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The effects of mexiletine, desipramine and fluoxetine in rat models involving central sensitization
Pain 69 (1997) 161–169
The effects of mexiletine, desipramine and fluoxetine in rat models involving central sensitization Mary-Frances Jett*, Jennifer McGuirk, Dan Waligora, John C. Hunter Institute of Pharmacology, Neurobiology Unit, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94304, USA Received 4 April 1996; revised version received 17 July 1996; accepted 30 July 1996
Abstract Drugs that are clinically effective (mexiletine and desipramine) or ineffective (fluoxetine) in the treatment of human neuropathic pain were evaluated for efficacy in rat models involving central sensitization (i.e., formalin model and the L5/L6 spinal nerve ligation model of neuropathic pain) using tests that differ in stimulus modality: noxious chemical stimulus (formalin model) as well as noxious (pin prick) and innocuous mechanical stimuli (application of von Frey filaments). Mexiletine (10–100 mg/kg, s.c.) significantly (P , 0.05) attenuated hyperalgesia in formalin-treated (60 mg/kg and 100 mg/kg) and neuropathic rats (100 mg/kg) as well as tactile allodynia in neuropathic rats (100 mg/kg). Desipramine (1–100 mg/kg, s.c.), on the other hand, reduced hyperalgesia significantly (P , 0.05) in formalin-treated (3, 10, 30 and 100 mg/kg) and neuropathic rats (10 mg/kg and 100 mg/kg), but did not reduce tactile allodynia in the neuropathic rats. Fluoxetine (3–30 mg/kg, s.c.) did not inhibit either hyperalgesia or allodynia in any of the tests employed. Fluoxetine, which is relatively ineffective in reducing neuropathic pain in humans, was also ineffective in reducing hyperalgesia and allodynia associated with central sensitization in rats. Thus, drugs which are effective in reducing human neuropathic pain consistently attenuated hyperalgesia in formalin-treated or neuropathic rats. Desipramine also distinguished mechanical hyperalgesia from tactile allodynia in rats rendered neuropathic by spinal nerve ligation. These data are consistent with the hypothesis that the neuronal mechanisms underlying these two manifestations of neuropathic pain are different Keywords: Formalin; Spinal nerve ligation; Neuropathic pain; Hyperalgesia; Allodynia; Mexiletine; Desipramine; Fluoxetine
1. Introduction Neuropathic pain resulting from peripheral nerve injury is a chronic and disabling condition, which is often refractory to conventional analgesics, such as opiates and nonsteroidal antiinflammatory drugs (Max et al., 1988a; Tanelian and Brose, 1991). In recent years, mexiletine, a class IB antiarrhythmic drug, has been used successfully to treat neuropathic pain in humans (Dejgard et al., 1988; Tanelian and Brose, 1991; Chabal et al., 1992). Certain antidepressants have also been shown in double-blind crossover studies to reduce neuropathic pain in humans (Max et al., 1987: Max et al., 1988b; Sindrup et al., 1990; Max et al., 1991; Max et al., 1992; Watson et al., 1992). Thus, amitriptyline which blocks neuronal re-uptake of both norepinephrine (NE) and 5-hydroxytryptamine (5-HT) is * Corresponding author. Roche Bioscience, 3401 Hillview Ave. (R2101), Palo Alto, CA 94304, USA. Tel.: +1 415 8521359; fax: +1 415 3547400; e-mail: [email protected]
effective in reducing pain associated with postherpetic neuralgia (Max et al., 1988b; Watson et al., 1992) and diabetic neuropathy (Max et al., 1987; Max et al., 1991; Max et al., 1992). Desipramine, a more selective, neuronal NE re-uptake inhibitor (Baldessarini, 1990) is efficacious in reducing pain associated with diabetic neuropathy (Sindrup et al., 1990; Max et al., 1991; Max et al., 1992). The efficacy of selective 5-HT uptake inhibitors (paroxetine, zimelidine and fluoxetine) in the treatment of neuropathic pain, however, is still in question. In head to head comparisons with amitriptyline, neither zimelidine (Watson et al., 1992) nor fluoxetine (Max et al., 1992) were found to significantly reduce neuropathic pain. On the other hand, paroxetine, another highly selective inhibitor of neuronal 5-HT re-uptake, was shown to alleviate pain associated with diabetic neuropathy (Sindrup et al., 1990). In general, then, antidepressants that block neuronal NE re-uptake alone or in conjunction with 5-HT are the most consistently effective therapies in relieving neuropathic pain.
0304-3959/97/$17.00 1997 International Association for the Study of Pain. Published by Elsevier Science Ireland Ltd. PII S0304-3959 (96 )0 3231-9
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The mechanism by which these pharmacologically distinct drugs ameliorate neuropathic pain in humans is not well understood. It is clear, however, that they must interfere in some manner with injury-induced sensitization of dorsal horn neurons (central sensitization), since this process is fundamental to the development of hyperalgesia and allodynia associated with neuropathic pain (Dubner and Ruda, 1992; Coderre et al., 1993b; Woolf and Doubell, 1994a). Central sensitization is provoked by nociceptive input that occurs concomitant with nerve injury and can be simulated by low frequency stimulation of C fiber afferents (Cook et al., 1987; Woolf et al., 1988). It is characterized by altered responsiveness of the dorsal horn neurons (Woolf, 1983; Coderre and Melzack, 1985; Cook et al., 1987; Woolf et al., 1994b), expansion of receptive fields (Woolf, 1983; Cook et al., 1987; Woolf et al., 1994b), and plasticity of neuronal connections (Basbaum and Wall, 1976; Devor and Wall, 1978; Woolf et al., 1992). To study the mechanism of analgesic action or the efficacy of novel analgesics, it is important to employ animal models which exhibit central sensitization. Several rodent models involving central sensitization have been developed. The formalin model, first described by Dubuisson and Dennis (1977), reflects both the phasic and tonic response to a noxious chemical stimulus. Injection of formalin provokes a bi-phasic response in which the phasic response is separated from the more enduring tonic response by an interphase (Wheeler-Aceto and Cowan, 1991; Coderre et al., 1993a; Jett and Michelson, 1996). Existing evidence suggests that the tonic component of the response reflects a state of central sensitization (Dickenson and Sullivan, 1987; Coderre et al., 1990), which we will term formalin-induced hyperalgesia. Both clomipramine (Ansuategui et al., 1989) and amitriptyline (Acton et al., 1992), NE/5-HT re-uptake inhibitors, attenuate the formalin-induced hyperalgesia. Neither mexiletine nor the selective re-uptake inhibitors (e.g., desipramine or fluoxetine), however, have been evaluated in the formalin test. Another rodent model involving central sensitization is the spinal nerve ligation (SNL) model of Kim and Chung (1992). In this model of chronic neuropathic pain in rats, two of the three spinal nerves (L5/L6) containing somatosensory afferent input from the sciatic nerve are ligated, leaving intact only those sciatic afferents that enter the spinal cord via L4 (Swett et al., 1991). Tight ligation of the spinal nerves provokes central sensitization reflected by tactile allodynia and thermal hyperalgesia of the affected hindpaw (Kim and Chung, 1992). Neither the class IB antiarrhythmics nor the antidepressants used to treat human neuropathic pain have been evaluated in this model, although amitriptyline (Ardid and Guilbaud, 1992; Koch et al., 1996), desipramine (Ardid and Guilbaud, 1992) and mexiletine (Koch et al., 1996) were shown to reduce hyperalgesia in rats rendered neuropathic by chronic constriction of the sciatic nerve (Bennett and
Xie, 1988; Tal and Bennett, 1994). Mexiletine has also been shown to attenuate allodynia-like symptoms in rats with ischemic spinal cord injury (Xu et al., 1992). The purpose of the present work was to evaluate mexiletine, desipramine and fluoxetine in rat models involving central sensitization (i.e., formalin and the SNL models) using tests that have different stimulus modalities: nociceptive (high-threshold) and non-nociceptive (low-threshold) stimuli. It is hoped that these studies will (i) identify tests in rat models that predict clinical efficacy in humans and (ii) provide insight into the mechanism underlying neuropathic pain. 2. Methods 2.1. Animals Adult male Sprague–Dawley rats were purchased from Harlan Sprague Dawley Inc. (San Diego, CA), housed at an average ambient temperature of 22°C with a 12-h light/ dark cycle for 7 days prior to the onset of experimentation. All procedures involving the rats were reviewed and approved by the Roche Bioscience Institutional Animal Care and Use Committee in accordance with Federal laws and the regulations of Roche Bioscience. 2.2. Animal surgery Spinal nerve ligation was carried out as described previously (Kim and Chung, 1992). Briefly, the rats (150 g) were anesthetized with sodium pentobarbital (70 mg/kg, i.p.), and the L5/L6 spinal nerves were tightly ligated (6.0 silk suture) just distal to the dorsal root ganglia. The incision was then closed in layers and the animals were allowed to recover for a period of at least 6 days before testing. 2.3. Behavioral testing Formalin-induced hyperalgesia was assessed in normal rats by measuring the incidence of pain-related, agitation behaviors (i.e., licking and flinching the affected paw, hopping and turning) in response to formalin treatment, using the automated detection system previously described (Jett and Michelson, 1996). Briefly, the rats were placed in individual polycarbonate tubes (i.d. 8 cm; length 16 cm) and allowed to acclimate for 15 min. The rats were removed from the tubes, administered formalin (50 ml of a 5% solution in phosphate buffered saline) subcutaneously into the plantar surface of the hindpaw and returned to the tubes. The tubes were then placed on load cells of the automated detection system and the response was monitored continuously for 60 min. As the rats within the tubes exhibit the agitation response to formalin treatment, the dynamic force that the rat exerts on the load cell changes, reflecting its behavioral change.
