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Reversal of experimental neuropathic pain by T-type calcium channel blockers
Pain 105 (2003) 159–168 www.elsevier.com/locate/pain
Reversal of experimental neuropathic pain by T-type calcium channel blockers Ahmet Dogrula, Luis R. Gardellb, Michael H. Ossipovb, F. Cankat Tulunayc, Josephine Laib, Frank Porrecab,* a b
Department of Pharmacology, Faculty of Medicine, Gulhane Medical Military Academy, Ankara, Turkey Department of Pharmacology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA c Department of Pharmacology, Faculty of Medicine, Ankara University, Ankara, Turkey Received 7 November 2002; received in revised form 16 April 2003; accepted 22 April 2003
Abstract Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca2þ current is associated with increased neuronal activity and blockade of N- and P-types, but not L-type, calcium channels have been found to block experimental neuropathic pain. While T-type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L5/L6 spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose-dependent blockade of both tactile and thermal hypersensitivities in nerve-injured rats; responses of sham-operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T-type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T-type calcium channel blockers may have significant potential in the treatment of neuropathic pain states. q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: T-type voltage sensing calcium channels; Neuropathic pain; Rat
1. Introduction Pain-related behaviors caused by peripheral nerve injury result partly from an increase in transduction sensitivity of primary afferents and increased activity within the central nervous system (Burgess et al., 2002; Coderre et al., 1993; Kajander and Bennett, 1992; Porreca et al., 2002). Peripheral nerve injury results in abnormal ectopic discharges from the injury site or the dorsal root ganglia (DRG), causing enhanced central input from the damaged primary afferent fibers and manifesting behaviorally as neuropathic pain (Kajander and Bennett, 1992; Yaksh and Chaplan, 1997; Gracely et al., 1992; Sheen and Chung, * Corresponding author. Tel.: þ 1-520-626-7421; fax: þ1-520-626-4182. E-mail address: [email protected] (F. Porreca).
1993; Yoon et al., 1996). Furthermore, injured small- and large-diameter primary afferent fibers show marked alterations in their pattern of excitability and conduction properties during evoked and spontaneous activity, reflecting alterations in the density and/or operating characteristics of multiple ion channels, including novel expression of ion channels (Devor and Seltzer, 1999; Millan, 1999; Yaksh and Chaplan, 1997). These differential alterations in the density and/or properties of specific ion channels upon injury to primary afferent fibers may be of functional importance in determining changes in primary afferent discharge and conduction (Gold et al., in press; Lai et al., 2002; Rasband et al., 2001; Schild and Kunze, 1997). Increases in local Ca2þ concentration at the site of injury or in the spinal cord may contribute to the development of neuropathic pain (Gold et al., 1994; Kawamata and Omote,
0304-3959/03/$20.00 q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0304-3959(03)00177-5
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1996). Five major types of voltage sensing calcium channels (VSCCs) have been identified (L-, T-, N-, P/Q- and Rtypes), with unique electrophysiologic and pharmacologic characteristics (Catterall, 2000; Bertolino and Llinas, 1992). The L- and N-type VSCCs have been implicated in the release of neurotransmitters and neuromodulators from sensory neurons in the spinal cord (Anwyl, 1991; Perney et al., 1986). Blockade of the N- and P/Q-type VSCCs, but not of the L-type VSCC, attenuated behavioral signs of neuropathic pain in animal models of nerve injury (Chaplan et al., 1994b; Matthews and Dickenson, 2001b; Nebe et al., 1999; Yamamoto and Sakashita, 1998; White and Cousins, 1998; Miljanich and Ramachandran, 1995; Xiao and Bennett, 1995). The T-type VSCC is thought to lower the threshold for action potentials and promote bursting activity and synaptic excitation, which are actions that would favor the development of enhanced pain (Matthews and Dickenson, 2001a; Sekizawa et al., 2000). However, until recently (Matthews and Dickenson, 2001a), the lack of selective antagonists for these channels has prevented evaluation of their contribution to the manifestation of neuropathic pain. Ethosuximide is relatively selective for T-type VSCC (Matthews and Dickenson, 2001a; Coulter et al., 1989), blocking T-type currents in rat sensory neurons at half the concentration needed to block the L-current (Kostyuk et al., 1992). In contrast, mibefradil demonstrates between tenfold and 100-fold selectivity in blocking T-type current over L-type current, indicating considerable selectivity(Jimenez et al., 2000; Monteil et al., 2000; Clozel et al., 1999; Ertel and Ertel, 1997; Ertel et al., 1997; Hermsmeyer et al., 1997). These compounds were thus employed here to explore the contributions of the T-type VSCC to experimental neuropathic pain.
2. Materials and methods Male Sprague –Dawley rats (Harlan, Indianapolis, IN), 200 –300 g at the time of testing, were maintained in a climate-controlled room on a 12-h light/dark cycle (lights on at 06:00 hours). Food and water were available ad libitum. All testing was performed in accordance with the policies and recommendations of the International Association for the Study of Pain and the National Institutes of Health guidelines for the handling and use of laboratory animals. These studies also received approval from the Institutional Animal Care and Use Committee of the University of Arizona. Mibefradil dihydrochloride was obtained as a gift from Roche (Basel, Switzerland). 2.1. Spinal nerve ligation Nerve injury was induced with the L5/L6 spinal nerve ligation (SNL) according to the procedure of Kim and Chung (1992). Anesthesia was induced with 2% halothane
in O2 at a rate of 2 l/min and maintained with 0.5% halothane in O2. After surgical preparation of the rats and exposure of the dorsal vertebral column from L4 to S2, the L5 and L6 spinal nerves were tightly ligated distal to the DRG with a 4-0 silk suture. The incision was closed and the animals were allowed to recover for 5 days. Rats that exhibited motor deficiency or failure to exhibit subsequent tactile hypersensitivity were excluded from further testing. Sham surgery was performed by exposing the spinal nerves without performing the ligation. Following recovery, rats were anesthetized as described above and implanted with catheters for intrathecal (i.th.) administration of drugs into the region of the lumbar spinal cord according to the method described by Yaksh and Rudy (1976). A PE-10 polyethylene tubing was inserted 8 cm into the vertebral canal through an incision made in the atlanto-occipital membrane and secured to the musculature at the incision. This length of tubing has been shown to consistently terminate at the level of the lumbar enlargement when used with rats within the weight range employed in the present studies. Misdirected catheters result in damage to the spinal cord or spinal nerves and also result in gross motor deficiencies or paralysis. These animals are not used, but immediately euthanized. The rats received 4.4 mg/kg of gentamycin intramuscularly and were allowed to recover for a period of at least 5 days prior to testing. Rats exhibiting motor deficiency were discarded from testing. Drugs administered i.th. were given in a volume of 5 ml, followed by a 9-ml flush. The intraperitoneal (i.p.) injections were made in a volume of 0.1 ml/kg and the intraplantar (i.pl.) injections were given