Cholinergic agents reduce allodynia after nerve injury in animals and may be useful in the treatment of neuropathic pain. Intrathecally administered neostigmine and neuronal nicotinic agonists are more potent in female than in male rats against acute thermal noxious stimuli. The purpose of this study was to determine whether there is also a sex difference in the antiallodynic effects of intrathecal cholinomimetic agents in two models of allodynia and to test their pharmacologic mechanisms.
Male and female rats with indwelling intrathecal catheters received injections of neostigmine, bethanechol (muscarinic agonist), RJR-2403 (neuronal nicotinic agonist) alone or with atropine (muscarinic antagonist), mecamylamine (nicotinic antagonist), phentolamine (alpha-adrenergic antagonist), or saline control. The effect of these agents was determined on mechanical allodynia produced by either intraplantar injection of capsaicin or ligation of spinal nerves.
Neostigmine and RJR-2403 but not bethanechol were more potent in female than in male rats in reducing allodynia after nerve injury, and antagonist studies were also consistent with a nicotinic component to explain this sex difference. Phentolamine did not reverse neostigmine's effect. In contrast, for capsaicin-induced allodynia, neostigmine plus mecamylamine but not neostigmine or RJR-2403 was more potent in female than in male rats.
These data demonstrate a sex difference of intrathecal neostigmine after nerve injury-induced allodynia similar to that observed in normal animals that received acute noxious thermal stimulation. However, this sex difference is not universal to all pain models because it was not present after intradermal capsaicin injection, nor is its interaction with spinal noradrenergic mechanisms consistent in all models.
SOME chronic painful conditions are more prevalent among women than men, 1particularly complex regional pain syndrome and chronic low back pain. Similarly, female rats are more likely than males to develop signs of neuropathic pain in certain models of neuropathic pain. 2In humans, sex differences in pain prevalence emerge at adolescence when hormonal status changes, 3and in animals, some studies suggest that hormonal status greatly influences behavioral and pharmacologic responses to stress and pain. 4We speculate that part of this hormonal dependence may, in part, reflect sex differences in spinal cholinergic activity and have demonstrated antinociception from intrathecal neostigmine, which is more potent in normal female than male rats exposed to acute noxious heat stimuli. 5That study suggested that, in the normal animal, there is an increased sensitivity to spinal nicotinic receptor activation in females compared with males, which explains the sex difference in sensitivity to neostigmine. Because the effect of a spinal nicotinic agonist was partially reversed by phentolamine, and because nicotinic agonists are known to stimulate norepinephrine release, 6we proposed that a nicotinic–noradrenergic interaction was present in females and not in males.
Chronic pain differs in physiology and pharmacology to responses to acute noxious somatic stimulation, and it is not clear whether results in normal animals would apply to those with hypersensitivity. The purpose of this study was to test whether a sex difference in sensitivity to intrathecal neostigmine exists in animals with mechanical allodynia after spinal nerve ligation 7and after intraplantar injection of capsaicin. 8We chose both models because they are well characterized anatomically and pharmacologically and are claimed to be predictive of response in patients with neuropathic pain. In addition, we chose the latter model because it can be tested in normal human volunteers.
Materials and Methods
All experiments were approved by the Animal Care and Use Committee of Wake Forest University School of Medicine. We did not assess or influence the hormonal cycle of the female rats in this study.
Allodynia Induced by Spinal Nerve Ligation
Male and female Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 150–180 g underwent spinal nerve ligation under halothane anesthesia as previously described. 7The left L5 and L6 spinal nerves were isolated adjacent to the vertebral column and tightly ligated with 6–0 silk sutures distal to the dorsal root ganglion. After surgery, animals were housed individually and allowed to recover for at least 2 weeks. Left paw mechanical allodynia was confirmed at this time by evaluation of hind paw withdrawal threshold in response to application of von Frey filaments, using an up–down method previously described. 9Only animals with withdrawal thresholds less than 4 g were used. An intrathecal catheter (polyethylene 10 tubing) was inserted as previously described 10through a small hole in the cisterna magnum and directed caudally such that the tip lay in the intrathecal space around the lumbar enlargement. Animals that showed neurologic deficits were immediately killed by an overdose of pentobarbital; the others were allowed to recover 4–5 days before drug testing. The animals were 10–12 weeks of age at the time of drug testing.
