Introduction

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Peripheral nerve injury can lead to a neuropathic pain state, termed tactile allodynia, in which innocuous tactile stimulation elicits pain behavior. Spinal administration of gabapentin, a novel anticonvulsant that binds to the ₂ subunits of the voltage-gated calcium channels in vitro (Marais et al., 2001), suppresses allodynia in neuropathic pain models without general analgesic effects (Hwang and Yaksh, 1997; Abdi et al., 1998; Field et al., 1999). In addition, the potencies of gabapentinoids against neuropathic pain correlate with their sterospecificity and binding affinities at the ₂ site (Suman-Chauhan et al., 1993; Dissanayake et al., 1997; Hwang and Yaksh, 1997). These observations have led to the hypothesis that nerve injury may cause changes in spinal ₂ subunit expression, which in turn results in an enhanced neuronal excitability that contributes to neuropathic pain development.

The ₂ subunit is important for functional assembly of the voltage-gated calcium channels. It is a glycoprotein consisting of covalently linked ₂- and -peptides that are encoded by the same gene (De Jongh et al., 1990). Except for a single transmembrane domain and five C-terminal amino acids, the majority of the ₂ subunit is extracellular. In vitro studies have indicated that the extracellular domain of the subunit is important for channel function and coexpression of the ₂ subunit with other calcium channel subunits results in enhanced calcium channel currents. This is accompanied by an increase in both the number of binding sites and their affinity for -conotoxin, a ligand for neuronal voltage-gated calcium channels (Mori et al., 1991; Williams et al., 1992; Brust et al., 1993; Gurnett et al., 1996). Three genes have been identified in mice that encode the ₂-1, ₂-2, and ₂-3 subunits, respectively (Klugbauer et al., 1999). The tissue-specific expression patterns of these subunits suggest that they may have diversified functions (Marais et al., 2001), and recent studies have suggested that the three ₂ subunits may contribute differentially to sensory information processing. In situ studies have shown that mRNA for the ₂-1 and ₂-2 subunits is expressed at high levels in small dorsal root ganglion (DRG) sensory neurons and at lower levels in large DRG neurons. Conversely, mRNA for the ₂-3 is relatively abundant in large DRG neurons and scarce in small sensory neurons (Yusaf et al., 2001b). Binding studies have shown that the ₂-1 and ₂-2, but not the ₂-3, subunits bind gabapentin with high affinities (Marais et al., 2001).

We have recently observed a marked up-regulation of the ₂-1 subunit in rat DRG that correlated tightly with gabapentin-sensitive tactile allodynia after spinal nerve ligation (Luo et al., 2001). This prompted the speculation that the ₂-1 subunit may play a role in gabapentin-sensitive tactile allodynia. However, tactile allodynia may be induced by a variety of nerve lesions, and it is not clear that these findings can be extrapolated to all nerve injury states that exhibit tactile allodynia. Indeed, data from clinical investigations have indicated that gabapentin sensitivity varies in neuropathic pain states arising from different types of nerve injury, suggesting that the mechanisms underlying the action of gabapentin in pain states of differing etiology may vary (Laird and Gidal, 2000). As a first step toward uncovering the potential role of the ₂-1 subunit in neuropathic pain, we investigated whether increased ₂-1 subunit expression is common to a range of nerve injury models that display tactile allodynia. We examined levels of the ₂-1 subunit in both the DRG and spinal cord of rats with mechanical injuries induced by spinal nerve ligation (SNL), spinal nerve transection (SNTx), or sciatic nerve chronic constriction injury (CCI), with a metabolic neuropathy induced by diabetes (DB) and with a toxic neuropathy induced by vincristine (VIN). In addition, we compared gabapentin sensitivity in these models and correlate that with the spinal cord and DRG ₂-1 subunit expression.

	Materials and Methods

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Materials. The monoclonal antibody raised against a human neuronal 2 peptide and its positive controls were derived from membrane extracts of human embryonic kidney (HEK) 293 cells overexpressing the human 2_b cDNAs and were provided by Merck Neuroscience Research Laboratories (La Jolla, CA). This antibody has been shown to specifically interact with the rat 2 subunit (Luo et al., 2001). Tris-acetate gels (NuPAGE) and buffers were obtained from Invitrogen (Carlsbad, CA). Horseradish peroxidase-labeled secondary antibodies (mouse IgG) and their substrates and enhancer solutions were from Pierce Chemical (Rockford, IL). The ECF Western blotting kit was from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Gabapentin was from Parke-Davis Pharmaceuticals (Ann Arbor, MI). Other chemicals were from Sigma-Aldrich (St. Louis, MO).