in a constant volume of 50 ml. The assessment of tactile hypersensitivity was determined according to the method described in detail by Chaplan et al. (1994a). A series of eight calibrated von Frey filaments with filament strengths placed at logarithmic intervals and ranging from 0.41 to 15 g were used. The probing was initiated with a filament strength of 2 g applied to the plantar aspect of the hindpaw of rats kept in suspended wire-mesh cages. If there was no response, then the stimulus was increased by one increment, otherwise it was decreased by one decrement. The stimulus was incrementally increased until a positive response was obtained, then decreased until a negative result was observed. This ‘up – down’ method was repeated until three changes in behavior were determined, and a withdrawal threshold based on the pattern of responses was calculated with the Dixon non-parametric statistic (Chaplan et al., 1994a; Dixon, 1980). When continuous positive responses occurred below the minimal stimulus (0.41 g) or when the maximal stimulus (i.e. 15 g) produced no response, then the cut-off values of 0.41 and 15, respectively, were assigned (Chaplan et al., 1994a; Dixon, 1980). The method of Hargreaves was employed to assess thermal hyperalgesia (Hargreaves et al., 1988). Rats were allowed to acclimate within Plexiglas enclosures on a clear glass plate maintained at 30 8C. A radiant heat source
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(i.e. high intensity projector lamp) was activated along with a timer and focused onto the plantar surface of the affected paw of nerve-injured or sham-operated rats. A motion sensor activated by paw withdrawal halted both lamp and timer. A maximal cut-off of 40 s was used to prevent tissue damage. In all tests, paw withdrawal thresholds and paw withdrawal latencies were obtained for the ligated and shamoperated groups before drug or vehicle injections. Rats were tested at 0, 15, 30, 45 and 60 min after injections. Within each of the treatment groups, post-injection means were compared with the baseline values by analysis of variance (ANOVA), followed by post hoc analysis of least significance difference for multiple comparisons. Comparisons between two means were performed by Student’s t-test. A probability level of 0.05 indicated significance. In order to generate a dose – response curve against tactile hypersensitivity, data were converted to %MPE (maximal possible effect) by the formula: %MPE ¼ 100 £ (test value 2 baseline value)/(15 g 2 baseline value). In order to generate a dose –response curve against thermal hyperalgesia, data were converted to %MPE by the formula: %MPE ¼ 100 £ (test latency 2 baseline latency)/(sham animal latency 2 baseline latency). The A50 dose (i.e. dose producing 50% MPE) and 95% confidence intervals were determined by linear regression analysis of the log dose – response curve. Experimenters were blinded to the experimental conditions. 2.2. Mean arterial blood pressure Rats were anesthetized with ketamine/xylazine (20:3; 100 mg/kg). A rectal probe was used to monitor body temperature, which was maintained by a feedback controlled heating pad. A polyethylene catheter (PE-50) was implanted into a carotid artery and connected to a Grass PT300 pressure transducer (Grass-Telefactor, West Warwick, RI). Data were transferred from the pressure transducer to a computer by means of an input/output port of the personal computer (PC) and captured, and heart rate, systolic, diastolic and mean arterial pressures are calculated with the JAD data acquisition software (Notocord, Paris, France). The effects of i.p. (5, 10 and 20 mg/kg) and i.pl. (30, 100 and 300 mg) administration of mibefradil on systolic and diastolic arterial blood pressures were evaluated.
3. Results 3.1. Blockade of tactile hypersensitivity Sham-operated rats displayed paw withdrawal thresholds to probing with von Frey filaments of 15 ^ 0 g. In contrast, rats with L5/L6 SNL demonstrated significantly lower paw withdrawal thresholds ðP , 0:05Þ, indicating tactile hypersensitivity. The mean paw withdrawal thresholds of rats with SNL ranged from 1.18 ^ 0.3 to 1.82 ^ 0.43 g. The i.p.
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injection of 5, 10 and 20 mg/kg of mibefradil reversed tactile hypersensitivity in a dose- and time-dependent fashion (Fig. 1). A peak effect of 14.13 ^ 0.55 g was obtained within 30 min of administration of the highest test dose of mibefradil (20 mg/kg, i.p.) (Fig. 1). The A50 for i.p. mibefradil was 7.44 mg/kg (95% CL 5.33– 10.4 mg/kg). Similarly, ethosuximide (50 – 300 mg/kg, i.p.) elicited a dose- and time-dependent reversal of tactile hypersensitivity. A maximal effect of 10.89 ^ 2.45 g was detected 30 min following the administration of the highest dose tested (300 mg/kg, i.p.) (Fig. 1). The A50 was 174 mg/kg (95% CL 58.1–524 mg/kg), i.p., indicating a significantly lower (23-fold) potency when compared with mibefradil against tactile hypersensitivity ðP # 0:05Þ (Fig. 1). The injection of 30, 100 and 300 mg of mibefradil into the plantar aspect of the injured hindlimb also produced dose-dependent reversal of tactile hypersensitivity, suggesting a local mechanism of action (Fig. 2). The maximal effect of mibefradil following i.pl. administration was 13.87 ^ 0.47 g, 15 min after the administration of 300 mg of mibefradil, and remained elevated 60 min postinjection (Fig. 2). The dose – effect curve generated from the data gathered at the time of peak effect yielded an A50 value of 92.4 mg (95% CL of 73.1– 117 mg) (Fig. 2). In contrast, injection of mibefradil (300 mg) into the hindpaw contralateral to the site of nerve injury was ineffective against tactile hypersensitivity of the hindpaw ipsilateral to SNL. The mean withdrawal threshold was 1.29 ^ 0.27 g, 30 min after injection and was not significantly different from baseline. Ethosuximide, injected i.pl. at a dosage of up to 500 mg, failed to produce any changes in paw withdrawal thresholds to light tactile stimuli (Fig. 2). The i.th. injection of mibefradil or of ethosuximide over the dose range of 10– 40 mg did not produce any significant changes in paw withdrawal threshold at any time (data not shown). 3.2. Blockade of thermal hyperalgesia SNL produced reliable thermal hyperalgesia in all groups tested. The mean baseline paw withdrawal latencies for all treatment groups ranged between 12.72 ^ 0.82 and 14.11 ^ 0.72 s after SNL, indicating consistency and reproducibility of the measurements among groups. These values were significantly less than the mean baseline paw withdrawal latency of 20.21 ^ 1.05 s displayed by the sham-operated rats ðP # 0:05Þ. The i.p. injection of mibefradil (5, 10 and 20 mg/kg) exerted a dose-dependent reversal of thermal hyperalgesia. The highest dose tested (20 mg/kg, i.p.) elicited an antihyperalgesic effect within 30 min of injection, as indicated by increased paw withdrawal latencies to 19.92 ^ 1.36 s, and antihyperalgesia was evident for 60 min (Fig. 3). The calculated i.p. A50 value of mibefradil was 11.9 mg/kg (95% CL 6.85– 20.5 mg/kg). Similarly, ethosuximide produced a dose-dependent antihyperalgesic effect over the dose range of 50 – 300 mg/kg, i.p. (Fig. 3). The highest dose of ethosuximide tested
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Fig. 1. Male Sprague– Dawley rats received L5/L6 SNL. Paw withdrawal thresholds to probing with von Frey filaments were determined prior to and at select time points after the i.p. injection of several doses of mibefradil (A) or ethosuximide (B). Both mibefradil and ethosuximide produced dose- and timedependent reversal of tactile hypersensitivity. Paw withdrawal thresholds at the time of peak effect (30 min) were converted to %MPE in order to generate dose–response curves (C). N ¼ 6 animals per group.