Each group of rats included six males and six females. Testing occurred at the same time during the day. Each animal received a maximum of two agonist injections separated by at least 4 days to avoid development of tolerance. The effect of the following cholinergic agonists to relieve mechanical allodynia in this model was evaluated by intrathecal cumulative dose injections (15-min intervals between doses): neostigmine (cholinesterase inhibitor and indirect agonist), 0.1, 0.3, 0.7, 1, 2, and 5 μg; bethanechol (nonselective muscarinic agonist), 10, 20, 50, 100, and 200 μg; RJR-2403 [(E)-N -methyl-4-(3-pyridinyl)-3-butene-1-amine fumarate, a specific neuronal nicotinic agonist acting on α4β2 receptors], 10, 20, 50, and 100 μg; carbachol (a nonspecific agonist with predominant muscarinic effect), 0.3, 0.7, 1, 2, and 5 μg. The dose producing a 50% return to presurgery withdrawal threshold (ED50) was calculated for each drug in male and female rats.
To confirm the sex difference revealed by agonist injections and to assess the role of muscarinic, nicotinic, and adrenergic receptors mediating the antiallodynic effect of neostigmine, rats received a single dose of neostigmine (1 μg) preceded by pretreatment with saline or one of the following antagonists: 10 μg atropine (muscarinic antagonist), 10 μg mecamylamine (nicotinic antagonist), or 30 μg phentolamine (α-adrenergic antagonist). Antagonist doses were chosen based on reports of blockade of responses to specific agonists in rats. 11,12
Allodynia Induced by Intraplantar Capsaicin Injection
Adult male and female Sprague Dawley rats were prepared with insertion of intrathecal catheters as previously described. Capsaicin (trans -8-methyl-N -vanillyl-6-nonenamide) was prepared in a concentration of 30 μg in 10 μl using the method described by Simone et al. 13Briefly, capsaicin powder was first dissolved in Tween 80, then in saline isotonic and heated to 70°C. The solution obtained was filtered and stored in sterile glass vials. Each group of rats included six males and six females tested at the same time, and each animal received only one injection in the left plantar surface of the hind paw. Each animal was lightly anesthetized under halothane and immediately after immobility received 30 μg intradermal capsaicin injected through a 28-gauge needle in the center of the plantar surface. This dose was chosen because it produces mechanical hyperalgesia that lasts at least 2 h. 8Recovery from anesthesia was rapid, and all of the animals displayed a nocifensive behavior that lasted < 5 min, then behaved normally and used the paw for locomotion. Hind-paw withdrawal thresholds were tested before capsaicin injection and every 30 min thereafter for 2 h, using von Frey filaments and the up–down method previously described. Care was taken to avoid direct contact of the injection point when measuring withdrawal threshold with the filaments.
Efficacy of spinal cholinergic agonists to prevent development of mechanical allodynia induced by capsaicin was assessed in male and female rats by intrathecal bolus injection of the following drugs before capsaicin injection: saline control, 1 μg neostigmine, 50 μg bethanechol, and 50 μg RJR-2403. In addition, the effect of 10 μg mecamylamine with 1 μg neostigmine was investigated. Because of spontaneous pain behavior observed with the combination of atropine and neostigmine and ongoing pain induced by capsaicin, we did not test an atropine/neostigmine combination in capsaicin studies.
The following drugs were used: neostigmine methylsulfate (Gensia, Irvine, CA), bethanechol chloride (Research Biochemicals International, Natick, MA), RJR-2403 (RJ Reynolds Tobacco Co., Winston-Salem, NC), atropine sulfate, mecamylamine, and phentolamine methaneosulfonate salt (Sigma, St. Louis, MO). All drugs were dissolved in normal saline and were injected intrathecally in a volume of 5 μl over 30 s followed by a 10-μl saline flush.
All data except raw withdrawal threshold (which is expressed in median ± 25th and 75th percentiles) are presented as mean ± SE. For raw withdrawal threshold data, effect of drug treatment over time was tested by the Kruskal-Wallis test followed by Dunn's test. For spinal nerve ligation data, withdrawal thresholds after experimental treatment were converted to percent maximum possible effect according to the formula:
All such transformed data were normally distributed. These data were analyzed by one- and two-way analysis of variance followed by Dunnett's test, with P < 0.05 considered significant. ED50was calculated by linear regression for each individual animal's dose response. For antagonist studies, the average percent maximum possible effect for 2 h after agonist injection was compared between saline control and antagonist pretreatment groups using one-way analysis of variance.
For capsaicin studies, the effect of antagonists alone were determined by t test. The percent change in threshold was calculated at each observational time compared with the postdrug baseline value. For simplicity, the results are shown as the average reduction in threshold over the 2 h after injection. Within each sex, saline control was compared with drug treatment over time by two-way repeated measures analysis of variance on raw data. Because sexes differed at baseline, sex differences were tested for each drug treatment by two-way repeated measures analysis of variance on the percent change from control over time after capsaicin injection.
