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Abstract |
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Nerve ligation injury may produce a pain syndrome that includes tactile allodynia. Reversal effects on tactile allodynia have been demonstrated after the intrathecal administration of
-aminobutyric acid (GABA) receptor agonists or cholinesterase inhibitors in rats. We examined the drug interactions between neostigmine and muscimol or baclofen in a rat model of nerve ligation injury. Rats were prepared with tight ligation of the left L5-6 spinal nerves and chronic intrathecal catheter implantation. Tactile allodynia was measured by applying von Frey filaments ipsilateral to the lesioned hindpaw. Thresholds for paw withdrawal were assessed. Neostigmine (0.3–10 µg), muscimol (0.1–10 µg), and baclofen (0.1–3.0 µg) were administered to obtain the dose-response curve and the 50% effective dose (ED50). Fractions of ED50 values were administered intrathecally to establish the ED50s of drug combinations (neostigmine-muscimol and neostigmine-baclofen). The drug interactions were performed. Intrathecal neostigmine, muscimol, baclofen, and their combinations produced a dose-dependent increase in withdrawal threshold of the lesioned hindpaw. Both analyses revealed a synergistic interaction for the neostigmine-muscimol combination, whereas the effect of the neostigmine-baclofen combination was additive. These results suggest that the activation of both muscarinic and GABAA receptors is required for synergistic interaction. IMPLICATIONS: This study indicates that drug interaction is synergistic for the neostigmine-muscimol combination, whereas the effect of the neostigmine-baclofen combination is additive. In a rat model of nerve ligation injury, neostigmine, muscimol, baclofen, and their combinations provide an antagonism on touch-evoked allodynia at the spinal level.
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Introduction |
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Peripheral nerve injury may result in a condition of extreme cutaneous sensitivity to normally innocuous mechanical stimuli, designated tactile allodynia. Unilateral ligation of L5 and L6 (fifth and sixth lumbar) spinal nerves produces some signs that seem representative of neuropathic pain (1,2). Tactile stimulus has more clinical significance than thermal stimulus, especially in some patients with a symptom of allodynia. Within a day or two, nerve injury-induced animals display profound and long-lasting tactile allodynia. Signs of tactile allodynia were most evident in the nerve ligation model among several experimental animal models (3). The spinal pharmacology at this nerve ligation-induced allodynia is distinct from that associated with acute nociceptive input. In general, cholinesterase inhibitors,
-2 adrenergic receptor agonists, N-methyl-D-aspartate receptor antagonists, -aminobutyric acid (GABA) receptor agonists, and adenosine receptor agonists have an antiallodynic effect at the level of the spinal cord after intrathecal administration to rats with nerve ligation injury (4–9). The intrathecal administration of neostigmine, muscimol, and baclofen has an antiallodynic effect in a dose-dependent manner, which is mediated by the spinal muscarinic and GABA receptor systems, respectively (4,7,9). Although the synergistic antiallodynic interaction of spinal neostigmine with morphine has been demonstrated in a neuropathic pain rat model (10), there have been no studies about the antiallodynic interaction between neostigmine and GABA receptor agonists in the spinal nerve ligation model. The present study was therefore designed to investigate the possible drug interactions by using fractional and isobolographic analyses between intrathecal neostigmine and muscimol or baclofen in a rat model of neuropathic pain. |
Methods |
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The following investigations were performed under a protocol approved by our animal care committee. The experiments were conducted in male Sprague-Dawley rats (weight 200–300 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 (1). Under halothane anesthesia, 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. After a 7-day postoperative period, implantation of the intrathecal catheter was performed as previously described (11) if the rat showed a withdrawal threshold of 
4.0 g by postoperative Day 7.