(300 mg/kg, i.p.) elicited an antihyperalgesic effect within 30 min of injection, as indicated by increased paw withdrawal latencies to 22.5 ^ 2.34 s (Fig. 5). The A50 value generated from data collected at the time of peak effect was 126 mg/kg (95% CL 60.1 – 265 mg/kg, i.p.) (Fig. 3). These data indicate that mibefradil has a significant, 11-fold greater potency than ethosuximide against thermal hyperalgesia ðP # 0:05Þ. Likewise, the i.pl. injection of mibefradil (30, 100 and 300 mg) into the injured hindlimb produced
a dose-dependent reversal of thermal hyperalgesia (Fig. 4), whereas the injection of the same doses into the hindpaw of sham-operated rats produced no change in paw withdrawal latency (data not shown). A maximal antihyperalgesic effect was found within 15 min and persisted for 60 min. The highest dose (300 mg) of mibefradil elicited a maximal antihyperalgesic effect within 15 min after injection, as indicated by the increase in paw withdrawal latencies to 20.0 ^ 1.0 s (Fig. 4). Importantly, the microinjection of 300 mg of mibefradil into the hindpaw contralateral to
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Fig. 2. Male Sprague–Dawley rats received L5/L6 SNL. Paw withdrawal thresholds to probing with von Frey filaments were determined prior to and at select time points after several doses of mibefradil or a maximal (500 mg) dose of ethosuximide (A). Injections were made s.c. into the plantar aspect of the injured hindpaw. Mibefradil produced dose- and time-dependent increases in paw withdrawal thresholds to tactile stimuli. The maximal dose of ethosuximide tested was not active against tactile hypersensitivity. Paw withdrawal thresholds at the time of peak effect of mibefradil (15 min) were converted to %MPE in order to generate dose–response curves (B). N ¼ 6 rats per group.
the injured paw did not produce any elevation in paw withdrawal latency of the hindpaw ipsilateral to SNL. The paw withdrawal latencies of the injured hindpaw did not change from the baseline value of 12.9 ^ 0.16 s over the 60-min observation period. Dose–response curves for the antihyperalgesic effect of mibefradil were constructed from data gathered at the time of peak effect. The A50 value after i.pl. injection was 72.0 mg (95% CL 62.0–83.6 mg) (Fig. 4). In contrast, the i.pl. injection of up to 500 mg of ethosuximide did not produce any changes in paw withdrawal latencies in SNL rats. The i.th. injection of 10–40 mg of either mibefradil or ethosuximide did not produce any change in paw withdrawal threshold at any time, indicating a lack of spinal antihyperalgesic activity (data not shown). In addition, the administration of 20 mg/kg i.p., 40 mg i.th. and 300 mg i.pl. of mibefradil to sham-operated rats did not increase paw withdrawal latencies beyond the respective baseline paw withdrawal latencies (Fig. 5), indicating an absence of antinociceptive activity.
3.3. Effects of mibefradil on arterial blood pressure Prior to drug administration, the animals had mean systolic and diastolic arterial blood pressures of 145.7 ^ 8.7 and 111 ^ 2.8 mmHg, respectively. The administration of mibefradil over the dose range of 5, 10 and 20 mg/kg i.p. induced a dose-dependent decrease in systolic and diastolic arterial blood pressures when compared with saline, whereas the i.pl. injection of the highest dose of mibefradil (300 mg) into hindlimb did not alter systolic and diastolic arterial pressures. The blood pressure lowering effects of mibefradil when given i.p. were found to begin within 15 min and remained over 60 min. The highest dose of mibefradil (20 mg/kg, i.p.) lowered systolic and diastolic arterial blood pressures to 93.7 ^ 2.8 and 64.6 ^ 4.2 mmHg, respectively, at 15 min following drug administration. The mean systolic and diastolic blood pressures were 143.8 ^ 4.7 and 112 ^ 1.78 mmHg, respectively, after the highest i.pl.
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Fig. 3. Male Sprague–Dawley rats received L5/L6 SNL. Paw withdrawal latencies to noxious radiant heat applied to the plantar aspect of the hindpaw were determined prior to and at select time points after the i.p. injection of several doses of mibefradil (A) or ethosuximide (B). Both mibefradil and ethosuximide produced dose- and time-dependent elevations in paw withdrawal latencies. The paw withdrawal latencies at the time of peak effect (30 min) were converted to %MPE in order to generate dose–response curves (C). N ¼ 6 animals per group.
dose of mibefradil (300 mg), which were not different from the control values.
4. Discussion The results of the present study indicate that the systemic administration of mibefradil and ethosuximide, both T-type VSCC blockers, effectively reverses the behavioral manifestation of neuropathic pain. This result is consistent with the electrophysiologic properties of the T-type VSCC
indicating that it may play a critical role in the modulation of neuronal excitation (Amir et al., 2002; Huguenard, 1996; Matthews and Dickenson, 2001a), and is therefore suited to modulate neuronal hyperexcitation after nerve injury. The T-type VSCCs, first described in peripheral sensory neurons of the DRG, are activated by subthreshold excitatory postsynaptic potentials (EPSPs), reducing threshold for the generation of action potentials and therefore contributing to the excitability of neuronal membranes (Carbone and Lux, 1984a,b; Huguenard, 1996; Magee et al., 1995; Markram and Sakmann, 1994; Matthews and Dickenson, 2001a;
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Fig. 4. Male Sprague– Dawley rats received L5/L6 SNL. Paw withdrawal latencies to noxious radiant heat applied to the plantar aspect of the injured hindpaw were determined prior to and at select time points after several doses of mibefradil or a maximal (500 mg) dose of ethosuximide (A). Injections were made s.c. into the plantar aspect of the injured hindpaw. Mibefradil produced dose- and time-dependent increases in paw withdrawal thresholds to noxious thermal stimuli. In contrast, the maximal dose of ethosuximide tested was not active against thermal hyperalgesia. Paw withdrawal latencies at the time of peak effect of mibefradil (15 min) were converted to %MPE in order to generate dose– response curves (B). N ¼ 6 rats per group.
Fig. 5. Sham-operated male Sprague–Dawley rats received mibefradil by i.p. injection (20 mg/kg), i.pl. injection (300 mg) or i.th. injection (40 mg). These doses represent the highest doses given to SNL rats. In addition, sham-operated rats also received saline i.p. as a reference control. Paw withdrawal latencies did not change from the pre-injection baseline values for any of the groups tested, and were identical to the responses of saline-injected rats, thus indicating a complete absence of antinociceptive activity. N ¼ 6 rats per group.