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These changes in dynamic force are detected and analyzed by the automated system. An event, the outcome measure of the automated system, is a change in dynamic force that exceeds an empirically determined threshold value (0.2 g) and occurs at intervals greater than 0.1 s. Events are expressed as events per 5-min interval. The sum of the events occurring in the tonic phase of the response to formalin reflects formalin-induced hyperalgesia. Mechanical hyperalgesia was assessed in SNL rats by a modification (Koch et al., 1996) of the pin prick test described previously (Tal and Bennett, 1994). Briefly, the rats were placed in clear plastic cages (height, 5 inches; length, 10 inches; width, 4 5/8 inches) fitted with wire mesh flooring and allowed to acclimate for 15 min. A diaper pin was employed to prick (5 times), but not penetrate, the plantar surfaces of the hindpaws. The reflex withdrawal duration (seconds) was then measured for the hindlimbs ipsilateral and contralateral to the site of ligation and used as the measure of mechanical hyperalgesia. To minimize the response variation, a randomized, cross-over design (4 × 4) was employed in which all rats received all treatments. Tactile allodynia was evaluated in SNL rats with a calibrated series of 8 von Frey filaments as described previously (Chaplan et al., 1994). Briefly, the rats were placed in clear plastic cages described above and allowed to acclimate for 15 min. The following filaments, listed by index numbers (log10 of the bending force (g)) were employed to test for allodynia: 3.61 (0.4 g), 3.84 (0.7 g), 4.08 (1.2 g), 4.31 (2.0 g), 4.56 (3.6 g), 4.74 (5.5 g), 4.93 (8.5 g) and 5.18 (15.1 g). Each filament was applied once to the mid-plantar surface of the affected hindpaw. It was applied in a perpendicular fashion and depressed slowly (4–6 s) until bending occurred. From the overall pattern of responses, a 50% withdrawal threshold (g) value was calculated, using the following formula:
Xf + kd) 50%Withdrawalthreshold(g) = 10(10000
where Xf is the value (in log units) of the final von Frey hair used; k is the pattern value (Chaplan et al., 1994) and d is the mean difference (in log units) between filaments: 0.223. The 50% withdrawal threshold to von Frey filaments (0.4–15.1 g) provides a measure of allodynia, since the innocuous mechanical stimulation produced by application of such filaments to the plantar surface of (i) a normal rat hindpaw or (ii) the hindpaw ipsilateral to the site of ligation in a SNL rat does not evoke a withdrawal response. 2.4. Data analysis In the formalin test, the phasic and tonic agitation responses to formalin were calculated as the sum of all events occurring between 0–10 and 15–60 min following formalin treatment, respectively. Each treatment group
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was then compared at each time point using a one-way ANOVA on the ranks (non-parametric technique, Kruskal–Wallis test). In the test of mechanical hyperalgesia, differences in the paw withdrawal duration for the ligated and sham hindpaws were calculated. The response differences for each treatment group were then compared at each time point using an ANOVA in a 4 × 4 latin square design on the ranks. In the tactile allodynia test, the 50% withdrawal threshold values for each treatment group were compared at each time point using a one-way ANOVA on the ranks (non-parametric technique, Kruskal–Wallis test). In all cases, pairwise comparisons of the drug-treated groups to the vehicle controls were performed using Dunnett’s t-tests on the ranks. The results were reported as mean ± SEM rather than median and quartile range (standard non-parametric summary statistics) because the mean and median values in the present work were similar and hence reporting the mean did not distort the summary of the data in any way. 2.5. Drugs and chemicals Desipramine was purchased from Research Biochemicals International (Natick, MA) and 37% formaldehyde (100% formalin) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Mexiletine and fluoxetine were prepared at the Institute of Organic Chemistry, Roche Bioscience, Palo Alto, CA. Drug doses (mg/kg) were calculated for the chemical base, excluding any contribution of the counter ion. The drugs were then dissolved in water and administered subcutaneously (,4 ml/kg) at 1–3 h before testing. 3. Results 3.1. Formalin-induced hyperalgesia and neuropathyinduced mechano-hypersensitivity Injection of 5% formalin into the plantar surface of the rat hindpaw elicited a bi-phasic agitation response, consisting of phasic and tonic responses (Fig. 1A). The agitation response included a variety of pain-related behaviors: paw flinching, paw licking, hopping and turning. The entire response lasted approximately 60 min with the tonic phase predominating (i.e., 15–60 min). The effects of mexiletine, desipramine and fluoxetine were assessed on the phasic and tonic responses, although only the tonic response involves a state of central sensitization and reflects a state of hyperalgesia. Mechano-hypersensitivity of the rat hindpaw resulting from tight ligation of L5/L6 spinal nerves was also evaluated over time (Fig. 1B). Mechanical hyperalgesia developed almost immediately after surgery and lasted for up to 84 days. Typically, the effects of drugs on mechanical hyperalgesia were evaluated between 30–40 days following ligation. Tactile allodynia was present by day 1 follow-
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the highest dose of mexiletine tested: vehicle, 74.5 ± 16.3 events versus mexiletine (100 mg/kg), 19.8 ± 5.6 events (P , 0.01). In the SNL neuropathy model, mexiletine (100 mg/kg, s.c.) significantly decreased mechanical hyperalgesia at 1 and 3 h following drug administration (Fig. 2B). Complete inhibition of mechanical hyperalgesia was not achieved at this dose. Mexiletine reduced tactile allodynia in a similar dose-dependent manner. Significant anti-allodynic effects occurred at 100 mg/kg, 1 h following dosing (Fig. 2C). Complete reversal of the allodynia was not, however, achieved under these conditions. Mexiletine did not affect the responsiveness of the paw
Fig. 1. Time courses of: (A) the formalin-induced agitation response (n = 8), where the mean number of events per 5-min interval ( ± SEM) are indicated (X); (B) neuropathy-induced mechanical hyperalgesia (n = 10); and (C) neuropathy-induced mechanical allodynia (n = 19). In B and C, the mean values ( ± SEM) for the responses of the hindpaw ipsilateral (X) and contralateral (W) to the ligation are reported. All responses of the ipsilateral paw are significantly (P , 0.05) different from those of the contralateral paw for both tests of mechano-hypersensitivity.