Animals. Rats (Sprague-Dawley; Harlan, Indianapolis, IN) were housed in separate cages and exposed to a 12-h day/night cycle with free access to food and water. All animal care and experiments were carried out according to protocols approved by the Institutional Animal Care Committee of the University of California, San Diego.

Neuropathic Lesions and Drug Administration. Spinal nerve ligation was induced by the procedure described by Kim and Chung (1992). Briefly, the left L5/6 lumbar spinal nerves of male Harlan rats (100-150 g) were exposed in halothane/oxygen-anesthetized rats and tightly ligated with 6.0 silk suture between their DRGs and the conjunction to form the sciatic nerve. Spinal nerve transection was performed at a similar location. Sham operations were performed in the same way except that spinal nerves were not ligated or transected.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Representative Western blots showing 2-1 subunit expression in dorsal spinal cord and DRG of rat neuropathic models and the allodynic states associated with these models at the times of tissue harvesting. Western blot analyses, as described under Materials and Methods, were performed on protein extracts of frozen dorsal spinal cord and DRG samples collected from Sprague-Dawley rats 1 week after SNL or SNTx, CCI of the sciatic nerve, or 4 weeks after the beginning of DB induction or VIN treatment. A-C, representative data from rats with mechanical, diabetes-, or vincristine-induced nerve injuries, respectively. Each blot represents the results from at least three independent experiments in each group. Membrane extracts of HEK cells overexpressing human 2b-1 subunit were used as positive controls shown as 2 bands because the -peptide was separated from the 2 subunit under denaturing conditions. S.C., spinal cord; C, contralateral or control; Ip, ipsilateral; D, diabetic; V, vincristine-treated. D, summarized PWTs to von Frey filament stimulation in sham (SNL, SNTx, and CCI)/control (DB and VIN) and injured animals at time points of tissue collection for Western blot analyses. The same sham controls were used for SNL and SNTx groups because the surgery procedures were almost identical except for the steps of nerve ligation or transection. Numbers in the parentheses represent the numbers of animals used in each group. The asterisks indicate significant changes compared with the control values (***, p < 0.001) as determined by unpaired two-tailed Student's t test.

Behavioral Testing. Tactile allodynia was tested as described previously (Chaplan et al., 1994). Briefly, after 15 min of acclimation, rats in a clear plastic cage with a wire mesh bottom were tested for the 50% PWT to von Frey filaments (Stoelting, Wood Dale, IL) using a modified up-down method of Dixon (1980). A filament with a calibrated 2.0-g buckling weight was applied to the left hindpaw plantar surface with a pressure causing the filament to bend. Absence of a paw lifting after 5 s led to the use of the next filament with increasing weight, and paw lifting indicated a positive response and led to the use of the next weaker filament. This paradigm continued until a total of six measurements, including the one before the first paw-lifting response had been made, or until four consecutive positive (assigned a score of 0.25 g) or five consecutive negative (assigned a score of 15 g) responses had occurred. The 50% response threshold was then calculated from the resulting scores as described previously (Luo et al., 2001).

Western Blot. Frozen tissue was pulverized and extracted in 50 mM Tris buffer, pH 8.0, containing 0.5% Triton, 150 mM NaCl, 1 mM EDTA, and protease inhibitors, and the cell extracts applied to electrophoresis in NuPAGE Tris-acetate gels under reducing conditions (0.05 M dithiothreitol) then electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). The 2 monoclonal antibodies in phosphate-buffered saline containing 0.1% Tween 20 were incubated with the membranes for 1 h at room temperature or overnight at 4°C after nonspecific binding sites were blocked with 5% low-fat milk. The antibody-protein complexes were detected by incubating the membrane with secondary antibodies labeled either with horseradish peroxidase or fluorescein for 1 h at room temperature followed by washing and addition of chemiluminescent reagents or of antifluorescein antibody and ECF substrate, respectively. Extracts of HEK293 cell membranes overexpressing the human neuronal ₂-1 gene were used as positive controls. Under reducing conditions, the -peptide separates from the 2 subunit (Jay et al., 1991) so the positive bands detected by the primary antibody reflect the 2 subunit only. Signal intensities were quantified by either densitometry within the linear range of the film sensitivity curve or a fluorescence scanning system (Storm; Molecular Dynamics, Sunnyvale, CA).

Statistical Analyses. Data were reported as means ± S.E.M. Unpaired Student's t tests were performed where significance was indicated by two-tailed p values: *p < 0.05, **p < 0.01, and ***p < 0.001.