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Sekizawa et al., 2000). It has been suggested that the development of behavioral manifestations of neuropathic pain is due, at least in part, to spontaneous ectopic discharge of primary afferent neurons and subsequent sensitization of dorsal horn neurons (Devor and Seltzer, 1999; Woolf and Thompson, 1991). The enhanced activity presents as increased neuronal responses to stimuli and also as spontaneous discharges that may originate from the site of injury, the DRG and also from remaining intact fibers (Devor and Seltzer, 1999; Kajander and Bennett, 1992). Accordingly, a fourfold to sixfold increase in primary afferent activity is detected almost immediately after nerve injury, but diminishes rapidly during the first week after injury while neuropathic pain persists (Han et al., 2000; Liu et al., 2000). The temporal disparity between ectopic activity and neuropathic pain states point to the possibility that other mechanisms may also be critical in the maintenance of the neuropathic state (Burgess et al., 2002; Porreca et al., 2002). Ectopic activity as well as enhanced release from primary afferents may be susceptible to blockade by mibefradil or ethosuximide since T-type VSCCs contribute to transmitter release and neuronal sensitization (Matthews and Dickenson, 2001a). In this study, ethosuximide was found to inhibit input spikes, indicative of diminished synaptic activity, probably through a reduction in exocytosis of neurotransmitter from primary afferent neurons. Furthermore, ethosuximide also inhibited N-methyl-D -aspartate (NMDA) mediated post-discharge spikes, which are indices of spinal sensitization and hypersensitivity (Matthews and Dickenson, 2001a). These mechanisms suggest a potential role for the T-type VSCC in the genesis of neuropathic pain. Consistent with this hypothesis, we found that systemic and locally applied mibefradil, and systemic ethosuximide, blocked tactile and thermal hypersensitivities after SNL. Furthermore, these results also agree with the observation that peripheral nerve injury results in an increase in the concentration of intracellular Ca2þ at the site of injury (Gold et al., 1994). White and Cousins (1998) reported that a subcutaneous (s.c.) injection of calcium chelating agent Quin 2 at the receptive field of an injured nerve blocked mechanical hyperalgesia and suggested a local calciumdependent mechanism in the receptive field of injured neurons. As mibefradil has strong cardiovascular effects, it might be argued that mibefradil-induced hypotension may interfere with paw withdrawal responses, and thus be misinterpreted as thermal antihyperalgesic and antiallodynic effects. However, it is unlikely that this reduction in mean arterial blood pressure contributes to blockade of thermal or tactile hypersensitivity, as the greatest dose of systemic mibefradil used in this study (i.e. 20 mg/kg, i.p.) exerted a nearly 40% reduction in systolic blood pressure, but did not induce any antinociceptive effect in thermal nociception in sham-operated animals. Additionally, local administration of mibefradil into the paw on the injured, but
not uninjured, side produced antiallodynic and antihyperalgesic actions. Local mibefradil did not affect blood pressure suggesting that the observed effects were not the result of changes in blood pressure. A further point here is that intrapaw mibefradil did not produce analgesia, suggesting that local blood flow changes were unlikely to be responsible for eliciting a direct response to noxious thermal stimuli. Previous studies have also shown that the hypotensive properties of antinociceptive agents do not produce false indication of antinociception in thermal tests (Michaluk et al., 1998; Dogrul et al., 1997). It should be noted, however, that in the present study, the systemic mibefradil blood pressure effects were determined in non-injured rats, rather than those with SNL, raising the possibility of local blood flow changes occurring either at the area of the injured nerves or in the paw as a potential mechanism by which the antiallodynic and antihyperalgesic actions might occur. Since mibefradil administered directly into the paw did produce antiallodynic and antihyperalgesic actions without hypotension, this possibility seems unlikely but remains to be investigated. Furthermore, the same dose given into the contralateral hindpaw also did not alter the enhanced behavioral response of the injured hindpaw. These data indicate that mibefradil acts at a local site of action rather than through systemic mechanisms. Finally, other studies have shown that the systemic administration of mibefradil did not block normal nocifensive responses to acute thermal noxious stimuli (Dogrul et al., 2001, 2002). In one contrasting study, 3 mg/kg, i.p. of mibefradil reportedly produced antinociception, based on a statistically significant elevation of paw withdrawal latency (Todorovic et al., 2002). However, the significant increase occurred at one time point (2 h after injection) and was very modest, reflecting a change of only 8– 19%. A higher dose (9 mg/kg) produced elevations of only 22%, 1 h after injection (Todorovic et al., 2002). The magnitude of these changes, and the absence of observed effects of much higher doses of mibefradil in the present study and in mice (Dogrul et al., 2001, 2002), suggest that mibefradil does not directly elicit antinociception. An interesting observation is that systemic, but not local, administration of ethosuximide was active against experimental neuropathic pain. It seems likely that the absence of activity of i.pl. ethosuximide against experimental neuropathic pain may be due to its low potency (24-fold) compared to mibefradil when given systemically. A surprising finding of the present study is that neither mibefradil nor ethosuximide blocks experimental neuropathic pain after spinal administration. The reasons for this are unknown. It is possible that the doses employed here were insufficient to generate an effect. Alternatively, it suggests that a peripheral, rather than central, site of action of these blockers underlies their effect on neuropathic pain states. The latter is also based on earlier observations by Matthews and Dickenson (2001a) who showed that an i.th.
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administration of up to 1000 mg of ethosuximide was required to produce a dose-dependent inhibition of dorsal horn units in halothane-anaesthetized sham-operated and SNL rats in response to electrical stimulation of Ab, Ad and c-fibers, or noxious and non-noxious tactile or thermal stimuli. It was also found that the degree of inhibition of evoked activity by ethosuximide did not differ among naive, sham-operated and SNL rats, leading to the suggestion that L5/L6 SNL produced little or no change in functionality of T-type VSCCs in the dorsal horn units (Matthews and Dickenson, 2001a). The peripheral activity of mibefradil shown in the present study points to the possibility that the biophysical properties, and/or the changes in expression or distribution of the T-type VSCC in the peripheral terminals of primary afferent fibers could differ substantially from those found centrally upon nerve injury. In this regard, three isoforms of the a1 subunit for T-type VSCC have been identified to date, namely a1G (CavT.1), a1H (CavT.2) and a1I (CavT.3) (Chemin et al., 2002; Lambert et al., 1997, 1998; Perez-Reyes et al., 1998). The results of the present study indicate that blockers of the T-type VSCCs may be useful in the management of neuropathic pain states. The major site of action of these compounds appears to be in the periphery. The contributions of the different isoforms of the T-type VSCC to manifestations of neuropathic pain remain to be elucidated and further, it is unclear if the composition of the subunits is altered in the nerve-injured state. Additionally, the effects of peripheral nerve injury on the physiologic and pharmacologic characteristics of these channels are also unknown. In spite of these unresolved issues, these results suggest that the T-type VSCC blockers such as mibefradil and ethosuximide could represent a new class of drugs for the treatment of chronic neuropathic pain.
