerance developing.
Behavioral testing was performed during the day portion of the circadian cycle (9:00 AM to 5:00 PM). To undertake these measurements of tactile threshold, rats were placed in individual plastic cages with wire mesh bottoms. After 20 min, mechanical threshold was measured by applying a von Frey hair to the midplantar surface of the lesioned hind paw until a positive sign for pain behavior was elicited. According to the method described by Chaplan et al. (18), tactile stimulus producing a 50% likelihood of withdrawal was determined by using the up-down method. A series of eight calibrated fine von Frey filaments (0.40, 0.70, 1.20, 2.00, 3.63, 5.50, 8.50, and 15.1 g) were presented serially to the lesioned hind paw in ascending order of strength with sufficient force to cause slight bending against the paw and held for 6 s. Brisk withdrawal or paw flinching were considered as positive responses. Measurements were taken before and 15, 30, 45, 60, 90, and 120 min after the intrathecal administration of the drug. In the absence of a response at a pressure of 15 g, animals were assigned to this cutoff value. Side effects were simply assessed by observing the presence of sedation, urination, and motor weakness. After injection, general posture, and ambulation were noted. Severe sedation was defined as a significant decrease in spontaneous activity and a loss of the orienting response to light touch stimulation. To study the motor abnormality of hind limb function, the absence of placing and stepping reflex (flaccidity) was noted as an index of motor weakness. Motor function was evaluated by observing the placing/stepping reflexes, righting reflex, and normal ambulation.
The first series of experiments defined the dose-response curves of intrathecally administered morphine and neostigmine from the %MPE. The data are reported for the peak effect. The ED50 value was obtained for each drug. In the second series of experiments, ED50 values and fractions (1/2, 1/4, and 1/8) of ED50 of each drug were administered concurrently in an equal dose ratio to establish the ED50 of morphine-neostigmine combinations. The dose-response curve was also obtained.
To determine whether the antiallodynic interaction of morphine and neostigmine is additive or synergistic, an equal dose ratio isobolographic analysis was performed by using the method of Tallarida and Murray (19). The ED50 doses of the individual drugs given in combination were calculated from the dose ratio our study. The theoretical additive dose combination was calculated by the method described by Tallarida and Murray (19) and Tallarida et al. (20). The experimentally derived values were determined as described above. The theoretical additive point lies on a line connecting the ED50 values of the individual drugs. Experimental values that lie below and to the left of this additive line are considered to be synergistic, whereas values that lie above and to the right of the line demonstrate a antagonistic interaction.
To obtain a value for describing the magnitude of the interaction, a total fraction value was calculated. The ED50 values of the drug given alone were assigned the number 1. Then, total fraction =MATH 1MATH 2 Values near 1 indicate an additive interaction, and values less than 1 imply a synergistic interaction.
The peak effect observed in each rat after drug delivery was recorded. This peak drug effect was then used to calculate a %MPE, and these data were used to plot a %MPE versus log dose curve. Withdrawal latency data from von Frey hair testing were obtained as the actual threshold in grams and were converted to %MPE using the formula: %MPE for antiallodynia =MATH 3 Analysis of the dose-response curves and statistics were obtained by using the pharmacologic software programs of Tallarida and Murray (19) and included calculation of the ED50 values and their 95% confidence intervals (CIs).
Statistical analysis of drug interactions was conducted according to procedures of Tallarida and Murray (19). The variances for the individual drugs in each mixture were calculated from the variance of total dose. CIs for each point were calculated from the variances of each component alone. The difference between the theoretical additive point and the experimentally derived ED50 was compared by using the Student’s t-test. For experimental values that were lower than theoretical additive values, a P value < 0.05 for the differences in both the X and Y directions was interpreted as a significant synergistic interaction.
After the spinal nerve ligation, all rats displayed a significant decrease in the mechanical threshold (from 15 g of preoperative value to <4.0 g of the mean baseline withdrawal threshold) necessary to evoke a brisk withdrawal response in the injured hind paw in response to von Frey hair stimulation. In all groups, the mean (± SD) withdrawal thresholds were in the range of 2.05 (± 0.86) g to 3.07 (± 1.13) g at the baseline measurement. The %MPE was maximally increased within 15 to 30 min and then gradually decreased to the baseline level. A somewhat longer antiallodynic time course was observed after intrathecal injection of neostigmine (3 and 10 μg) and morphine (10 and 30 μg). Even with the dose of morphine 30 μg, mean %MPE was <65% at the peak time. Similar patterns of time course were shown in the morphine-neostigmine combination groups. Intrathecal administration of morphine and neostigmine alone and in combination resulted in a dose-dependent increase in antiallodynic effect (Fig. 1). The ED50 values, slopes, and 95% CIs for morphine, neostigmine, and combination are presented in Table 1. The experimentally determined mixture ED50 (CI) was 0.11 (0.08–0.16) μg for neostigmine and 1.52 (0.83–2.75) μg for morphine (P < 0.05). The theoretical additive ED50 (± SEM) was calculated to be 0.44 (± 0.08) μg for neostigmine and 6 (± 1.07) μg for morphine. According to the fractional method used here, fractions of each drug are both 0.13, and total fractional value less than 1 indicate a synergistic interaction (Table 2).
Some rats showed mild to moderate motor weakness and sedation and frequent urination, but there was no severe motor weakness or sedation in all rats. The incidence and magnitude of side effects were generally reduced when combined. A dose-related pattern of side effects was shown in the neostigmine and morphine (only sedation component) groups (Table 3). At antiallodynic doses used, none of the drugs obtunded the brisk hind paw withdrawal induced by the higher von Frey hair stimuli. After intrathecal injection of 10 μg of neostigmine, a moderate motor weakness was observed in only two rats and the residual effect lasted for more than 3 h.