ing surgery, remained fairly constant for up to 28 days and resolved progressively over time (Fig. 1C). The effects of drugs on tactile allodynia were evaluated on days 7–28 following ligation, since the therapeutic window was greatest during this period. 3.2. Effects of mexiletine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Mexiletine inhibited formalin-induced hyperalgesia in a dose-dependent manner, with significant reductions in the response occurring at 60 and 100 mg/kg, s.c. (Fig. 2A). Near complete inhibition of the tonic agitation response (i.e., 80% reduction) was achieved at the highest dose of mexiletine employed (100 mg/kg). Mexiletine significantly inhibited the phasic response to formalin only at
Fig. 2. Effect of mexiletine on formalin-induced and neuropathy-induced responses to different stimulus modalities. Vehicle (white) or mexiletine at 10 (wide diagonal lines), 30 (find diagonal lines), 60 (bi-directional, diagonal lines) or 100 mg/kg (black) were administered subcutaneously 1 h before testing began. The mean responses ( ± SEM) are reported for: (A) the tonic agitation response to formalin (n = 7–8), following administration of vehicle or mexiletine at 10, 30, 60 or 100 mg/kg; (B) neuropathy-induced mechanical hyperalgesia (n = 16), following administration of vehicle or mexiletine at 10, 30 and 100 mg/kg; and (C) tactile allodynia (n = 9), following administration of vehicle or mexiletine at 10, 30 and 100 mg/kg. Significance of differences from vehicle control at the P , 0.05, 0.01 and 0.001 levels is indicated by single, double and triple asterisks, respectively.
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the phasic component of the formalin test at all doses tested: vehicle, 61.2 ± 4.5 events versus desipramine at 1 mg/kg, 41.0 ± 5.6 events (P , 0.01); 3 mg/kg, 42.5 ± 5.6 events (P , 0.05); 10 mg/kg, 25.9 ± 4.6 events (P , 0.001); 30 mg/kg, 14.2 ± 5.7 events (P , 0.001) and 100 mg/kg, 17.3 ± 5.7 events (P , 0.001). In the SNL model of neuropathic pain, desipramine (30 and 100 mg/kg, s.c.) significantly decreased mechanical hyperalgesia 1 h after drug administration (Fig. 3B). Significant anti-hyperalgesic effects of desipramine were also seen 3 h after dosing. By contrast, desipramine (10–100 mg/kg, s.c.) was ineffective in reducing tactile allodynia (Fig. 3C). Under the conditions used, desipramine did not affect the responsiveness of the hindpaw contralateral to the site nerve ligation in the test of mechanical hyperalgesia or the general behavior of the rats.
Fig. 3. Effect of desipramine on formalin-induced and neuropathyinduced responses to different stimulus modalities. Vehicle (white) or desipramine at 1 (cross-hatched lines), 3 (small dots), 10 (wide diagonal lines), 30 (fine diagonal lines) or 100 mg/kg (black) were administered subcutaneously 1 h before testing began. Mean values ( ± SEM) are reported for: (A) the tonic agitation response to formalin (n = 10), following administration of vehicle or desipramine at 1, 3, 10, 30 or 100 mg/kg; (B) mechanical hyperalgesia (n = 16), following administration of vehicle or desipramine at 1, 10 or 100 mg/kg: and (C) tactile allodynia (n = 9–10), following administration of 1, 10 or 100 mg/kg. Significance of differences from vehicle control at the P , 0.05, 0.01 and 0.001 levels is indicated by single, double and triple asterisks, respectively.
contralateral to the site of ligation in the test of mechanical hyperalgesia or the general behavior of the rats, under the conditions used. 3.3. Effects of desipramine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Desipramine significantly reduced formalin-induced hyperalgesia at doses of 3–100 mg/kg, s.c. with near complete inhibition (91%) being achieved at 100 mg/kg (Fig. 3A). Desipramine also significantly inhibited
Fig. 4. Effect of fluoxetine on formalin-induced and neuropathy-induced responses to different stimulus modalities. Vehicle (white) or fluoxetine at 3 (small dots), 10 (wide diagonal lines) or 30 mg/kg (fine diagonal lines) were administered subcutaneously 1 h before testing. The mean values ( ± SEM) are reported for: (A) the agitation response to formalin (n = 7–8); (B) mechanical hyperalgesia (n = 16); and (C) tactile allodynia (n = 8–9).
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3.4. Effects of fluoxetine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Fluoxetine (3–30 mg/kg, s.c.) did not affect the hyperalgesia or the allodynia in either of the models employed (Fig. 4A–C). Under the conditions used, the drug did not affect the responsiveness of the hindpaw contralateral to the site of ligation in the test of mechanical hyperalgesia or the general behavior of the rats. 4. Discussion In the present work, the anti-hyperalgesic and anti-allodynic effects of mexiletine, desipramine and fluoxetine were examined in rat models involving central sensitization using tests that differ by the stimulus modality employed. The drugs were selected because of (i) their clinical efficacy (mexiletine and desipramine) or lack of efficacy (fluoxetine) as analgesics in the treatment of human neuropathic pain and (ii) fundamental differences in their pharmacological activities. 4.1. Formalin-induced hyperalgesia and neuropathyinduced mechano-hypersensitivity Formalin injection into the plantar surface of the rat hindpaw elicited a characteristic bi-phasic agitation response in our automated assay. We have shown previously (Jett and Michelson, 1996) that the agitation response corresponds well with other formalin-induced, pain-related behaviors, such as flinching and licking (Wheeler-Aceto and Cowan, 1991) the affected paw as well as a weighted-scores measure (Dubuisson and Dennis, 1977; Coderre et al., 1993a). In the present study, we focused on the tonic agitation response (15–60 min) rather than the phasic response (0–10 min), since the tonic response involves central sensitization (Dickenson and Sullivan, 1987; Coderre et al., 1990) and reflects a state of hyperalgesia. Following tight ligation of the L5/L6 spinal nerves, mechanical hyperalgesia and tactile allodynia developed rapidly and lasted for weeks to months, depending on the test employed to monitor the mechano-hypersensitivity. Mechanical hyperalgesia, as evinced by an exaggerated response to the pin prick, was very long lasting and resembled that observed in rats rendered neuropathic by a chronic constriction injury to the sciatic nerve (Koch et al., 1996). The magnitude of tactile allodynia in the SNL model was greatest during the first 2–3 weeks following nerve ligation and then declined substantially over the next 5–6 weeks. These results are consistent with those described for SNL model by others (Kim and Chung, 1992; Kim et al., 1993; Chaplan et al., 1994; Kinnman and Levine, 1995). The fact that the time courses of mechanical hyperalgesia and tactile allodynia in SNL
rats differ is also consistent with differences in their pathophysiology. For example, noxious, high-threshold stimuli such as a pin pricks and innocuous, low-threshold, tactile stimuli such as the application of small caliber, von Frey filaments activate different afferent fibers (C/Ad and Ab fibers, respectively) which in turn project to different regions within the dorsal horn of the spinal cord (Light and Perl, 1979; Sugiura et al., 1986; Fields, 1987; Woolf and Doubell, 1994a). 