Allodynia Induced by Spinal Nerve Ligation
Before nerve ligation, withdrawal threshold to von Frey filament testing was lower in female than in male animals (P < 0.01). Spinal nerve ligation resulted in a decrease in withdrawal threshold within 5 days to a similar level in both sexes, and this allodynia was stable over at least 3 weeks. Intrathecal injection of neostigmine increased withdrawal threshold more in female than in male rats (fig. 1). This was true when data were analyzed as raw withdrawal threshold or percent return to presurgery control.
There were significant sex differences in dose response for antiallodynia for neostigmine, the mixed agonist, carbachol, and the nicotinic agonist RJR-2403 (fig. 2). RJR-2403 showed no significant effect in males. ED50could be calculated for neostigmine and carbachol, and this analysis confirmed increased potency in females compared with males. For neostigmine, ED50was 0.46 ± 0.01 μg in females compared with 1.3 ± 0.1 μg in males (P < 0.05), and for carbachol, ED50was 0.46 ± 0.01 μg in females compared with 2.7 ± 0.1 μg in males (P < 0.05). In contrast, there was only a nonsignificant trend (P = 0.08) for a sex difference in the response to the muscarinic non–subtype-selective agonist bethanechol (fig. 2). Bethanechol response did not differ at doses > 10 μg in females (e.g. , there was an apparent plateau) but not in males (fig. 2).
Intrathecal injection of saline did not alter the antiallodynic effect of neostigmine in either sex (data not shown). In contrast, atropine completely prevented the antiallodynic effect of 1 μg neostigmine in both sexes (fig. 3). This combination also resulted in spontaneous vocalization and intermittent agitation. There was a sex difference in the effects mecamylamine, which significantly reduced neostigmine's effect in females but not in males. Phentolamine did not alter neostigmine's effect in either males or females (fig. 3). Atropine alone did not affect withdrawal threshold in females (median, 1.8 g before and 1.0 g after atropine) or males (median, 1.6 g before and 0.9 g after atropine). Similarly, mecamylamine alone did not affect withdrawal threshold in females (median, 2.0 g before and 1.1 g after mecamylamine) or males (median, 1.4 g before and 1.9 g after mecamylamine).
Allodynia Induced by Intraplantar Capsaicin Injection
Intraplantar injection of capsaicin produced a rapid-onset, sustained reduction in withdrawal threshold in both sexes (fig. 4). However, male and female control animals differed in withdrawal threshold at baseline and after capsaicin (fig. 4), and the sexes were therefore compared by percent change from precapsaicin threshold. By this analysis, the average percent reduction in withdrawal threshold over the 2 h after capsaicin injection was 74 ± 5.3% in females and 64 ± 6.7% in males (P = 0.47, not significant). Drug pretreatment increased withdrawal threshold significantly only in female rats that received bethanechol (table 1). Intrathecal injection of saline had no effect on capsaicin-induced allodynia in either sex. In contrast, neostigmine diminished allodynia from capsaicin to a similar degree in males and females (fig. 5). The nicotinic agonist RJR-2403 reversed capsaicin-induced allodynia to a similar degree in males and females. Bethanechol reversed capsaicin-induced allodynia only in females but also affected withdrawal threshold before capsaicin in this group, making interpretation of this difference difficult. To examine further the role of muscarinic receptors, a combination of neostigmine and mecamylamine was administered. This did not affect withdrawal threshold in either sex before capsaicin (table 1), but significantly more effectively blocked capsaicin-induced hypersensitivity in females than in males (fig. 5).
Given the difficulty in the treatment of many patients with neuropathic pain syndromes, which are often considered to be less responsive to opioids, 14there has been considerable interest in development of novel, nonopioid analgesics for this use. At the same time, multiple animal models of hyperalgesia and allodynia have been described, and the anatomic, physiologic, and molecular changes responsible for their altered sensitivity have been explored. Of particular interest have been models that display allodynia to touch, because this is a common and bothersome feature of patients with neuropathic pain. We observed efficacy of intrathecal neostigmine in two such models—spinal nerve ligation and intraplantar capsaicin injection—but in only one of these models was neostigmine more potent in female than in male rats. Intrathecal neostigmine produces intense nausea at doses producing analgesia, 15and, although its use has been anecdotally reported in patients with chronic pain, 16its usefulness in clinical treatment is uncertain. However, the current descriptive study of the role of spinal cholinergic stimulation in alleviating mechanical allodynia leads to testable hypotheses regarding the pathophysiology of these hypersensitive states and sex differences in these processes and may lead to clinical development of more useful analgesics.