There are two important observations in our study. First, the intrathecal administration of morphine and neostigmine produced a significant synergistic effect with regard to a spinally mediated mechanical allodynia. Second, an intrathecal injection of morphine produced a mild to moderate reduction of mechanical allodynia at the analgesic dose range. These data suggest that mechanical allodynia may be, in part, mediated by the spinal opioid system in this animal model.
Some investigators have suggested that morphine is ineffective against neuropathic pain in both clinical (10) and animal studies (7,11–13), whereas others, although in clinical studies, have reported that opioids may alleviate neuropathic pain, but all these studies have methodological limitations, and they measured only analgesic effects on neuropathic pain, not the mechanical allodynic component (14,15). Two studies demonstrated that morphine administered intraperitoneally or intracerebroventricularly was effective on the mechanical allodynia in a rat model with ligation of the L5-6 nerve roots, whereas intrathecal morphine was not (12,13). In our study, intrathecal morphine produced a mild to moderate reduction of mechanical allodynia at the analgesic dose range.
Contrary to our findings, some groups have reported that intrathecal morphine has little or no effect against mechanical hypersensitivity in this model (21). The exact mechanism is not known, but this discrepancy may be explained by several reasons. First, we measured the only tactile component of allodynia. Second, we used younger rats for the nerve ligation procedure (age, 6–7 weeks). According to our experience, the younger the rats, the more sensitive the response and the better the occurrence rate of allodynia once the touch-evoked allodynia develops after the spinal nerve ligation. Third, there may be some methodological differences, such as drugs used, skills of experimenter, subjective guideline for positive response, and test time in a behavioral study. Studies using an animal model of neuropathic pain involving the tight ligation of the L5-6 nerve roots have shown that intrathecally administered morphine lacks efficacy against mechanical allodynia (7,13), whereas α2 agonists, such as clonidine, are effective (7). Nicholas et al. (7) reported that intrathecal morphine did not alter allodynia at doses up to 100 μg and a high-efficacy μ-opioid agonist produced a significant, dose-related antiallodynic action. In addition, this antiallodynic efficacy of morphine was regulated by cholecystokinin in the same rat model of neuropathy. In contrast, morphine sulfate in doses of 90–150 μg evoked a prominent behavioral syndrome suggestive of a pain state (allodynia) (21). Clinical reports have suggested this phenomenon may also occur in humans after large doses of intrathecal morphine. 1 Because Aβ fibers are not known to contain opioid receptors, any observed opioid-mediated antiallodynic activity most likely occurs at postsynaptic sites. In addition, any observed antiallodynic action of spinal opioids would require either a high-efficacy opioid agonist or large doses of a lower-efficacy opioid, because the receptor pool available for spinal antiallodynic actions of opioids (i.e., postsynaptic opioid receptors) is only 40% of the total μ receptor population in the spinal dorsal horn (13).
We have previously shown that intrathecal neostigmine produced a dose-dependent antagonism on touch-evoked allodynia and that this antiallodynic action is likely mediated by the spinal muscarinic system, especially at the M1 receptor subtype (5). A previous study suggested that muscarinic cholinergic stimulation of spinal sites results in the activation of local lumbar cholinergic circuits that may modulate the local cholinergic transmission (4). It appears that acetylcholinesterase inhibitors could act by the acetylcholine released from these intrinsic spinal cholinergic terminals. In our previous study, some rats given neostigmine showed a dose-related pattern of motor weakness and other side effects such as salivation, tremor, and urination (5). In our study, some of the rats showed mild to moderate side effects; however, none showed severe motor weakness and sedation. We think that each test used here is a very gross test with no quantification and the side effects in animal studies are difficult to evaluate without blinded objective scoring and quantification. Therefore, we simply assessed the motor dysfunction by checking only the presence of each component. In Table 3, there are inadequate data to determine the degree of interaction of these drugs for side effects. Although the total number of rats showing sedation in the combination group (5 of 24) is less than that of morphine (7 of 24) or neostigmine (9 of 24), there may be a synergistic interaction in producing sedation. More worrisome is that the major side effect of neostigmine in humans, and a major side effect of morphine is nausea, which cannot be evaluated in the rat.
Naguib and Yaksh (16) reported that the antinociceptive effects of coadministered drugs at the spinal level were mediated by independent receptor systems and that there was a reduction in dose for either drug, suggesting synergistic interaction. It is possible, however, that the enhanced effect resulted from a decreased clearance, changes in agonist affinity, and functional interactions. Although the redistribution of the present drugs was not examined, we do not believe that these results reflect an altered clearance of either drug. In a similar study with α2 and μ agonists, clearance was not altered (23). In addition, there was no apparent increase in the duration of action in our study. If there is a functional receptor interaction, we would anticipate that the appearance of motor weakness and urination (with neostigmine) or sedation (with morphine) would have been similarly enhanced. Failure to observe such an enhancement likely would exclude a facilitation of the receptor interaction. Despite the above possible explanations, the exact mechanisms are not known.
In conclusion, we demonstrated that intrathecal morphine produced a mild to moderate antagonism on touch-evoked allodynia at the spinal level in a rat model of neuropathic pain and that the antiallodynic interaction between morphine and neostigmine, when coadministered, was synergistic.
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