Under halothane anesthesia, the rats were placed in a stereotaxic head holder. Intrathecal polyethylene tubings were passed caudally from the cisterna magna to the spinal cord level of lumbar enlargement. The correct placement of catheter was verified by the injection of 2% lidocaine 20 µL before testing. Only animals with no evidence of neurologic deficit after the operation were studied. All of the pharmacologic experiments were conducted at least 2 wk after spinal nerve ligation because tactile allodynia develops within 1 wk after surgery and it lasts for 6 to 8 wk. For intrathecal administration, the drugs were given by using a Hamilton microinjection syringe over a 60-s interval in a volume of 10 or 20 µL (in combination groups), followed by a 10-µL flush. The experimenters were blinded to the drug. For the determination of time to peak effect and the dose (ED50) estimated to produce 50% maximal possible effect (MPE) for each drug, neostigmine (Research Biochemicals International [RBI], Natick, MA), muscimol (RBI), and baclofen (RBI) were administered intrathecally. The doses of 0.3, 1, 3, and 10 µg were injected for neostigmine, 0.1, 0.3, 1, 3, and 10 µg for muscimol, and 0.1, 0.3, 1, and 3 µg for baclofen, respectively. When the drug combinations were given, the intrathecal injections were concurrent, because the times of the peak effect of intrathecal neostigmine, muscimol, and baclofen coincided. For the evaluation of antagonistic effect in each group, muscarinic antagonist pirenzepine (RBI) 3 µg was administered 10 min after injections of each drug and their equipotent combination. All drugs were dissolved in 0.9% sodium chloride solution. The rats were randomized by crossing-over a new drug protocol when receiving multiple drug injections. There was at least a 5-day interval between drug injections of successive experiments to minimize any possibility of tolerance development and to eliminate the residual effects of a drug. Each animal received a total of three injections.
Behavioral testing was performed during the day portion of the circadian rhythm (9:00 AM to 3:00 PM). To undertake these measurements of a tactile threshold, the rats were placed in an individual plastic cage with a wire mesh bottom. After 20 min, tactile threshold was measured by applying a von Frey hair to the midplantar surface of the hindpaw ipsilateral to the nerve injury until a positive sign for pain behavior was elicited (12). In the absence of a response at 15-g pressure, the animals were assigned to this cutoff value. According to the method described by Chaplan et al. (12), the tactile stimulus producing a 50% likelihood of withdrawal was determined by using the up-down method. Measurements were taken before and 15, 30, 45, 60, 90, 120, and 180 min after an intrathecal dose of the drug(s). Side effects were simply assessed by observing the presence of sedation, urination, and motor weakness. Severe sedation was defined as a significant decrease in spontaneous activity and a loss of the orienting response to the light touch stimulation. Motor weakness was evaluated by observing the righting and placing/stepping reflexes, abnormal weight bearing, and abnormal ambulation.
The first series of experiments defined the dose-response curves of intrathecally administered neostigmine, muscimol, baclofen, and their combinations from the mean %MPE. In the second series of experiments, ED50 fractions of each drug were administered concurrently to establish the ED50 of neostigmine-muscimol and neostigmine-baclofen combinations. Thereafter, drug interactions between two drugs were assessed isobolographically. In the third series of experiments, to investigate a possible mechanism of the spinal interaction between neostigmine and muscimol or baclofen in the presence of blockade of the spinal cholinergic muscarinic system, muscarinic antagonist pirenzepine 3 µg was delivered intrathecally 10 min after injection of each drug or combination of two drugs. The maximal decrease from the peak effect for each group was assessed and compared to peak %MPE in the same group.
To determine whether the drug interactions between neostigmine and GABA agonists are additive or synergistic, an equal dose ratio isobolographic analysis was performed by using the method of Tallarida and Murray (13). Individual ED50 values for each agonist were resolved from the combination dose required to cause 50% MPE and were plotted on the isobologram as the experimental combination dose. ED50 was defined separately for each drug. Fractions (1/2, 1/4, 1/8, and 1/16) of the ED50 of each drug were then administered concurrently, and the ED50 of the total dose of the mixture (neostigmine-muscimol and neostigmine-baclofen combination) was determined. The theoretical additive dose combination was calculated by the method described by Tallarida et al. (13,14). Experimental values were compared with theoretical additive values as defined by the theoretical additive line. 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 an antagonistic interaction. To obtain a value for describing the nature and the magnitude of drug interaction, total fraction values (see Table 1) were calculated.
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The ED50 values of the drug given alone were assigned the number 1. Then, for example, total fraction = (ED50 dose of neostigmine in combination/ED50 value for neostigmine alone) + (ED50 dose of muscimol in combination/ED50 value for muscimol alone). Values near 1 indicate an additive interaction, and values <1 imply a synergistic interaction (15). The peak drug effect was used to calculate a %MPE, and these data were used to plot a %MPE versus log dose curve. Withdrawal threshold data from von Frey hair testing were obtained as the actual threshold in grams and were converted to %MPE by using the formula: %MPE for antiallodynia = ([postdrug threshold - baseline threshold]/[15 g - baseline threshold]) x 100, where postdrug threshold = the largest threshold observed after intrathecal injection. The cutoff value was defined as a stimulus intensity of 15 g for the tactile threshold (i.e., %MPE = 100).