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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. Kostyuk PG, Molokanova EA, Pronchuk NF, Savchenko AN, Verkhratsky AN. Different action of ethosuximide on low- and high-threshold calcium currents in rat sensory neurons. Neuroscience 1992;51:755– 8. Lai J, Gold MS, Kim C, Bian D, Ossipov MH, Hunter JC, Porreca F. Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, Nav1.8. Pain 2002;95:143–52. Lambert RC, Maulet Y, Mouton J, Beattie R, Volsen S, De Waard M, Feltz A. T-type Ca2þ current properties are not modified by Ca2þ channel beta subunit depletion in nodosus ganglion neurons. J Neurosci 1997; 17:6621–8. Lambert RC, McKenna F, Maulet Y, Talley EM, Bayliss DA, Cribbs LL, Lee JH, Perez-Reyes E, Feltz A. Low-voltage-activated Ca2þ currents are generated by members of the CavT subunit family (alpha1G/H) in rat primary sensory neurons. J Neurosci 1998;18:8605 –13. Liu X, Eschenfelder S, Blenk KH, Janig W, Habler H. Spontaneous activity of axotomized afferent neurons after L5 spinal nerve injury in rats. Pain 2000;84:309–18. Magee JC, Christofi G, Miyakawa H, Christie B, Lasser-Ross N, Johnston D. Subthreshold synaptic activation of voltage-gated Ca2þ channels mediates a localized Ca2þ influx into the dendrites of hippocampal pyramidal neurons. J Neurophysiol 1995;74:1335–42. Markram H, Sakmann B. Calcium transients in dendrites of neocortical neurons evoked by single subthreshold excitatory postsynaptic potentials via low-voltage-activated calcium channels. Proc Natl Acad Sci USA 1994;91:5207–11. Matthews EA, Dickenson AH. Effects of ethosuximide, a T-type Ca(2þ) channel blocker, on dorsal horn neuronal responses in rats. Eur J Pharmacol 2001a;415:141– 9. Matthews EA, Dickenson AH. Effects of spinally delivered N- and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model of neuropathy. Pain 2001b;92:235–46. Michaluk J, Karolewicz B, Antkiewicz-Michaluk L, Vetulani J. Effects of various Ca2þ channel antagonists on morphine analgesia, tolerance and dependence, and on blood pressure in the rat. Eur J Pharmacol 1998; 352:189–97. Miljanich GP, Ramachandran J. Antagonists of neuronal calcium channels: structure, function, and therapeutic implications. Annu Rev Pharmacol Toxicol 1995;35:707–34. Millan MJ. The induction of pain: an integrative review. Prog Neurobiol 1999;57:1–164. Monteil A, Chemin J, Bourinet E, Mennessier G, Lory P, Nargeot J. Molecular and functional properties of the human alpha(1G) subunit that forms T-type calcium channels. J Biol Chem 2000;275:6090–100. Nebe J, Ebersberger A, Vanegas H, Schaible HG. Effects of omegaagatoxin IVA, a P-type calcium channel antagonist, on the development
of spinal neuronal hyperexcitability caused by knee inflammation in rats. J Neurophysiol 1999;81:2620– 6. Perez-Reyes E, Cribbs LL, Daud A, Lacerda AE, Barclay J, Williamson MP, Fox M, Rees M, Lee JH. Molecular characterization of a neuronal low-voltage-activated T-type calcium channel. Nature 1998;391: 896 –900. Perney TM, Hirning LD, Leeman SE, Miller RJ. Multiple calcium channels mediate neurotransmitter release from peripheral neurons. Proc Natl Acad Sci USA 1986;83:6656 –9. Porreca F, Ossipov MH, Gebhart GF. Chronic pain and medullary descending facilitation. Trends Neurosci 2002;25:319– 25. Rasband MN, Park EW, Vanderah TW, Lai J, Porreca F, Trimmer JS. Distinct potassium channels on pain-sensing neurons. Proc Natl Acad Sci USA 2001;98:13373–8. Schild JH, Kunze DL. Experimental and modeling study of Na þ current heterogeneity in rat nodose neurons and its impact on neuronal discharge. J Neurophysiol 1997;78:3198–209. Sekizawa SI, French AS, Torkkeli PH. Low-voltage-activated calcium current does not regulate the firing behavior in paired mechanosensory neurons with different adaptation properties. J Neurophysiol 2000;83: 746 –53. 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. Todorovic SM, Meyenburg A, Jevtovic-Todorovic V. Mechanical and thermal antinociception in rats following systemic administration of mibefradil, a T-type calcium channel blocker. Brain Res 2002;951: 336 –40. White DM, Cousins MJ. Effect of subcutaneous administration of calcium channel blockers on nerve injury-induced hyperalgesia. Brain Res 1998;801:50–8. Woolf CJ, Thompson SW. The induction and maintenance of central sensitization is dependent on N-methyl-D -aspartic acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states. Pain 1991;44:293–9. Xiao WH, Bennett GJ. Synthetic omega-conopeptides applied to the site of nerve injury suppress neuropathic pains in rats. J Pharmacol Exp Ther 1995;274:666–72. Yaksh TL, Chaplan SR. Physiology and pharmacology of neuropathic pain. Anesthesiol Clin N Am 1997;15:335–53. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6. Yamamoto T, Sakashita Y. Differential effects of intrathecally administered N-, P-type voltage-sensitive calcium channel blockers upon two models of experimental mononeuropathy in the rat. Brain Res 1998;794: 329 –32. Yoon YW, Na HS, Chung JM. Contributions of injured and intact afferents to neuropathic pain in an experimental rat model. Pain 1996; 64:27–36.
Reversal of experimental neuropathic pain by T-type calcium channel blockers Ahmet Dogrula, Luis R. Gardellb, Michael H. Ossipovb, F. Cankat Tulunayc, Josephine Laib, Frank Porrecab,* a b
Department of Pharmacology, Faculty of Medicine, Gulhane Medical Military Academy, Ankara, Turkey Department of Pharmacology, The University of Arizona Health Sciences Center, Tucson, AZ 85724, USA c Department of Pharmacology, Faculty of Medicine, Ankara University, Ankara, Turkey Received 7 November 2002; received in revised form 16 April 2003; accepted 22 April 2003
Abstract Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca2þ current is associated with increased neuronal activity and blockade of N- and P-types, but not L-type, calcium channels have been found to block experimental neuropathic pain. While T-type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L5/L6 spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose-dependent blockade of both tactile and thermal hypersensitivities in nerve-injured rats; responses of sham-operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T-type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T-type calcium channel blockers may have significant potential in the treatment of neuropathic pain states. q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: T-type voltage sensing calcium channels; Neuropathic pain; Rat
1. Introduction Pain-related behaviors caused by peripheral nerve injury result partly from an increase in transduction sensitivity of primary afferents and increased activity within the central nervous system (Burgess et al., 2002; Coderre et al., 1993; Kajander and Bennett, 1992; Porreca et al., 2002). Peripheral nerve injury results in abnormal ectopic discharges from the injury site or the dorsal root ganglia (DRG), causing enhanced central input from the damaged primary afferent fibers and manifesting behaviorally as neuropathic pain (Kajander and Bennett, 1992; Yaksh and Chaplan, 1997; Gracely et al., 1992; Sheen and Chung, * Corresponding author. Tel.: þ 1-520-626-7421; fax: þ1-520-626-4182. E-mail address: [email protected] (F. Porreca).
1993; Yoon et al., 1996). Furthermore, injured small- and large-diameter primary afferent fibers show marked alterations in their pattern of excitability and conduction properties during evoked and spontaneous activity, reflecting alterations in the density and/or operating characteristics of multiple ion channels, including novel expression of ion channels (Devor and Seltzer, 1999; Millan, 1999; Yaksh and Chaplan, 1997). These differential alterations in the density and/or properties of specific ion channels upon injury to primary afferent fibers may be of functional importance in determining changes in primary afferent discharge and conduction (Gold et al., in press; Lai et al., 2002; Rasband et al., 2001; Schild and Kunze, 1997). Increases in local Ca2þ concentration at the site of injury or in the spinal cord may contribute to the development of neuropathic pain (Gold et al., 1994; Kawamata and Omote,
0304-3959/03/$20.00 q 2003 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0304-3959(03)00177-5
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1996). Five major types of voltage sensing calcium channels (VSCCs) have been identified (L-, T-, N-, P/Q- and Rtypes), with unique electrophysiologic and pharmacologic characteristics (Catterall, 2000; Bertolino and Llinas, 1992). The L- and N-type VSCCs have been implicated in the release of neurotransmitters and neuromodulators from sensory neurons in the spinal cord (Anwyl, 1991; Perney et al., 1986). Blockade of the N- and P/Q-type VSCCs, but not of the L-type VSCC, attenuated behavioral signs of neuropathic pain in animal models of nerve injury (Chaplan et al., 1994b; Matthews and Dickenson, 2001b; Nebe et al., 1999; Yamamoto and Sakashita, 1998; White and Cousins, 1998; Miljanich and Ramachandran, 1995; Xiao and Bennett, 1995). The T-type VSCC is thought to lower the threshold for action potentials and promote bursting activity and synaptic excitation, which are actions that would favor the development of enhanced pain (Matthews and Dickenson, 2001a; Sekizawa et al., 2000). However, until recently (Matthews and Dickenson, 2001a), the lack of selective antagonists for these channels has prevented evaluation of their contribution to the manifestation of neuropathic pain. Ethosuximide is relatively selective for T-type VSCC (Matthews and Dickenson, 2001a; Coulter et al., 1989), blocking T-type currents in rat sensory neurons at half the concentration needed to block the L-current (Kostyuk et al., 1992). In contrast, mibefradil demonstrates between tenfold and 100-fold selectivity in blocking T-type current over L-type current, indicating considerable selectivity(Jimenez et al., 2000; Monteil et al., 2000; Clozel et al., 1999; Ertel and Ertel, 1997; Ertel et al., 1997; Hermsmeyer et al., 1997). These compounds were thus employed here to explore the contributions of the T-type VSCC to experimental neuropathic pain.