4.2. Effects of mexiletine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Mexiletine, a sodium channel blocker with demonstrated efficacy as a class IB antiarrhythmic (Bigger and Hoffman, 1990) and analgesic in the treatment of human neuropathic pain (Dejgard et al., 1988; Tanelian and Brose, 1991; Chabal et al., 1992), reduced both formalinand neuropathy-induced hyperalgesia as well as neuropathy-induced allodynia. Our results are consistent with previous studies showing that systemic administration of mexiletine decreases both the spontaneous activity and the mechanical sensitivity of rat sciatic neuromas (Chabal et al., 1989), and reduces mechanical hyperalgesia (pin prick test) in rats with a chronic constriction injury (Koch et al., 1996). It is unlikely that the anti-hyperalgesic and anti-allodynic actions of mexiletine demonstrated in the present work are due to nerve block or non-specific behavioral effects of the drug, under the conditions used. For example, we have previously shown that mexiletine does not significantly affect spinal reflexes, as measured in the tail flick test, at doses up to 100 mg/kg in normal rats (Hedley et al., 1995). In addition, we have shown here that mexiletine specifically affects the paw ipsilateral to the spinal nerve ligation in the test of mechanical hyperalgesia. Whether mexiletine and other class IB antiarrhythmic drugs exert their analgesic effects via peripheral, central or mixed mechanisms is not known. Electrophysiological studies aimed at elucidating the site at which lidocaine and tocainide, class Ib antiarrhythmic drugs, exert their antinociceptive action provide compelling evidence in support of a central site of action (Woolf and Wiesenfeld-Hallin, 1985). Following nerve injury, peripheral mechanisms may develop which are responsive to sodium channel blockade. For example, class Ib antiarrhythmic drugs decrease (i) spontaneous neuronal discharge from rat neuromas (Wall and Gutnick, 1974; Chabal et al., 1989; Devor et al., 1992), (ii) tonic Ad- and C-fiber discharge following acute injury in an in vitro rabbit corneal preparation (Tanelian and MacIver, 1991) and (iii) tap-induced phantom limb pain in humans following local instillation (Nystrom and Hagbarth, 1981). Recently, we have also shown that the quaternary amine of lidocaine, QX-314, which does not access the central nervous system, blocks injury-
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induced neuronal activity (Omana-Zapata et al., 1995) and cold allodynia in rats with a chronic constriction injury to the sciatic nerve (Hunter et al., 1995). Thus, mexiletine and other class Ib antiarrhythmic drugs may act via both central and peripheral mechanisms. 4.3. Effects of desipramine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Desipramine, a tricyclic antidepressant which selectively blocks neuronal NE re-uptake (Baldessarini, 1990), exhibited differential effects depending on the model and the stimulus modality employed. Systemically administered desipramine inhibited both formalin- and neuropathy-induced hyperalgesia. The anti-hyperalgesic effects of desipramine observed in the formalin test at low doses (,10 mg/kg) may be mediated by NE acting through central adrenergic receptors, since the effects of desipramine on NE re-uptake is maximal at 10 mg/kg (Wong et al., 1975; Baldessarini, 1990). This possibility is consistent with the work of others showing that agonists at both the a1 adrenoceptors (Tasker and Melzack, 1989; Tasker et al., 1992) and a2 adrenoceptors (Pertovaara et al., 1990) reduce the tonic response to formalin injection. It is also consistent with the observation that both acute (Acton et al., 1992) and chronic (Ansuategui et al., 1989) administration of tricyclic antidepressants (amitriptyline and clomipramine), which inhibit both NE and 5-HT reuptake, decreases formalin-induced hyperalgesia. These same drugs have also been shown to ameliorate mechanical hyperalgesia in rats rendered neuropathic by chronic constriction of the sciatic nerve following acute (Ardid and Guilbaud, 1992; Koch et al., 1996) and chronic drug administration (Ardid and Guilbaud, 1992). Thus, the anti-hyperalgesic effects of desipramine may be, in part, dependent on adrenergic mechanisms. It is difficult, however, to explain all the actions of desipramine in this way. For example, maximal inhibition of formalin- and neuropathy-induced hyperalgesia required doses far in excess of those needed to inhibit NE re-uptake. The basis of these low-potency effects is unknown. Desipramine, like mexiletine, is a sodium channel blocker (Delpon et al., 1993) and may act via sodium channel blockade at the doses employed in the present study. Systemic administration of desipramine did not alleviate neuropathy-induced tactile allodynia in rats rendered neuropathic by SNL. As such, desipramine appears to distinguish mechanical hyperalgesia from tactile allodynia. This result is important because it provides further evidence that the neuronal mechanism underlying tactile allodynia is different from that underlying mechanical hyperalgesia. One explanation of the results is that systemically administered desipramine provides a sympathomimetic drive which antagonizes the analgesic effects of the drug. This explanation is consistent with reports that
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sympathetic stimulation augments neuropathy-induced mechano-hypersensitivity in the SNL model (Kim et al., 1993; Kinnman and Levine, 1995). Further studies will be necessary to clearly elucidate the actions of desipramine in this experimental model. Insight in this area will help us explain why some patients from seemingly homogeneous populations of patients do not respond to this drug (Sindrup et al., 1990; Max et al., 1991; Max et al., 1992). 4.4. Effects of fluoxetine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Fluoxetine, the selective 5-HT re-uptake inhibitor (Wong et al., 1975), was ineffective in reducing the nociceptive responses in all the tests employed at doses that have been shown to block 5-HT re-uptake maximally (Wong et al., 1975). Similarly, fluoxetine is not consistently effective in reducing either neuropathic pain (Watson and Evans, 1985; Max et al., 1992) or post-operative dental pain (Gordon et al., 1994). Our results suggest that 5-HT does not appear to play a major role in the maintenance of hyperalgesia or allodynia in the rat models involving central sensitization used in this work. 5. Summary Drugs that have been shown to be effective in the treatment of patients experiencing neuropathic pain are effective in reducing the hyperalgesia induced by formalin treatment and SNL in rats. As such, these tests may be used to explore the mechanisms of central sensitization as well as to evaluate the effect of potential analgesics on painful neuropathies. Mechanistically, desipramine differentially affects mechanical hyperalgesia and tactile allodynia. Understanding this difference may give us insight into central and/or peripheral mechanisms underlying central sensitization and thus the basis of neuropathic pain.
Acknowledgements The authors wish to thank Claudia Kermeen and Mohamed Khabbaz for having helped surgically pre-pare the neuropathic rats, Lois Kellerman and Sophie Chiu in the Department of Biomathematics for their help in statistically analyzing the data. We are also grateful for the assistance of Kristi Bezik in preparing the manuscript. References Acton, J., McKenna, J.E. and Melzack, R., Amitriptyline produces analgesia in the formalin pain test, Exp. Neurol., 117 (1992) 94– 96. Ansuategui, M., Naharro, L. and Feria, M., Noradrenergic and opioidergic influences on the antinociceptive effect of clomipramine in the formalin test in rats, Psychopharmacology, 98 (1989) 93–96.
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M.-F. Jett et al. / Pain 69 (1997) 161–169 Tanelian, D.L. and Brose, W.G., Neuropathic pain can be relieved by drugs that are use-dependent sodium channel blockers: lidocaine, carbamazepine, and mexiletine, Anesthesiology, 74 (1991) 949–951. Tanelian, D.L. and MacIver, M.B., Analgesic concentrations of lidocaine suppress tonic A-delta and C fiber discharges produced by acute injury, Anesthesiology, 74 (1991) 934–936. Tasker, R.A.R. and Melzack, R., Different alpha-receptor subtypes are involved in colonidine-produced analgesia in different pain tests, Life Sci., 44 (1989) 9–17. Tasker, R.A.R., Connell, B.J. and Yole, M.J., Systemic injection of alpha-1 adrenergic agonists produce antinociception in the formalin test, Pain, 49 (1992) 383–391. Wall, P.D.T. and Gutnick, M., Properties of afferent nerve impulses originating from a neuroma, Nature, 248 (1974) 740–743. Watson and Evans, 1985 Please supple full reference. Watson, C.P.N., Chipman, M., Reed, K., Evans, R.J. and Birkett, N., Amitriptyline versus maprotiline in postherpetic neuralgia: a randomized, double-blind, crossover trial, Pain, 48 (1992) 29–36. Wheeler-Aceto, H. and Cowan, A., Standardization of the rat paw formalin test for the evaluation of analgesics, Psychopharmacology, 104 (1991) 35–44. Wong, T.D., Bymaster, F.P., Horng, J.S. and Molloy, B.B., New selective inhibitor for uptake of serotonin into synaptosomes of rat brain: 3(p-trifluoromethylphenoxy)-N-methyl-3-phenylpropylamine, J. Pharmacol. Exp. Ther., 193 (1975) 804–811.