Statistical analysis of drug interactions was conducted (13). Dose response data were graphically presented as mean %MPE ± SEM for the peak effects. The ED50 values, slopes, and 95% confidence intervals (CIs) were assessed using a dose-response analysis. Variances and their 95% CIs for the theoretical ED50 may also be calculated from the variances of each component administered alone (14). The difference between the theoretical additive value and the experimentally derived ED50 value was compared by using a Student’s t-test. For experimental values that were less 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. The smallest antagonistic effect was compared with the peak agonistic effect for each group by paired t-test. A P value < 0.05 was considered to be statistically significant.
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Results |
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Normal rats all showed withdrawal threshold values of >5 g. After spinal nerve ligation, most rats displayed significant decreases in the mechanical threshold (from 15 g of preoperative values to a mean baseline of <4.0 g) but showed normal general behavior and weight gain. After catheter implantation in the animals with nerve ligations, the mean ± SE withdrawal thresholds for evoking hindpaw were in the range of 1.99 ± 0.17 g to 3.69 ± 0.5 g at the baseline measurement in all groups. All agonists tested produced a dose-dependent antiallodynia in the von Frey hair test. The time-effect courses as a function of the intrathecal doses of three agonists were similar in general. The maximal effects occurred within 30 min for all doses tested in each group. A similar pattern of time-effect course was shown in two combination groups (graphs not shown). Although not indicated here, there were a dose-dependent increase in the magnitude and a dose-dependent prolongation in the duration of the effect. The intrathecal administration of neostigmine, muscimol, baclofen, and their combination resulted in a dose-dependent antiallodynic effect, as illustrated in Figure 1. Although not closely paralleled, the slopes of the combination groups were shifted to the left side compared with that of each agonist.
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The ED50 values and slopes (95% CIs) are as follows: 0.3 µg (0.1–0.8) and 36.4 µg (21.1–51.7) for neostigmine (n = 20); 0.25 µg (0.1–0.6) and 33.8 µg (19.6–48.0) for muscimol (n = 30); 0.1 µg (0.03–0.4) and 36.3 µg (16.4–56.2) for baclofen (n = 25); 0.04 µg (0.02–0.08) and 50 µg (22.7–77.2) for neostigmine-muscimol combination (n = 40); and 0.17 µg (0.06–0.52) and 30.1 µg (-13–76.4) for neostigmine-baclofen combination (n = 20), respectively.
However, the dose-response curves did not deviate significantly from parallelism, thus suggesting a common final mechanism of action for antiallodynic activity.
As shown in Figure 2, a synergistic effect was found in the neostigmine-muscimol combination, whereas only additive interaction was observed in the neostigmine-baclofen combination. The experimentally determined neostigmine-muscimol mixture ED50 (±SEM) was 0.02 µg (±0.008) for neostigmine and 0.016 µg (±0.007) for muscimol. The theoretical additive ED50 was calculated to be 0.15 µg (±0.06) for neostigmine and 0.13 µg (±0.05) for muscimol. The experimental value of neostigmine-muscimol combination was significantly more than the calculated additive value (P < 0.05). Likewise, the experimentally determined neostigmine-baclofen mixture ED50 (±SEM) was 0.13 µg (±0.08) for neostigmine and 0.04 µg (±0.027) for baclofen. The theoretical additive ED50 was calculated to be 0.15 µg (±0.22) for neostigmine and 0.05 µg (±0.07) for baclofen. A graphic illustration on the isobologram shows that the standard errors of these two points do overlap, which means there is additive interaction. According to the fractional method used here, total fractions were 0.14 for the neostigmine-muscimol combination and 0.83 for the neostigmine-baclofen combination, respectively (Table 1).
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Posttreatment with pirenzepine remarkably reduced the maximally increased withdrawal thresholds produced by neostigmine alone and neostigmine with GABA agonists (Fig. 3). Over the first half-hour, the agonists (alone or in combination) produced peak effects. Thirty minutes later, however, antagonism by pirenzepine produced a significant reversal effect. In rats posttreated with pirenzepine, leading to a partial blockade of the action of spinal cord acetylcholine, either muscimol or baclofen alone was ineffective in altering the paw withdrawal response. The magnitude of reduction from the peak effect of each drug or combinations was significantly greater in three groups of neostigmine alone and neostigmine with GABA agonists (P < 0.01), but not in those of muscimol and baclofen.