2. Materials and methods Male Sprague –Dawley rats (Harlan, Indianapolis, IN), 200 –300 g at the time of testing, were maintained in a climate-controlled room on a 12-h light/dark cycle (lights on at 06:00 hours). Food and water were available ad libitum. All testing was performed in accordance with the policies and recommendations of the International Association for the Study of Pain and the National Institutes of Health guidelines for the handling and use of laboratory animals. These studies also received approval from the Institutional Animal Care and Use Committee of the University of Arizona. Mibefradil dihydrochloride was obtained as a gift from Roche (Basel, Switzerland). 2.1. Spinal nerve ligation Nerve injury was induced with the L5/L6 spinal nerve ligation (SNL) according to the procedure of Kim and Chung (1992). Anesthesia was induced with 2% halothane
in O2 at a rate of 2 l/min and maintained with 0.5% halothane in O2. After surgical preparation of the rats and exposure of the dorsal vertebral column from L4 to S2, the L5 and L6 spinal nerves were tightly ligated distal to the DRG with a 4-0 silk suture. The incision was closed and the animals were allowed to recover for 5 days. Rats that exhibited motor deficiency or failure to exhibit subsequent tactile hypersensitivity were excluded from further testing. Sham surgery was performed by exposing the spinal nerves without performing the ligation. Following recovery, rats were anesthetized as described above and implanted with catheters for intrathecal (i.th.) administration of drugs into the region of the lumbar spinal cord according to the method described by Yaksh and Rudy (1976). A PE-10 polyethylene tubing was inserted 8 cm into the vertebral canal through an incision made in the atlanto-occipital membrane and secured to the musculature at the incision. This length of tubing has been shown to consistently terminate at the level of the lumbar enlargement when used with rats within the weight range employed in the present studies. Misdirected catheters result in damage to the spinal cord or spinal nerves and also result in gross motor deficiencies or paralysis. These animals are not used, but immediately euthanized. The rats received 4.4 mg/kg of gentamycin intramuscularly and were allowed to recover for a period of at least 5 days prior to testing. Rats exhibiting motor deficiency were discarded from testing. Drugs administered i.th. were given in a volume of 5 ml, followed by a 9-ml flush. The intraperitoneal (i.p.) injections were made in a volume of 0.1 ml/kg and the intraplantar (i.pl.) injections were given in a constant volume of 50 ml. The assessment of tactile hypersensitivity was determined according to the method described in detail by Chaplan et al. (1994a). A series of eight calibrated von Frey filaments with filament strengths placed at logarithmic intervals and ranging from 0.41 to 15 g were used. The probing was initiated with a filament strength of 2 g applied to the plantar aspect of the hindpaw of rats kept in suspended wire-mesh cages. If there was no response, then the stimulus was increased by one increment, otherwise it was decreased by one decrement. The stimulus was incrementally increased until a positive response was obtained, then decreased until a negative result was observed. This ‘up – down’ method was repeated until three changes in behavior were determined, and a withdrawal threshold based on the pattern of responses was calculated with the Dixon non-parametric statistic (Chaplan et al., 1994a; Dixon, 1980). When continuous positive responses occurred below the minimal stimulus (0.41 g) or when the maximal stimulus (i.e. 15 g) produced no response, then the cut-off values of 0.41 and 15, respectively, were assigned (Chaplan et al., 1994a; Dixon, 1980). The method of Hargreaves was employed to assess thermal hyperalgesia (Hargreaves et al., 1988). Rats were allowed to acclimate within Plexiglas enclosures on a clear glass plate maintained at 30 8C. A radiant heat source
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(i.e. high intensity projector lamp) was activated along with a timer and focused onto the plantar surface of the affected paw of nerve-injured or sham-operated rats. A motion sensor activated by paw withdrawal halted both lamp and timer. A maximal cut-off of 40 s was used to prevent tissue damage. In all tests, paw withdrawal thresholds and paw withdrawal latencies were obtained for the ligated and shamoperated groups before drug or vehicle injections. Rats were tested at 0, 15, 30, 45 and 60 min after injections. Within each of the treatment groups, post-injection means were compared with the baseline values by analysis of variance (ANOVA), followed by post hoc analysis of least significance difference for multiple comparisons. Comparisons between two means were performed by Student’s t-test. A probability level of 0.05 indicated significance. In order to generate a dose – response curve against tactile hypersensitivity, data were converted to %MPE (maximal possible effect) by the formula: %MPE ¼ 100 £ (test value 2 baseline value)/(15 g 2 baseline value). In order to generate a dose –response curve against thermal hyperalgesia, data were converted to %MPE by the formula: %MPE ¼ 100 £ (test latency 2 baseline latency)/(sham animal latency 2 baseline latency). The A50 dose (i.e. dose producing 50% MPE) and 95% confidence intervals were determined by linear regression analysis of the log dose – response curve. Experimenters were blinded to the experimental conditions. 2.2. Mean arterial blood pressure Rats were anesthetized with ketamine/xylazine (20:3; 100 mg/kg). A rectal probe was used to monitor body temperature, which was maintained by a feedback controlled heating pad. A polyethylene catheter (PE-50) was implanted into a carotid artery and connected to a Grass PT300 pressure transducer (Grass-Telefactor, West Warwick, RI). Data were transferred from the pressure transducer to a computer by means of an input/output port of the personal computer (PC) and captured, and heart rate, systolic, diastolic and mean arterial pressures are calculated with the JAD data acquisition software (Notocord, Paris, France). The effects of i.p. (5, 10 and 20 mg/kg) and i.pl. (30, 100 and 300 mg) administration of mibefradil on systolic and diastolic arterial blood pressures were evaluated.
3. Results 3.1. Blockade of tactile hypersensitivity Sham-operated rats displayed paw withdrawal thresholds to probing with von Frey filaments of 15 ^ 0 g. In contrast, rats with L5/L6 SNL demonstrated significantly lower paw withdrawal thresholds ðP , 0:05Þ, indicating tactile hypersensitivity. The mean paw withdrawal thresholds of rats with SNL ranged from 1.18 ^ 0.3 to 1.82 ^ 0.43 g. The i.p.
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injection of 5, 10 and 20 mg/kg of mibefradil reversed tactile hypersensitivity in a dose- and time-dependent fashion (Fig. 1). A peak effect of 14.13 ^ 0.55 g was obtained within 30 min of administration of the highest test dose of mibefradil (20 mg/kg, i.p.) (Fig. 1). The A50 for i.p. mibefradil was 7.44 mg/kg (95% CL 5.33– 10.4 mg/kg). Similarly, ethosuximide (50 – 300 mg/kg, i.p.) elicited a dose- and time-dependent reversal of tactile hypersensitivity. A maximal effect of 10.89 ^ 2.45 g was detected 30 min following the administration of the highest dose tested (300 mg/kg, i.p.) (Fig. 1). The A50 was 174 mg/kg (95% CL 58.1–524 mg/kg), i.p., indicating a significantly lower (23-fold) potency when compared with mibefradil against tactile hypersensitivity ðP # 0:05Þ (Fig. 1). The injection of 30, 100 and 300 mg of mibefradil into the plantar aspect of the injured hindlimb also produced dose-dependent reversal of tactile hypersensitivity, suggesting a local mechanism of action (Fig. 2). The maximal effect of mibefradil following i.pl. administration was 13.87 ^ 0.47 g, 15 min after the administration of 300 mg of mibefradil, and remained elevated 60 min postinjection (Fig. 2). The dose – effect curve generated from the data gathered at the time of peak effect yielded an A50 value of 92.4 mg (95% CL of 73.1– 117 mg) (Fig. 2). In contrast, injection of mibefradil (300 mg) into the hindpaw contralateral to the site of nerve injury was ineffective against tactile hypersensitivity of the hindpaw ipsilateral to SNL. The mean withdrawal threshold was 1.29 ^ 0.27 g, 30 min after injection and was not significantly different from baseline. Ethosuximide, injected i.pl. at a dosage of up to 500 mg, failed to produce any changes in paw withdrawal thresholds to light tactile stimuli (Fig. 2). The i.th. injection of mibefradil or of ethosuximide over the dose range of 10– 40 mg did not produce any significant changes in paw withdrawal threshold at any time (data not shown). 3.2. Blockade of thermal hyperalgesia SNL produced reliable thermal hyperalgesia in all groups tested. The mean baseline paw withdrawal latencies for all treatment groups ranged between 12.72 ^ 0.82 and 14.11 ^ 0.72 s after SNL, indicating consistency and reproducibility of the measurements among groups. These values were significantly less than the mean baseline paw withdrawal latency of 20.21 ^ 1.05 s displayed by the sham-operated rats ðP # 0:05Þ. The i.p. injection of mibefradil (5, 10 and 20 mg/kg) exerted a dose-dependent reversal of thermal hyperalgesia. The highest dose tested (20 mg/kg, i.p.) elicited an antihyperalgesic effect within 30 min of injection, as indicated by increased paw withdrawal latencies to 19.92 ^ 1.36 s, and antihyperalgesia was evident for 60 min (Fig. 3). The calculated i.p. A50 value of mibefradil was 11.9 mg/kg (95% CL 6.85– 20.5 mg/kg). Similarly, ethosuximide produced a dose-dependent antihyperalgesic effect over the dose range of 50 – 300 mg/kg, i.p. (Fig. 3). The highest dose of ethosuximide tested
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Fig. 1. Male Sprague– Dawley rats received L5/L6 SNL. Paw withdrawal thresholds to probing with von Frey filaments were determined prior to and at select time points after the i.p. injection of several doses of mibefradil (A) or ethosuximide (B). Both mibefradil and ethosuximide produced dose- and timedependent reversal of tactile hypersensitivity. Paw withdrawal thresholds at the time of peak effect (30 min) were converted to %MPE in order to generate dose–response curves (C). N ¼ 6 animals per group.