	Results

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Up-Regulation of DRG and/or Spinal Cord ₂-1 Subunit Was Induced by Mechanical and Diabetes-, but Not Vincristine-Induced, Neuropathies. Our previous experiments have shown up-regulation of the ₂-1 subunit in DRG and spinal cord of rats with spinal nerve ligation injuries (Luo et al., 2001). To test whether DRG and spinal cord ₂-1 subunit up-regulation also occurs in other neuropathic pain models manifesting similar allodynic states, we compared ₂-1 subunit levels in DRG and dorsal spinal cord of rats with neuropathies derived from SNL, SNTx, CCI, DB, and VIN. The time points chosen in each category (1 week after mechanical injuries, 4 weeks after the initiation of diabetes and vintristine treatment) corresponded to times when significant tactile allodynia occurs in these neuropathic rats as shown in Fig. 1D. Our Western blot data indicated that only the mechanical peripheral nerve injuries caused significant ₂-1 subunit up-regulation in DRG ipsilateral to the injury compared with that in DRG from the contralateral side and sham-operated rats. The degree of DRG ₂-1 up-regulation seemed to correlate with the severity of the injuries because that induced by CCI was much less than that induced by SNL and SNTx (Figs. 1A and 2A). DRG ₂-1 subunit expression in DB and VIN rats was not significantly increased compared with levels in DRG of matched control rats (Figs. 1, B and C, and 2A). In dorsal spinal cord, only SNL and DB caused a significant increase in ₂-1 subunit expression compared with that in sham and control rats, respectively (Fig. 1, A and B, and 2B). A summary of Western blot data is included in Table 1.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Summarized 2-1 subunit levels in DRG and dorsal spinal cord of rats with different neuropathies. A and B, 2-1 protein levels in DRG and dorsal spinal cord, respectively, of neuropathic rats. Data shown are means ± S.E.M. of the percentage of changes in DRG or spinal cord ipsilateral to SNL, SNTx, and CCI compared with that in contralateral sides (chosen as 100%), or in DB and VIN rats compared with that in control rats (chosen as 100%). The values in the parentheses indicate the number of independent animals for each group. The asterisks indicate significant changes compared with the values from sham/control animals (*, p < 0.05; and ***, p < 0.001) as determined by unpaired two-tailed Student's t test.

View this table: [in this window] [in a new window]   TABLE 1 Summarized data of allodynia, gabapentin sensitivity, and calcium channel 2-1 subunit expression in neuropathic pain models Western blot data were obtained from tissue collected at time points when behavioral pharmacology data were collected as indicated in Fig. 4. The number of plus signs in each column represents the relative degree of significant 2-1 subunit up-regulation compared within DRG or spinal cord samples only; Minus sign indicates no significant change.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Time-dependent changes of DRG 2-1 subunit expression in nerve-ligated rats. Frozen DRG collected from rats with SNL and CCI neuropathies at the designated times were extracted and the 2-1 subunit levels were examined with Western blot analyses as described. Data are presented as percentages of 2-1 subunit levels in DRG ipsilateral to the nerve injuries compared with that in contralateral DRG, chosen as 100% and reported as means ± S.E.M. from independent animals as indicated in the parentheses. The asterisks indicate significant changes compared with the control values (*, p < 0.05; **, p < 0.01; and ***, p < 0.001) determined by unpaired two-tailed Student's t test.