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All doses of neostigmine, muscimol, and baclofen produced antiallodynic responses without any severe motor weakness. Some rats showed a mild-to-moderate motor weakness, sedation, and frequent urination in all groups, but no severe motor weakness or sedation was observed in all rats. The incidence and magnitude of side effects were considerably reduced after several doses in two combination groups (Table 2). Even after the posttreatment with pirenzepine, side effects did not return to the previous level of posttreatment. After intrathecal injections of either 10 µg of neostigmine and muscimol or 3 µg of baclofen, a moderate motor weakness was observed in five rats (three in the Neostigmine group, one in the Muscimol group, and one in the Baclofen group). No other adverse effects were noted.
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Discussion |
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There are two important observations in this study. First, the intrathecal coadministration of neostigmine and muscimol produced a significant synergistic effect with regard to a spinally mediated tactile allodynia. Second, GABAA receptor agonist was more efficient in reducing tactile allodynia than GABAB receptor agonist when coadministered with intrathecal neostigmine. These results suggest that the activation of a combination of muscarinic and GABAA receptors was required for the synergistic interaction.
Previous observations indicate that intrathecal baclofen and muscimol antagonize the allodynic response to tactile stimuli without a marked motor dysfunction in a dose-dependent manner. The selective antagonism confirms that the antiallodynic effects are mediated by spinal GABAA and GABAB receptor subtypes (7).
Baclofen is a potent GABAmimetic. It is selective for the GABAB receptor and is inactive at the GABAA receptor, whereas muscimol is a potent GABAmimetic selective for the GABAA subtype. The GABAA receptor is a part of the chloride ionophore complex. In the spinal cord, the GABA-synthesizing enzyme or GABA itself is present in large concentrations in the superficial dorsal horn, and this location is presynaptic in relation to primary afferent terminals. Neuropathic pain is characterized by the fact that it may be activated by low threshold mechanical input, which is transmitted by A-ß fibers to produce allodynia. Most GABAergic neurons are interneurons, with short axons and local connections. Peripheral nerve injury may induce a down-regulation of inhibitory transmitters and receptors; thus, the administration of GABA agonists may reverse a tactile allodynia induced by spinal nerve ligation (16). The distribution of the two receptor subtypes is quite different. GABAB receptors are located both presynaptically on nerve terminals and postsynaptically in the dorsal horn of the spinal cord. In the spinal cord dorsal horn, 50% of GABAB binding sites are associated with small-diameter primary afferent fibers (17). Activation of a GABAA receptor leads to an increase in chloride conductance and subsequent hyperpolarization of the postsynaptic membrane. There exists the possibility for a large number of interactions with other drugs that may alter intracellular cyclic AMP levels, such as opioids (µ and 
), 2-adrenoceptors, ß-adrenoceptors, GABAB, muscarinic, and cholinergic receptors (18).
The ligand-gated chloride channels, including those of GABAA and glycine receptors, are common in dorsal root ganglia and dorsal horn neurons (19). At spinal levels, GABAA receptors on primary afferent terminals gate a chloride channel that allows efflux of chloride (20), with a net effect, therefore, of depolarizing primary afferent terminals.
In our study, there was a significant difference between muscimol and baclofen in a drug interaction with neostigmine. Neostigmine affects on the surface receptors linked to G-proteins by a muscarinic receptor, whereas baclofen activates GABAB receptor through similar action of second messenger systems. Both GABAB and muscarinic receptors inhibit pain signaling via second messenger systems. Therefore, the activation of any of these receptors increases the potassium conductance of the membrane and inhibits the neurons through hyperpolarization (21). In contrast, the GABAA receptor increases chloride conductance (20). Consequently, two different effector systems are involved in the action of neostigmine-muscimol combination. This might explain, at least in part, such difference (synergistic versus additive) in two combination groups. Synergy is defined by the points of the isobole and the 95% CI occurring well below the line of additivity. Overlapping of the confidence intervals with the additive line indicates additivity (22). In this current study, the interaction between neostigmine and baclofen was additive inasmuch as the isobole for the combinations remained within the zone of additivity defined by the upper and lower CIs of the ED50 for each drug alone.