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Woolf, C.J., Evidence for a central component of post-injury pain hypersensitivity, Nature, 306 (1983) 686–688. Woolf, C.J. and Wiesenfeld-Hallin, Z., The systemic administration of local anaesthetics produces a selective depression of C-afferent fibre evoked activity in the spinal cord, Pain, 23 (1985) 361–374. Woolf, C.J., Thompson, W.N. and King, A.E., Prolonged primary afferent induced alterations in dorsal horn neurons, an intracellular analysis in vivo and in vitro, J. Physiol., 83 (1988) 255–266. Woolf, C.J., Shortland, P. and Coggeshall, R.E., Peripheral nerve injury triggers central sprouting of myelinated afferents, Nature, 355 (1992) 75–77. Woolf, C.J. and Doubell, T.P., The pathophysiology of chronic painincreased sensitivity to low threshold Ab-fibre inputs, Curr. Opin. Neurobiol., 4 (1994a) 525–534. Woolf, C.J., Shortland, P. and Sivilotti, L.G., Sensitization of high mechanothreshold superficial dorsal horn and flexor motor neurones following chemosensitive primary afferent activation, Pain, 58 (1994b) 141–155. Xu, X.-J., Hao, J.-X., Seiger, A., Staffan, A., Lindblom, U. and Wiesenfeld-Hallin, Z., Systemic mexiletine relieves chronic allodynia like symptoms in rats with ischemic spinal cord injury, Anesth. Analg., 74 (1992) 649–652.
The effects of mexiletine, desipramine and fluoxetine in rat models involving central sensitization Mary-Frances Jett*, Jennifer McGuirk, Dan Waligora, John C. Hunter Institute of Pharmacology, Neurobiology Unit, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, CA 94304, USA Received 4 April 1996; revised version received 17 July 1996; accepted 30 July 1996
Abstract Drugs that are clinically effective (mexiletine and desipramine) or ineffective (fluoxetine) in the treatment of human neuropathic pain were evaluated for efficacy in rat models involving central sensitization (i.e., formalin model and the L5/L6 spinal nerve ligation model of neuropathic pain) using tests that differ in stimulus modality: noxious chemical stimulus (formalin model) as well as noxious (pin prick) and innocuous mechanical stimuli (application of von Frey filaments). Mexiletine (10–100 mg/kg, s.c.) significantly (P , 0.05) attenuated hyperalgesia in formalin-treated (60 mg/kg and 100 mg/kg) and neuropathic rats (100 mg/kg) as well as tactile allodynia in neuropathic rats (100 mg/kg). Desipramine (1–100 mg/kg, s.c.), on the other hand, reduced hyperalgesia significantly (P , 0.05) in formalin-treated (3, 10, 30 and 100 mg/kg) and neuropathic rats (10 mg/kg and 100 mg/kg), but did not reduce tactile allodynia in the neuropathic rats. Fluoxetine (3–30 mg/kg, s.c.) did not inhibit either hyperalgesia or allodynia in any of the tests employed. Fluoxetine, which is relatively ineffective in reducing neuropathic pain in humans, was also ineffective in reducing hyperalgesia and allodynia associated with central sensitization in rats. Thus, drugs which are effective in reducing human neuropathic pain consistently attenuated hyperalgesia in formalin-treated or neuropathic rats. Desipramine also distinguished mechanical hyperalgesia from tactile allodynia in rats rendered neuropathic by spinal nerve ligation. These data are consistent with the hypothesis that the neuronal mechanisms underlying these two manifestations of neuropathic pain are different Keywords: Formalin; Spinal nerve ligation; Neuropathic pain; Hyperalgesia; Allodynia; Mexiletine; Desipramine; Fluoxetine
1. Introduction Neuropathic pain resulting from peripheral nerve injury is a chronic and disabling condition, which is often refractory to conventional analgesics, such as opiates and nonsteroidal antiinflammatory drugs (Max et al., 1988a; Tanelian and Brose, 1991). In recent years, mexiletine, a class IB antiarrhythmic drug, has been used successfully to treat neuropathic pain in humans (Dejgard et al., 1988; Tanelian and Brose, 1991; Chabal et al., 1992). Certain antidepressants have also been shown in double-blind crossover studies to reduce neuropathic pain in humans (Max et al., 1987: Max et al., 1988b; Sindrup et al., 1990; Max et al., 1991; Max et al., 1992; Watson et al., 1992). Thus, amitriptyline which blocks neuronal re-uptake of both norepinephrine (NE) and 5-hydroxytryptamine (5-HT) is * Corresponding author. Roche Bioscience, 3401 Hillview Ave. (R2101), Palo Alto, CA 94304, USA. Tel.: +1 415 8521359; fax: +1 415 3547400; e-mail: [email protected]
effective in reducing pain associated with postherpetic neuralgia (Max et al., 1988b; Watson et al., 1992) and diabetic neuropathy (Max et al., 1987; Max et al., 1991; Max et al., 1992). Desipramine, a more selective, neuronal NE re-uptake inhibitor (Baldessarini, 1990) is efficacious in reducing pain associated with diabetic neuropathy (Sindrup et al., 1990; Max et al., 1991; Max et al., 1992). The efficacy of selective 5-HT uptake inhibitors (paroxetine, zimelidine and fluoxetine) in the treatment of neuropathic pain, however, is still in question. In head to head comparisons with amitriptyline, neither zimelidine (Watson et al., 1992) nor fluoxetine (Max et al., 1992) were found to significantly reduce neuropathic pain. On the other hand, paroxetine, another highly selective inhibitor of neuronal 5-HT re-uptake, was shown to alleviate pain associated with diabetic neuropathy (Sindrup et al., 1990). In general, then, antidepressants that block neuronal NE re-uptake alone or in conjunction with 5-HT are the most consistently effective therapies in relieving neuropathic pain.