(300 mg/kg, i.p.) elicited an antihyperalgesic effect within 30 min of injection, as indicated by increased paw withdrawal latencies to 22.5 ^ 2.34 s (Fig. 5). The A50 value generated from data collected at the time of peak effect was 126 mg/kg (95% CL 60.1 – 265 mg/kg, i.p.) (Fig. 3). These data indicate that mibefradil has a significant, 11-fold greater potency than ethosuximide against thermal hyperalgesia ðP # 0:05Þ. Likewise, the i.pl. injection of mibefradil (30, 100 and 300 mg) into the injured hindlimb produced
a dose-dependent reversal of thermal hyperalgesia (Fig. 4), whereas the injection of the same doses into the hindpaw of sham-operated rats produced no change in paw withdrawal latency (data not shown). A maximal antihyperalgesic effect was found within 15 min and persisted for 60 min. The highest dose (300 mg) of mibefradil elicited a maximal antihyperalgesic effect within 15 min after injection, as indicated by the increase in paw withdrawal latencies to 20.0 ^ 1.0 s (Fig. 4). Importantly, the microinjection of 300 mg of mibefradil into the hindpaw contralateral to
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Fig. 2. Male Sprague–Dawley rats received L5/L6 SNL. Paw withdrawal thresholds to probing with von Frey filaments were determined prior to and at select time points after several doses of mibefradil or a maximal (500 mg) dose of ethosuximide (A). Injections were made s.c. into the plantar aspect of the injured hindpaw. Mibefradil produced dose- and time-dependent increases in paw withdrawal thresholds to tactile stimuli. The maximal dose of ethosuximide tested was not active against tactile hypersensitivity. Paw withdrawal thresholds at the time of peak effect of mibefradil (15 min) were converted to %MPE in order to generate dose–response curves (B). N ¼ 6 rats per group.
the injured paw did not produce any elevation in paw withdrawal latency of the hindpaw ipsilateral to SNL. The paw withdrawal latencies of the injured hindpaw did not change from the baseline value of 12.9 ^ 0.16 s over the 60-min observation period. Dose–response curves for the antihyperalgesic effect of mibefradil were constructed from data gathered at the time of peak effect. The A50 value after i.pl. injection was 72.0 mg (95% CL 62.0–83.6 mg) (Fig. 4). In contrast, the i.pl. injection of up to 500 mg of ethosuximide did not produce any changes in paw withdrawal latencies in SNL rats. The i.th. injection of 10–40 mg of either mibefradil or ethosuximide did not produce any change in paw withdrawal threshold at any time, indicating a lack of spinal antihyperalgesic activity (data not shown). In addition, the administration of 20 mg/kg i.p., 40 mg i.th. and 300 mg i.pl. of mibefradil to sham-operated rats did not increase paw withdrawal latencies beyond the respective baseline paw withdrawal latencies (Fig. 5), indicating an absence of antinociceptive activity.
3.3. Effects of mibefradil on arterial blood pressure Prior to drug administration, the animals had mean systolic and diastolic arterial blood pressures of 145.7 ^ 8.7 and 111 ^ 2.8 mmHg, respectively. The administration of mibefradil over the dose range of 5, 10 and 20 mg/kg i.p. induced a dose-dependent decrease in systolic and diastolic arterial blood pressures when compared with saline, whereas the i.pl. injection of the highest dose of mibefradil (300 mg) into hindlimb did not alter systolic and diastolic arterial pressures. The blood pressure lowering effects of mibefradil when given i.p. were found to begin within 15 min and remained over 60 min. The highest dose of mibefradil (20 mg/kg, i.p.) lowered systolic and diastolic arterial blood pressures to 93.7 ^ 2.8 and 64.6 ^ 4.2 mmHg, respectively, at 15 min following drug administration. The mean systolic and diastolic blood pressures were 143.8 ^ 4.7 and 112 ^ 1.78 mmHg, respectively, after the highest i.pl.
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Fig. 3. Male Sprague–Dawley rats received L5/L6 SNL. Paw withdrawal latencies to noxious radiant heat applied to the plantar aspect of the hindpaw were determined prior to and at select time points after the i.p. injection of several doses of mibefradil (A) or ethosuximide (B). Both mibefradil and ethosuximide produced dose- and time-dependent elevations in paw withdrawal latencies. The paw withdrawal latencies at the time of peak effect (30 min) were converted to %MPE in order to generate dose–response curves (C). N ¼ 6 animals per group.
dose of mibefradil (300 mg), which were not different from the control values.
4. Discussion The results of the present study indicate that the systemic administration of mibefradil and ethosuximide, both T-type VSCC blockers, effectively reverses the behavioral manifestation of neuropathic pain. This result is consistent with the electrophysiologic properties of the T-type VSCC
indicating that it may play a critical role in the modulation of neuronal excitation (Amir et al., 2002; Huguenard, 1996; Matthews and Dickenson, 2001a), and is therefore suited to modulate neuronal hyperexcitation after nerve injury. The T-type VSCCs, first described in peripheral sensory neurons of the DRG, are activated by subthreshold excitatory postsynaptic potentials (EPSPs), reducing threshold for the generation of action potentials and therefore contributing to the excitability of neuronal membranes (Carbone and Lux, 1984a,b; Huguenard, 1996; Magee et al., 1995; Markram and Sakmann, 1994; Matthews and Dickenson, 2001a;
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Fig. 4. Male Sprague– Dawley rats received L5/L6 SNL. Paw withdrawal latencies to noxious radiant heat applied to the plantar aspect of the injured hindpaw were determined prior to and at select time points after several doses of mibefradil or a maximal (500 mg) dose of ethosuximide (A). Injections were made s.c. into the plantar aspect of the injured hindpaw. Mibefradil produced dose- and time-dependent increases in paw withdrawal thresholds to noxious thermal stimuli. In contrast, the maximal dose of ethosuximide tested was not active against thermal hyperalgesia. Paw withdrawal latencies at the time of peak effect of mibefradil (15 min) were converted to %MPE in order to generate dose– response curves (B). N ¼ 6 rats per group.