After peripheral nerve injury, a low-intensity mechanical stimulus can evoke prominent pain behavior, mediated by the activation of low-threshold mechanoreceptors subserved by large, myelinated afferent fibers (23). A previous study suggested that muscarinic stimulation of critical spinal sites resulted in the activation of local lumbar cholinergic circuits that might modulate the local transmission of the afferent allodynic information (4). There is considerable evidence that the muscarinic cholinergic system of the lumbar spinal cord is intrinsic (24,25) and the cholinergic innervation of the intermediolateral cell column is from a source intrinsic to the spinal cord (25). These spinal cholinergic terminals are presynaptic to primary afferents.
We hypothesized that antiallodynic effects of the coadministered drugs at the spinal level were mediated by the independent receptor systems and 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 redistribution of the present drugs was not examined, we do not believe that these results reflect on the altered clearance of either drug because there was no apparent increase in the duration of action in the combination groups. With regard to changes in agonist affinity, Kenakin (26) described that increases in slopes might reflect increased efficacy. In our experiments, the slope was increased in the neostigmine-muscimol combination group and was shifted to the left (Fig. 1), which explains a synergistic interaction. If there were a functional receptor interaction, we would anticipate that the appearance of motor weakness and urination or sedation would have been similarly enhanced. Although there is a synergism in the allodynic component, failure to observe such enhancement likely excludes a facilitation of the receptor interaction (Table 2). However, because the receptors of the sensory (especially pain) component are mainly located in the dorsal horn of the spinal cord, and because the GABAA receptor may interact with muscarinic or cholinergic receptors (18), the possibility of a functional receptor interaction in our experiments is probably increased. Despite the above possible explanations, the exact mechanisms are not yet known.
To investigate the reversal effect of each group on the tactile allodynia, we performed antagonistic studies in the blockade of the spinal muscarinic system in all groups. In our experiment, we chose a pirenzepine because the antagonist effect was most effective in the reversal of tactile allodynia in the spinal nerve ligation injury model (7). Posttreatment with pirenzepine remarkably reduced the maximal effect of intrathecal neostigmine and combinations with GABA agonists, whereas it could not reduce those of intrathecal baclofen and muscimol alone. These findings may suggest that the spinal cholinergic system is necessary for the optimal function of the spinal GABAergic system, especially the GABAA receptor, in inducing a synergistic effect.
Although not systematically quantified, intrathecal neostigmine or GABA agonists resulted in a dose-dependent reduction in spontaneous activity. In our study, some of the rats showed a moderate degree of side effects, but none showed severe motor weakness and sedation. In Table 2, it is not easy to determine the degree of interaction of these drugs for side effects. In some patients with neuropathic pain, especially involving a symptom of tactile allodynia, intrathecal administration of this combination could be helpful and valuable in some cases, and might be another or an adjuvant method of treatment in a malignant pain syndrome; but further study is needed before assuring clinical safety.
In conclusion, intrathecal neostigmine, muscimol, and baclofen alone produced a dose-dependent antiallodynia without any severe side effect. Intrathecal neostigmine produced a synergistic interaction with muscimol, but not with baclofen, in a rat model of nerve ligation injury. These results suggest that the activation of a combination of muscarinic and GABAA receptors is required for the synergistic interaction of tactile allodynia.
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Acknowledgments |
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This work was supported in part by Grant 99-153 from Asan Life Science Institute, Seoul, Korea.
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Footnotes |
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Presented in part at the annual meeting for the American Society of Anesthesiologists, Dallas, TX, October 11, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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- Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992; 50: 355–63.[Web of Science][Medline]
- Sheen K, Chung JM. Signs of neuropathic pain depend on signals from injured nerve fibers in a rat model. Brain Res 1993; 610: 62–8.[Web of Science][Medline]
- Kim KJ, Yoon YW, Chung JM. Comparison of three rodent neuropathic pain models. Exp Brain Res 1997; 113: 200–6.[Web of Science][Medline]
- Hwang JH, Hwang KS, Leem JK, et al. The antiallodynic effects of intrathecal cholinesterase inhibitors in a rat model of neuropathic pain. Anesthesiology 1999; 90: 492–9.[Web of Science][Medline]
-
Yaksh TL, Pogrel JW, Lee YW, Chaplan SR. Reversal of nerve-ligation induced allodynia by spinal alpha-2 adrenoceptor agonists. J Pharmacol Exp Ther 1995; 272: 207–14.