0304-3959/97/$17.00 1997 International Association for the Study of Pain. Published by Elsevier Science Ireland Ltd. PII S0304-3959 (96 )0 3231-9
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The mechanism by which these pharmacologically distinct drugs ameliorate neuropathic pain in humans is not well understood. It is clear, however, that they must interfere in some manner with injury-induced sensitization of dorsal horn neurons (central sensitization), since this process is fundamental to the development of hyperalgesia and allodynia associated with neuropathic pain (Dubner and Ruda, 1992; Coderre et al., 1993b; Woolf and Doubell, 1994a). Central sensitization is provoked by nociceptive input that occurs concomitant with nerve injury and can be simulated by low frequency stimulation of C fiber afferents (Cook et al., 1987; Woolf et al., 1988). It is characterized by altered responsiveness of the dorsal horn neurons (Woolf, 1983; Coderre and Melzack, 1985; Cook et al., 1987; Woolf et al., 1994b), expansion of receptive fields (Woolf, 1983; Cook et al., 1987; Woolf et al., 1994b), and plasticity of neuronal connections (Basbaum and Wall, 1976; Devor and Wall, 1978; Woolf et al., 1992). To study the mechanism of analgesic action or the efficacy of novel analgesics, it is important to employ animal models which exhibit central sensitization. Several rodent models involving central sensitization have been developed. The formalin model, first described by Dubuisson and Dennis (1977), reflects both the phasic and tonic response to a noxious chemical stimulus. Injection of formalin provokes a bi-phasic response in which the phasic response is separated from the more enduring tonic response by an interphase (Wheeler-Aceto and Cowan, 1991; Coderre et al., 1993a; Jett and Michelson, 1996). Existing evidence suggests that the tonic component of the response reflects a state of central sensitization (Dickenson and Sullivan, 1987; Coderre et al., 1990), which we will term formalin-induced hyperalgesia. Both clomipramine (Ansuategui et al., 1989) and amitriptyline (Acton et al., 1992), NE/5-HT re-uptake inhibitors, attenuate the formalin-induced hyperalgesia. Neither mexiletine nor the selective re-uptake inhibitors (e.g., desipramine or fluoxetine), however, have been evaluated in the formalin test. Another rodent model involving central sensitization is the spinal nerve ligation (SNL) model of Kim and Chung (1992). In this model of chronic neuropathic pain in rats, two of the three spinal nerves (L5/L6) containing somatosensory afferent input from the sciatic nerve are ligated, leaving intact only those sciatic afferents that enter the spinal cord via L4 (Swett et al., 1991). Tight ligation of the spinal nerves provokes central sensitization reflected by tactile allodynia and thermal hyperalgesia of the affected hindpaw (Kim and Chung, 1992). Neither the class IB antiarrhythmics nor the antidepressants used to treat human neuropathic pain have been evaluated in this model, although amitriptyline (Ardid and Guilbaud, 1992; Koch et al., 1996), desipramine (Ardid and Guilbaud, 1992) and mexiletine (Koch et al., 1996) were shown to reduce hyperalgesia in rats rendered neuropathic by chronic constriction of the sciatic nerve (Bennett and
Xie, 1988; Tal and Bennett, 1994). Mexiletine has also been shown to attenuate allodynia-like symptoms in rats with ischemic spinal cord injury (Xu et al., 1992). The purpose of the present work was to evaluate mexiletine, desipramine and fluoxetine in rat models involving central sensitization (i.e., formalin and the SNL models) using tests that have different stimulus modalities: nociceptive (high-threshold) and non-nociceptive (low-threshold) stimuli. It is hoped that these studies will (i) identify tests in rat models that predict clinical efficacy in humans and (ii) provide insight into the mechanism underlying neuropathic pain. 2. Methods 2.1. Animals Adult male Sprague–Dawley rats were purchased from Harlan Sprague Dawley Inc. (San Diego, CA), housed at an average ambient temperature of 22°C with a 12-h light/ dark cycle for 7 days prior to the onset of experimentation. All procedures involving the rats were reviewed and approved by the Roche Bioscience Institutional Animal Care and Use Committee in accordance with Federal laws and the regulations of Roche Bioscience. 2.2. Animal surgery Spinal nerve ligation was carried out as described previously (Kim and Chung, 1992). Briefly, the rats (150 g) were anesthetized with sodium pentobarbital (70 mg/kg, i.p.), and the L5/L6 spinal nerves were tightly ligated (6.0 silk suture) just distal to the dorsal root ganglia. The incision was then closed in layers and the animals were allowed to recover for a period of at least 6 days before testing. 2.3. Behavioral testing Formalin-induced hyperalgesia was assessed in normal rats by measuring the incidence of pain-related, agitation behaviors (i.e., licking and flinching the affected paw, hopping and turning) in response to formalin treatment, using the automated detection system previously described (Jett and Michelson, 1996). Briefly, the rats were placed in individual polycarbonate tubes (i.d. 8 cm; length 16 cm) and allowed to acclimate for 15 min. The rats were removed from the tubes, administered formalin (50 ml of a 5% solution in phosphate buffered saline) subcutaneously into the plantar surface of the hindpaw and returned to the tubes. The tubes were then placed on load cells of the automated detection system and the response was monitored continuously for 60 min. As the rats within the tubes exhibit the agitation response to formalin treatment, the dynamic force that the rat exerts on the load cell changes, reflecting its behavioral change.
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These changes in dynamic force are detected and analyzed by the automated system. An event, the outcome measure of the automated system, is a change in dynamic force that exceeds an empirically determined threshold value (0.2 g) and occurs at intervals greater than 0.1 s. Events are expressed as events per 5-min interval. The sum of the events occurring in the tonic phase of the response to formalin reflects formalin-induced hyperalgesia. Mechanical hyperalgesia was assessed in SNL rats by a modification (Koch et al., 1996) of the pin prick test described previously (Tal and Bennett, 1994). Briefly, the rats were placed in clear plastic cages (height, 5 inches; length, 10 inches; width, 4 5/8 inches) fitted with wire mesh flooring and allowed to acclimate for 15 min. A diaper pin was employed to prick (5 times), but not penetrate, the plantar surfaces of the hindpaws. The reflex withdrawal duration (seconds) was then measured for the hindlimbs ipsilateral and contralateral to the site of ligation and used as the measure of mechanical hyperalgesia. To minimize the response variation, a randomized, cross-over design (4 × 4) was employed in which all rats received all treatments. Tactile allodynia was evaluated in SNL rats with a calibrated series of 8 von Frey filaments as described previously (Chaplan et al., 1994). Briefly, the rats were placed in clear plastic cages described above and allowed to acclimate for 15 min. The following filaments, listed by index numbers (log10 of the bending force (g)) were employed to test for allodynia: 3.61 (0.4 g), 3.84 (0.7 g), 4.08 (1.2 g), 4.31 (2.0 g), 4.56 (3.6 g), 4.74 (5.5 g), 4.93 (8.5 g) and 5.18 (15.1 g). Each filament was applied once to the mid-plantar surface of the affected hindpaw. It was applied in a perpendicular fashion and depressed slowly (4–6 s) until bending occurred. From the overall pattern of responses, a 50% withdrawal threshold (g) value was calculated, using the following formula:
Xf + kd) 50%Withdrawalthreshold(g) = 10(10000
where Xf is the value (in log units) of the final von Frey hair used; k is the pattern value (Chaplan et al., 1994) and d is the mean difference (in log units) between filaments: 0.223. The 50% withdrawal threshold to von Frey filaments (0.4–15.1 g) provides a measure of allodynia, since the innocuous mechanical stimulation produced by application of such filaments to the plantar surface of (i) a normal rat hindpaw or (ii) the hindpaw ipsilateral to the site of ligation in a SNL rat does not evoke a withdrawal response. 2.4. Data analysis In the formalin test, the phasic and tonic agitation responses to formalin were calculated as the sum of all events occurring between 0–10 and 15–60 min following formalin treatment, respectively. Each treatment group
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was then compared at each time point using a one-way ANOVA on the ranks (non-parametric technique, Kruskal–Wallis test). In the test of mechanical hyperalgesia, differences in the paw withdrawal duration for the ligated and sham hindpaws were calculated. The response differences for each treatment group were then compared at each time point using an ANOVA in a 4 × 4 latin square design on the ranks. In the tactile allodynia test, the 50% withdrawal threshold values for each treatment group were compared at each time point using a one-way ANOVA on the ranks (non-parametric technique, Kruskal–Wallis test). In all cases, pairwise comparisons of the drug-treated groups to the vehicle controls were performed using Dunnett’s t-tests on the ranks. The results were reported as mean ± SEM rather than median and quartile range (standard non-parametric summary statistics) because the mean and median values in the present work were similar and hence reporting the mean did not distort the summary of the data in any way. 2.5. Drugs and chemicals Desipramine was purchased from Research Biochemicals International (Natick, MA) and 37% formaldehyde (100% formalin) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Mexiletine and fluoxetine were prepared at the Institute of Organic Chemistry, Roche Bioscience, Palo Alto, CA. Drug doses (mg/kg) were calculated for the chemical base, excluding any contribution of the counter ion. The drugs were then dissolved in water and administered subcutaneously (,4 ml/kg) at 1–3 h before testing. 3. Results 3.1. Formalin-induced hyperalgesia and neuropathyinduced mechano-hypersensitivity Injection of 5% formalin into the plantar surface of the rat hindpaw elicited a bi-phasic agitation response, consisting of phasic and tonic responses (Fig. 1A). The agitation response included a variety of pain-related behaviors: paw flinching, paw licking, hopping and turning. The entire response lasted approximately 60 min with the tonic phase predominating (i.e., 15–60 min). The effects of mexiletine, desipramine and fluoxetine were assessed on the phasic and tonic responses, although only the tonic response involves a state of central sensitization and reflects a state of hyperalgesia. Mechano-hypersensitivity of the rat hindpaw resulting from tight ligation of L5/L6 spinal nerves was also evaluated over time (Fig. 1B). Mechanical hyperalgesia developed almost immediately after surgery and lasted for up to 84 days. Typically, the effects of drugs on mechanical hyperalgesia were evaluated between 30–40 days following ligation. Tactile allodynia was present by day 1 follow-
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the highest dose of mexiletine tested: vehicle, 74.5 ± 16.3 events versus mexiletine (100 mg/kg), 19.8 ± 5.6 events (P , 0.01). In the SNL neuropathy model, mexiletine (100 mg/kg, s.c.) significantly decreased mechanical hyperalgesia at 1 and 3 h following drug administration (Fig. 2B). Complete inhibition of mechanical hyperalgesia was not achieved at this dose. Mexiletine reduced tactile allodynia in a similar dose-dependent manner. Significant anti-allodynic effects occurred at 100 mg/kg, 1 h following dosing (Fig. 2C). Complete reversal of the allodynia was not, however, achieved under these conditions. Mexiletine did not affect the responsiveness of the paw
Fig. 1. Time courses of: (A) the formalin-induced agitation response (n = 8), where the mean number of events per 5-min interval ( ± SEM) are indicated (X); (B) neuropathy-induced mechanical hyperalgesia (n = 10); and (C) neuropathy-induced mechanical allodynia (n = 19). In B and C, the mean values ( ± SEM) for the responses of the hindpaw ipsilateral (X) and contralateral (W) to the ligation are reported. All responses of the ipsilateral paw are significantly (P , 0.05) different from those of the contralateral paw for both tests of mechano-hypersensitivity.