Fig. 5. Sham-operated male Sprague–Dawley rats received mibefradil by i.p. injection (20 mg/kg), i.pl. injection (300 mg) or i.th. injection (40 mg). These doses represent the highest doses given to SNL rats. In addition, sham-operated rats also received saline i.p. as a reference control. Paw withdrawal latencies did not change from the pre-injection baseline values for any of the groups tested, and were identical to the responses of saline-injected rats, thus indicating a complete absence of antinociceptive activity. N ¼ 6 rats per group.
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Sekizawa et al., 2000). It has been suggested that the development of behavioral manifestations of neuropathic pain is due, at least in part, to spontaneous ectopic discharge of primary afferent neurons and subsequent sensitization of dorsal horn neurons (Devor and Seltzer, 1999; Woolf and Thompson, 1991). The enhanced activity presents as increased neuronal responses to stimuli and also as spontaneous discharges that may originate from the site of injury, the DRG and also from remaining intact fibers (Devor and Seltzer, 1999; Kajander and Bennett, 1992). Accordingly, a fourfold to sixfold increase in primary afferent activity is detected almost immediately after nerve injury, but diminishes rapidly during the first week after injury while neuropathic pain persists (Han et al., 2000; Liu et al., 2000). The temporal disparity between ectopic activity and neuropathic pain states point to the possibility that other mechanisms may also be critical in the maintenance of the neuropathic state (Burgess et al., 2002; Porreca et al., 2002). Ectopic activity as well as enhanced release from primary afferents may be susceptible to blockade by mibefradil or ethosuximide since T-type VSCCs contribute to transmitter release and neuronal sensitization (Matthews and Dickenson, 2001a). In this study, ethosuximide was found to inhibit input spikes, indicative of diminished synaptic activity, probably through a reduction in exocytosis of neurotransmitter from primary afferent neurons. Furthermore, ethosuximide also inhibited N-methyl-D -aspartate (NMDA) mediated post-discharge spikes, which are indices of spinal sensitization and hypersensitivity (Matthews and Dickenson, 2001a). These mechanisms suggest a potential role for the T-type VSCC in the genesis of neuropathic pain. Consistent with this hypothesis, we found that systemic and locally applied mibefradil, and systemic ethosuximide, blocked tactile and thermal hypersensitivities after SNL. Furthermore, these results also agree with the observation that peripheral nerve injury results in an increase in the concentration of intracellular Ca2þ at the site of injury (Gold et al., 1994). White and Cousins (1998) reported that a subcutaneous (s.c.) injection of calcium chelating agent Quin 2 at the receptive field of an injured nerve blocked mechanical hyperalgesia and suggested a local calciumdependent mechanism in the receptive field of injured neurons. As mibefradil has strong cardiovascular effects, it might be argued that mibefradil-induced hypotension may interfere with paw withdrawal responses, and thus be misinterpreted as thermal antihyperalgesic and antiallodynic effects. However, it is unlikely that this reduction in mean arterial blood pressure contributes to blockade of thermal or tactile hypersensitivity, as the greatest dose of systemic mibefradil used in this study (i.e. 20 mg/kg, i.p.) exerted a nearly 40% reduction in systolic blood pressure, but did not induce any antinociceptive effect in thermal nociception in sham-operated animals. Additionally, local administration of mibefradil into the paw on the injured, but
not uninjured, side produced antiallodynic and antihyperalgesic actions. Local mibefradil did not affect blood pressure suggesting that the observed effects were not the result of changes in blood pressure. A further point here is that intrapaw mibefradil did not produce analgesia, suggesting that local blood flow changes were unlikely to be responsible for eliciting a direct response to noxious thermal stimuli. Previous studies have also shown that the hypotensive properties of antinociceptive agents do not produce false indication of antinociception in thermal tests (Michaluk et al., 1998; Dogrul et al., 1997). It should be noted, however, that in the present study, the systemic mibefradil blood pressure effects were determined in non-injured rats, rather than those with SNL, raising the possibility of local blood flow changes occurring either at the area of the injured nerves or in the paw as a potential mechanism by which the antiallodynic and antihyperalgesic actions might occur. Since mibefradil administered directly into the paw did produce antiallodynic and antihyperalgesic actions without hypotension, this possibility seems unlikely but remains to be investigated. Furthermore, the same dose given into the contralateral hindpaw also did not alter the enhanced behavioral response of the injured hindpaw. These data indicate that mibefradil acts at a local site of action rather than through systemic mechanisms. Finally, other studies have shown that the systemic administration of mibefradil did not block normal nocifensive responses to acute thermal noxious stimuli (Dogrul et al., 2001, 2002). In one contrasting study, 3 mg/kg, i.p. of mibefradil reportedly produced antinociception, based on a statistically significant elevation of paw withdrawal latency (Todorovic et al., 2002). However, the significant increase occurred at one time point (2 h after injection) and was very modest, reflecting a change of only 8– 19%. A higher dose (9 mg/kg) produced elevations of only 22%, 1 h after injection (Todorovic et al., 2002). The magnitude of these changes, and the absence of observed effects of much higher doses of mibefradil in the present study and in mice (Dogrul et al., 2001, 2002), suggest that mibefradil does not directly elicit antinociception. An interesting observation is that systemic, but not local, administration of ethosuximide was active against experimental neuropathic pain. It seems likely that the absence of activity of i.pl. ethosuximide against experimental neuropathic pain may be due to its low potency (24-fold) compared to mibefradil when given systemically. A surprising finding of the present study is that neither mibefradil nor ethosuximide blocks experimental neuropathic pain after spinal administration. The reasons for this are unknown. It is possible that the doses employed here were insufficient to generate an effect. Alternatively, it suggests that a peripheral, rather than central, site of action of these blockers underlies their effect on neuropathic pain states. The latter is also based on earlier observations by Matthews and Dickenson (2001a) who showed that an i.th.
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administration of up to 1000 mg of ethosuximide was required to produce a dose-dependent inhibition of dorsal horn units in halothane-anaesthetized sham-operated and SNL rats in response to electrical stimulation of Ab, Ad and c-fibers, or noxious and non-noxious tactile or thermal stimuli. It was also found that the degree of inhibition of evoked activity by ethosuximide did not differ among naive, sham-operated and SNL rats, leading to the suggestion that L5/L6 SNL produced little or no change in functionality of T-type VSCCs in the dorsal horn units (Matthews and Dickenson, 2001a). The peripheral activity of mibefradil shown in the present study points to the possibility that the biophysical properties, and/or the changes in expression or distribution of the T-type VSCC in the peripheral terminals of primary afferent fibers could differ substantially from those found centrally upon nerve injury. In this regard, three isoforms of the a1 subunit for T-type VSCC have been identified to date, namely a1G (CavT.1), a1H (CavT.2) and a1I (CavT.3) (Chemin et al., 2002; Lambert et al., 1997, 1998; Perez-Reyes et al., 1998). The results of the present study indicate that blockers of the T-type VSCCs may be useful in the management of neuropathic pain states. The major site of action of these compounds appears to be in the periphery. The contributions of the different isoforms of the T-type VSCC to manifestations of neuropathic pain remain to be elucidated and further, it is unclear if the composition of the subunits is altered in the nerve-injured state. Additionally, the effects of peripheral nerve injury on the physiologic and pharmacologic characteristics of these channels are also unknown. In spite of these unresolved issues, these results suggest that the T-type VSCC blockers such as mibefradil and ethosuximide could represent a new class of drugs for the treatment of chronic neuropathic pain.
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