[Abstract/Free Full Text] -
Chaplan SR, Malmberg AB, Yaksh TL. Efficacy of spinal NMDA receptor antagonism in formalin hyperalgesia and nerve injury evoked allodynia in the rat. J Pharmacol Exp Ther 1997; 280: 829–38.
[Abstract/Free Full Text] - Hwang JH, Yaksh TL. The effect of spinal GABA receptor agonists on tactile allodynia in a surgically induced neuropathic pain model in the rat. Pain 1997; 70: 15–22.[Web of Science][Medline]
-
Lee YW, Yaksh TL. Pharmacology of the spinal adenosine receptor which mediates the antiallodynic action of intrathecal adenosine agonists. J Pharmacol Exp Ther 1996; 277: 1642–8.
[Abstract/Free Full Text] - Lavand’homme PM, Pan HL, Eisenach JC. Intrathecal neostigmine, but not sympathectomy, relieves mechanical allodynia in a rat model of neuropathic pain. Anesthesiology 1998; 89: 493–9.[Web of Science][Medline]
-
Hwang JH, Hwang KS, Choi Y, et al. An analysis of drug interaction between morphine and neostigmine in rats with nerve-ligation injury. Anesth Analg 2000; 90: 421–6.
[Abstract/Free Full Text] - Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976; 17: 1031–6.[Medline]
- Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 1994; 53: 55–63.[Web of Science][Medline]
- Tallarida RJ, Murray RB. Manual of pharmacologic calculations with computer programs. 2nd ed. New York: Springer-Verlag, 1987.
- Tallarida RJ, Porreca F, Cowan A. Statistical analysis of drug-drug and site-site interactions with isobolograms. Life Sci 1989; 45: 947–61.[Web of Science][Medline]
- Berenbaum MC. Synergy, additivism, and antagonism in immunosuppression: a critical review. Clin Exp Immunol 1977; 28: 1–18.[Web of Science][Medline]
- Woolf CJ. Molecular signals responsible for the reorganization of the synaptic circuitry of the dorsal horn after peripheral nerve injury: the mechanisms of tactile allodynia. In: Borsook D, ed. Molecular neurobiology of pain. Seattle: IASP Press, 1997: 171–200.
- Price GW, Kelly JS, Bowery NG. The location of GABAB receptor binding sites in mammalian spinal cord. Synapse 1987; 1: 530–8.[Web of Science][Medline]
-
Moss SJ, Smart TG, Blackstone CD, Huganir RL. Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation. Science 1992; 257: 661–5.
[Abstract/Free Full Text] - Grenningloh G, Rienitz A, Schmitt B, et al. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature 1987; 328: 215–20.[Medline]
- Schmidt RF, Senges J, Zimmermann M. Presynaptic depolarization of cutaneous mechanoreceptor afferents after mechanical skin stimulation. Exp Brain Res 1967; 3: 234–47.[Web of Science][Medline]
- Dougherty PM, Staats PS. Intrathecal drug therapy for chronic pain: from basic science to clinical practice. Anesthesiology 1999; 91: 1891–918.[Web of Science][Medline]
-
Roerig SC, Fujimoto JM, Tseng LF. Comparisons of descending pain inhibitory pathways activated by ß-endorphin and morphine as characterized by supraspinal and spinal antinociceptive interactions in mice. J Pharmacol Exp Ther 1988; 247: 1107–13.
[Abstract/Free Full Text] - Campbell JN, Raja SN, Meyer RA, Mackinnon SE. Myelinated afferents signal the hyperalgesia associated with nerve injury. Pain 1988; 32: 89–94.[Web of Science][Medline]
- Sherriff FE, Henderson Z, Morrison JF. Further evidence for the absence of a descending cholinergic projection from the brainstem to the spinal cord in the rat. Neurosci Lett 1991; 128: 52–6.[Web of Science][Medline]
- Barber RP, Phelps PE, Houser CR, et al. The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study. J Comp Neurol 1984; 229: 329–46.[Web of Science][Medline]
- Kenakin TP. Pharmacologic analysis of drug-receptor interaction. New York: Raven Press, 1987.
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