ing surgery, remained fairly constant for up to 28 days and resolved progressively over time (Fig. 1C). The effects of drugs on tactile allodynia were evaluated on days 7–28 following ligation, since the therapeutic window was greatest during this period. 3.2. Effects of mexiletine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Mexiletine inhibited formalin-induced hyperalgesia in a dose-dependent manner, with significant reductions in the response occurring at 60 and 100 mg/kg, s.c. (Fig. 2A). Near complete inhibition of the tonic agitation response (i.e., 80% reduction) was achieved at the highest dose of mexiletine employed (100 mg/kg). Mexiletine significantly inhibited the phasic response to formalin only at
Fig. 2. Effect of mexiletine on formalin-induced and neuropathy-induced responses to different stimulus modalities. Vehicle (white) or mexiletine at 10 (wide diagonal lines), 30 (find diagonal lines), 60 (bi-directional, diagonal lines) or 100 mg/kg (black) were administered subcutaneously 1 h before testing began. The mean responses ( ± SEM) are reported for: (A) the tonic agitation response to formalin (n = 7–8), following administration of vehicle or mexiletine at 10, 30, 60 or 100 mg/kg; (B) neuropathy-induced mechanical hyperalgesia (n = 16), following administration of vehicle or mexiletine at 10, 30 and 100 mg/kg; and (C) tactile allodynia (n = 9), following administration of vehicle or mexiletine at 10, 30 and 100 mg/kg. Significance of differences from vehicle control at the P , 0.05, 0.01 and 0.001 levels is indicated by single, double and triple asterisks, respectively.
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the phasic component of the formalin test at all doses tested: vehicle, 61.2 ± 4.5 events versus desipramine at 1 mg/kg, 41.0 ± 5.6 events (P , 0.01); 3 mg/kg, 42.5 ± 5.6 events (P , 0.05); 10 mg/kg, 25.9 ± 4.6 events (P , 0.001); 30 mg/kg, 14.2 ± 5.7 events (P , 0.001) and 100 mg/kg, 17.3 ± 5.7 events (P , 0.001). In the SNL model of neuropathic pain, desipramine (30 and 100 mg/kg, s.c.) significantly decreased mechanical hyperalgesia 1 h after drug administration (Fig. 3B). Significant anti-hyperalgesic effects of desipramine were also seen 3 h after dosing. By contrast, desipramine (10–100 mg/kg, s.c.) was ineffective in reducing tactile allodynia (Fig. 3C). Under the conditions used, desipramine did not affect the responsiveness of the hindpaw contralateral to the site nerve ligation in the test of mechanical hyperalgesia or the general behavior of the rats.
Fig. 3. Effect of desipramine on formalin-induced and neuropathyinduced responses to different stimulus modalities. Vehicle (white) or desipramine at 1 (cross-hatched lines), 3 (small dots), 10 (wide diagonal lines), 30 (fine diagonal lines) or 100 mg/kg (black) were administered subcutaneously 1 h before testing began. Mean values ( ± SEM) are reported for: (A) the tonic agitation response to formalin (n = 10), following administration of vehicle or desipramine at 1, 3, 10, 30 or 100 mg/kg; (B) mechanical hyperalgesia (n = 16), following administration of vehicle or desipramine at 1, 10 or 100 mg/kg: and (C) tactile allodynia (n = 9–10), following administration of 1, 10 or 100 mg/kg. Significance of differences from vehicle control at the P , 0.05, 0.01 and 0.001 levels is indicated by single, double and triple asterisks, respectively.
contralateral to the site of ligation in the test of mechanical hyperalgesia or the general behavior of the rats, under the conditions used. 3.3. Effects of desipramine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Desipramine significantly reduced formalin-induced hyperalgesia at doses of 3–100 mg/kg, s.c. with near complete inhibition (91%) being achieved at 100 mg/kg (Fig. 3A). Desipramine also significantly inhibited
Fig. 4. Effect of fluoxetine on formalin-induced and neuropathy-induced responses to different stimulus modalities. Vehicle (white) or fluoxetine at 3 (small dots), 10 (wide diagonal lines) or 30 mg/kg (fine diagonal lines) were administered subcutaneously 1 h before testing. The mean values ( ± SEM) are reported for: (A) the agitation response to formalin (n = 7–8); (B) mechanical hyperalgesia (n = 16); and (C) tactile allodynia (n = 8–9).