In our companion article in this issue, 5we describe an increased sensitivity to the antinociceptive effect of intrathecal neostigmine in normal female compared with male rats. This sex difference is associated with a combined muscarinic and nicotinic component in females but only a muscarinic component in males. The current results support a similar sex difference in the antiallodynic effect of spinal cholinergic agents in the spinal nerve ligation model of chronic pain. Thus, stimulation of spinal muscarinic receptors reduces allodynia in this model in both sexes, as evidenced by similar effects in each sex to the muscarinic agonist bethanechol and reduction in neostigmine's effect by atropine. The nicotinic component is only present in the female, as evidenced indirectly by increased potency of the mixed agonist carbachol in females and, more directly, by increased potency of the neuronal nicotinic agonist RJR-2403 and by mecamylamine sensitivity of neostigmine's antiallodynic effect only in this sex.
In contrast to our observations in normal animals, 5in this nerve injury model, neostigmine's analgesic effect in females was not dependent on spinal noradrenergic stimulation because it was not appreciably diminished by phentolamine. Because α2-adrenergic agonists are extremely effective in this model, 17and because norepinephrine, the endogenous ligand for this receptor, is released from nicotinic receptor stimulation, 6we anticipated that increased noradrenergic stimulation after intrathecal neostigmine would be responsible for its increased potency in females. According to the current results, this is not the case. Although the reasons for this change in pharmacology after nerve injury are unclear, these results suggest that nicotinic and α2-adrenergic agonists act via different mechanisms in the spinal cord to relieve allodynia in this model, and their interaction may therefore be supraadditive.
Intradermal injection of capsaicin results in mechanical allodynia in humans 13and rats 8and is of interest because this allodynia results from spinal, not peripheral, sensitization 18dependent on N -methyl-D-aspartate receptor activation 19and can be used for studies in human volunteers of these processes. 20Intrathecal neostigmine reduces allodynia after intraplantar capsaicin injection, but its pharmacology is completely different from that of the nerve injury model. In complete contrast to the results after nerve injury, the antiallodynic effect of intrathecal neostigmine is the same in female and in male rats, as is the antiallodynic effect of the neuronal nicotinic receptor agonist RJR-2403. The muscarinic agonist bethanechol, which shows no sex difference in effect against nerve injury–induced allodynia, is effective only in females after capsaicin injection, although interpretation of this result is complicated by effects of bethanechol before capsaicin injection. The redundant experiment testing the muscarinic component (neostigmine plus mecamylamine) demonstrated a significantly greater effect in females than in males. Although only single doses were studied in these experiments, they were chosen based on their efficacy in other models (including the nerve injury model in the current study) and have been shown to be effective as selective agonists and antagonists at these doses after intrathecal injection in rats. Thus, there was a shared muscarinic mechanism to neostigmine's action in both sexes to acute noxious stimulation 5and to allodynia after nerve injury, whereas there seems to be a shared nicotinic mechanism to neostigmine's action in both sexes to allodynia after capsaicin injection.
The reasons for this difference in pharmacology in the different models are unclear. Spinal nicotinic receptor stimulation results in excitatory amino acid release. 21It is conceivable that this acute release may lead to desensitization of N -methyl-D-aspartate receptors, which is important in the capsaicin model of acute allodynia is less so in the chronic setting after nerve injury or in response to acute noxious stimulation. It should be noted that the timing of drug testing in the current study differed in the two models of hypersensitivity: pretreatment in the capsaicin model and posttreatment in the nerve injury model. This reflects the different utility of the models, with capsaicin representing an acute central plasticity that can be tested in human volunteers when capsaicin is administered at the time of maximum drug effect, and with nerve injury representing a model of established hypersensitivity. Regardless of the cause, these results suggest that a sex difference in response to spinal cholinergic agents for neuropathic pain may not be appreciated by screening with intradermal capsaicin testing in healthy volunteers.
In summary, intrathecal neostigmine reduces mechanical allodynia in two well-characterized rodent models. Allodynia after nerve injury is reduced by intrathecal neostigmine with a greater potency in females than in males, reflecting a mixture of nicotinic and muscarinic effects in females compared with a purely muscarinic effect in males. In contrast, allodynia after capsaicin is reduced by intrathecal neostigmine with similar potency in females and males, reflecting a mixture of nicotinic and muscarinic effects in females and a nicotinic effect in males. These results suggest that spinal cholinergic receptor stimulation affects allodynia in male and female rats by fundamentally different processes in these two models and suggests caution in extrapolating the results of one model to the other, or from either model to patients with chronic pain.