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3.4. Effects of fluoxetine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Fluoxetine (3–30 mg/kg, s.c.) did not affect the hyperalgesia or the allodynia in either of the models employed (Fig. 4A–C). Under the conditions used, the drug did not affect the responsiveness of the hindpaw contralateral to the site of ligation in the test of mechanical hyperalgesia or the general behavior of the rats. 4. Discussion In the present work, the anti-hyperalgesic and anti-allodynic effects of mexiletine, desipramine and fluoxetine were examined in rat models involving central sensitization using tests that differ by the stimulus modality employed. The drugs were selected because of (i) their clinical efficacy (mexiletine and desipramine) or lack of efficacy (fluoxetine) as analgesics in the treatment of human neuropathic pain and (ii) fundamental differences in their pharmacological activities. 4.1. Formalin-induced hyperalgesia and neuropathyinduced mechano-hypersensitivity Formalin injection into the plantar surface of the rat hindpaw elicited a characteristic bi-phasic agitation response in our automated assay. We have shown previously (Jett and Michelson, 1996) that the agitation response corresponds well with other formalin-induced, pain-related behaviors, such as flinching and licking (Wheeler-Aceto and Cowan, 1991) the affected paw as well as a weighted-scores measure (Dubuisson and Dennis, 1977; Coderre et al., 1993a). In the present study, we focused on the tonic agitation response (15–60 min) rather than the phasic response (0–10 min), since the tonic response involves central sensitization (Dickenson and Sullivan, 1987; Coderre et al., 1990) and reflects a state of hyperalgesia. Following tight ligation of the L5/L6 spinal nerves, mechanical hyperalgesia and tactile allodynia developed rapidly and lasted for weeks to months, depending on the test employed to monitor the mechano-hypersensitivity. Mechanical hyperalgesia, as evinced by an exaggerated response to the pin prick, was very long lasting and resembled that observed in rats rendered neuropathic by a chronic constriction injury to the sciatic nerve (Koch et al., 1996). The magnitude of tactile allodynia in the SNL model was greatest during the first 2–3 weeks following nerve ligation and then declined substantially over the next 5–6 weeks. These results are consistent with those described for SNL model by others (Kim and Chung, 1992; Kim et al., 1993; Chaplan et al., 1994; Kinnman and Levine, 1995). The fact that the time courses of mechanical hyperalgesia and tactile allodynia in SNL
rats differ is also consistent with differences in their pathophysiology. For example, noxious, high-threshold stimuli such as a pin pricks and innocuous, low-threshold, tactile stimuli such as the application of small caliber, von Frey filaments activate different afferent fibers (C/Ad and Ab fibers, respectively) which in turn project to different regions within the dorsal horn of the spinal cord (Light and Perl, 1979; Sugiura et al., 1986; Fields, 1987; Woolf and Doubell, 1994a). 4.2. Effects of mexiletine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Mexiletine, a sodium channel blocker with demonstrated efficacy as a class IB antiarrhythmic (Bigger and Hoffman, 1990) and analgesic in the treatment of human neuropathic pain (Dejgard et al., 1988; Tanelian and Brose, 1991; Chabal et al., 1992), reduced both formalinand neuropathy-induced hyperalgesia as well as neuropathy-induced allodynia. Our results are consistent with previous studies showing that systemic administration of mexiletine decreases both the spontaneous activity and the mechanical sensitivity of rat sciatic neuromas (Chabal et al., 1989), and reduces mechanical hyperalgesia (pin prick test) in rats with a chronic constriction injury (Koch et al., 1996). It is unlikely that the anti-hyperalgesic and anti-allodynic actions of mexiletine demonstrated in the present work are due to nerve block or non-specific behavioral effects of the drug, under the conditions used. For example, we have previously shown that mexiletine does not significantly affect spinal reflexes, as measured in the tail flick test, at doses up to 100 mg/kg in normal rats (Hedley et al., 1995). In addition, we have shown here that mexiletine specifically affects the paw ipsilateral to the spinal nerve ligation in the test of mechanical hyperalgesia. Whether mexiletine and other class IB antiarrhythmic drugs exert their analgesic effects via peripheral, central or mixed mechanisms is not known. Electrophysiological studies aimed at elucidating the site at which lidocaine and tocainide, class Ib antiarrhythmic drugs, exert their antinociceptive action provide compelling evidence in support of a central site of action (Woolf and Wiesenfeld-Hallin, 1985). Following nerve injury, peripheral mechanisms may develop which are responsive to sodium channel blockade. For example, class Ib antiarrhythmic drugs decrease (i) spontaneous neuronal discharge from rat neuromas (Wall and Gutnick, 1974; Chabal et al., 1989; Devor et al., 1992), (ii) tonic Ad- and C-fiber discharge following acute injury in an in vitro rabbit corneal preparation (Tanelian and MacIver, 1991) and (iii) tap-induced phantom limb pain in humans following local instillation (Nystrom and Hagbarth, 1981). Recently, we have also shown that the quaternary amine of lidocaine, QX-314, which does not access the central nervous system, blocks injury-
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induced neuronal activity (Omana-Zapata et al., 1995) and cold allodynia in rats with a chronic constriction injury to the sciatic nerve (Hunter et al., 1995). Thus, mexiletine and other class Ib antiarrhythmic drugs may act via both central and peripheral mechanisms. 4.3. Effects of desipramine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Desipramine, a tricyclic antidepressant which selectively blocks neuronal NE re-uptake (Baldessarini, 1990), exhibited differential effects depending on the model and the stimulus modality employed. Systemically administered desipramine inhibited both formalin- and neuropathy-induced hyperalgesia. The anti-hyperalgesic effects of desipramine observed in the formalin test at low doses (,10 mg/kg) may be mediated by NE acting through central adrenergic receptors, since the effects of desipramine on NE re-uptake is maximal at 10 mg/kg (Wong et al., 1975; Baldessarini, 1990). This possibility is consistent with the work of others showing that agonists at both the a1 adrenoceptors (Tasker and Melzack, 1989; Tasker et al., 1992) and a2 adrenoceptors (Pertovaara et al., 1990) reduce the tonic response to formalin injection. It is also consistent with the observation that both acute (Acton et al., 1992) and chronic (Ansuategui et al., 1989) administration of tricyclic antidepressants (amitriptyline and clomipramine), which inhibit both NE and 5-HT reuptake, decreases formalin-induced hyperalgesia. These same drugs have also been shown to ameliorate mechanical hyperalgesia in rats rendered neuropathic by chronic constriction of the sciatic nerve following acute (Ardid and Guilbaud, 1992; Koch et al., 1996) and chronic drug administration (Ardid and Guilbaud, 1992). Thus, the anti-hyperalgesic effects of desipramine may be, in part, dependent on adrenergic mechanisms. It is difficult, however, to explain all the actions of desipramine in this way. For example, maximal inhibition of formalin- and neuropathy-induced hyperalgesia required doses far in excess of those needed to inhibit NE re-uptake. The basis of these low-potency effects is unknown. Desipramine, like mexiletine, is a sodium channel blocker (Delpon et al., 1993) and may act via sodium channel blockade at the doses employed in the present study. Systemic administration of desipramine did not alleviate neuropathy-induced tactile allodynia in rats rendered neuropathic by SNL. As such, desipramine appears to distinguish mechanical hyperalgesia from tactile allodynia. This result is important because it provides further evidence that the neuronal mechanism underlying tactile allodynia is different from that underlying mechanical hyperalgesia. One explanation of the results is that systemically administered desipramine provides a sympathomimetic drive which antagonizes the analgesic effects of the drug. This explanation is consistent with reports that
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sympathetic stimulation augments neuropathy-induced mechano-hypersensitivity in the SNL model (Kim et al., 1993; Kinnman and Levine, 1995). Further studies will be necessary to clearly elucidate the actions of desipramine in this experimental model. Insight in this area will help us explain why some patients from seemingly homogeneous populations of patients do not respond to this drug (Sindrup et al., 1990; Max et al., 1991; Max et al., 1992). 4.4. Effects of fluoxetine on formalin-induced hyperalgesia and neuropathy-induced mechanohypersensitivity Fluoxetine, the selective 5-HT re-uptake inhibitor (Wong et al., 1975), was ineffective in reducing the nociceptive responses in all the tests employed at doses that have been shown to block 5-HT re-uptake maximally (Wong et al., 1975). Similarly, fluoxetine is not consistently effective in reducing either neuropathic pain (Watson and Evans, 1985; Max et al., 1992) or post-operative dental pain (Gordon et al., 1994). Our results suggest that 5-HT does not appear to play a major role in the maintenance of hyperalgesia or allodynia in the rat models involving central sensitization used in this work. 5. Summary Drugs that have been shown to be effective in the treatment of patients experiencing neuropathic pain are effective in reducing the hyperalgesia induced by formalin treatment and SNL in rats. As such, these tests may be used to explore the mechanisms of central sensitization as well as to evaluate the effect of potential analgesics on painful neuropathies. Mechanistically, desipramine differentially affects mechanical hyperalgesia and tactile allodynia. Understanding this difference may give us insight into central and/or peripheral mechanisms underlying central sensitization and thus the basis of neuropathic pain.
Acknowledgements The authors wish to thank Claudia Kermeen and Mohamed Khabbaz for having helped surgically pre-pare the neuropathic rats, Lois Kellerman and Sophie Chiu in the Department of Biomathematics for their help in statistically analyzing the data. We are also grateful for the assistance of Kristi Bezik in preparing the manuscript. References Acton, J., McKenna, J.E. and Melzack, R., Amitriptyline produces analgesia in the formalin pain test, Exp. Neurol., 117 (1992) 94– 96. Ansuategui, M., Naharro, L. and Feria, M., Noradrenergic and opioidergic influences on the antinociceptive effect of clomipramine in the formalin test in rats, Psychopharmacology, 98 (1989) 93–96.
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