Development of Tactile Allodynia and Its Gabapentin Sensitivity in Neuropathic Pain Models. All neuropathic models developed tactile allodynia at the time of tissue harvesting (Fig. 1D) and before gabapentin treatment (Fig. 4; Table 1). To examine whether antiallodynic effects of gabapentin correlate with expression levels of spinal cord and DRG ₂-1 subunit in these neuropathic models, we compared the effects of 50 mg/kg intraperitoneal gabapentin on fully developed tactile allodynia in these models. The gabapentin treatment resulted in similar therapeutic profiles in all the animal models tested (Fig. 4, A-D) except in the vincristine-treated animals (Fig. 4E). The antiallodynic effects of the drug were evident as early as 15 to 30 min after drug administration in some models and a complete reversal of the allodynic states was observed about 60 to 90 min after the treatment in all the models sensitive to gabapentin treatment. This antiallodynic efficacy of intraperitoneal gabapentin was similar to that reported in the SNL model (Hunter et al., 1997; Abdi et al., 1998). To test whether the insensitivity of VIN animals to the gabapentin treatment was due to inadequate dosing, we treated VIN rats with 100 and 300 mg/kg intraperitoneal gabapentin and failed to see an allodynia reversal in these animals. We observed mild sedation in animals 30 min after the treatment with 300 mg/kg gabapentin, consistent with reported findings from Hunter et al. (1997). These data are summarized in Table 1.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effects of gabapentin on tactile allodynia of different neuropathic pain models. Gabapentin (50 mg/kg) () or saline () was given intraperitoneally to neuropathic rats when maximum tactile allodynia had occurred. In VIN rats (E), 300 mg/kg gabapentin () was administered into the same group of rats (n = 5) 2 days after the administration of 100 mg/kg gabapentin (). PWTs to von Frey filament stimulation were measured in the injured paws immediately before and at indicated times after the drug administration and reported as means ± S.E.M. from independent animals in each treatment group as indicated below. In sham-operated animals, the PWT to similar stimulation is between 10 to 15 g (Fig. 1D; Luo et al., 2001). A, rats with SNL for 1 week (n = 5). B, rats with SNTx for 1 week (n = 6). C, rats with CCI for 2 weeks (n = 6). D and E, rats at 4 weeks after the beginning of diabetes induction (n = 4) or vincristine treatment (n = 5 in each dose group), respectively. The asterisks indicate significant changes compared with saline-treated values (*, p < 0.05; **, p < 0.01; and ***, p < 0.001) determined by unpaired two-tailed Student's t test.

	Discussion

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Our data indicate that ₂-1 subunit expression is up-regulated significantly in spinal cord dorsal horn (post-SNL and DB) and/or DRG (post-SNL, SNTx, and CCI) of selected, but not all, types of rat neuropathic pain models examined, suggesting an injury type-specific regulation of the subunit. In addition, antiallodynic effects of gabapentin were observed only in models with significant spinal cord and/or DRG ₂-1 subunit up-regulation, even though tactile allodynia developed in all examined neuropathic pain models. This supports the hypothesis that elevation of the ₂-1 subunit may underlie the antiallodynic action of gabapentin. However, it also seems that not all allodynic states share a common mechanism involving the ₂-1 subunit.

Our previous studies led us to speculate that injury-induced DRG ₂-1 subunit expression is regulated by factors transported retrogradely from the peripheral nerve because only injuries to the peripheral, but not central, axons caused dramatic up-regulation of the ₂-1 subunit (Luo et al., 2001). This could arise either because DRG ₂-1 subunit expression is suppressed by factors from innervated tissue and interruption of this negative inhibition would thus induce DRG ₂ subunit up-regulation or because nerve injury factors generated at the injury site activate ₂-1 subunit expression.

In the present studies, we found that the magnitude of DRG ₂-1 subunit expression in rats with mechanical injuries to peripheral nerves varied with the type of lesion. Some important differences in each of the three models could account for this distinction. The SNL model caused a greater induction than SNTx, despite both injuries being performed at a similar site and equally restricting any putative target-derived inhibitory factors. This would suggest that factors related to the injury site regulate DRG ₂-1 subunit expression. The smallest induction of DRG ₂-1 subunit was seen after CCI at all the time points examined (Fig. 3), in which the injury is performed more distally in the sciatic nerve. This indicates that the weakest induction by CCI on DRG ₂-1 expression at early time points (for example, 1 week post-CCI as shown in Figs. 1A and 2A) is not due to lacking of injury factors that would require more time to reach DRG. Because CCI often results in fewer number of damaged DRG neurons and leaves some intact axons that may continue to retrogradely transport target-derived factors from the periphery (Shubayev and Myers, 2001), our data suggest that DRG ₂-1 subunit expression after mechanical injury is likely linked to the number of damaged DRG neurons, or determined by a balance between supply of target-derived inhibitory and injury site-derived stimulatory factors, or both.

In contrast to mechanical nerve injury, neither DB nor VIN neuropathy altered expression of the ₂-1 subunit in the DRG. Others have recently reported increased mRNA levels for the ₂-1 subunit in the DRG of diabetic rats (Yusaf et al., 2001a), but in the present studies, any increase in mRNA expression did not result in a detectable increase of the protein. Although both DB and VIN have been shown to impede retrograde axonal transport (Jakobsen et al., 1981; Macfarlane et al., 1997), there is neither complete loss of retrograde transport mechanisms nor a specific lesion site in the nerve in either model. Thus, restriction of retrograde axonal transport alone is not sufficient to induce changes in DRG ₂-1 subunit expression.

The pattern of ₂-1 subunit expression in the spinal cord of nerve-injured rats did not correlate with that seen in the DRG, with induction of protein being apparent in the SNL, but not SNTx and CCI, mechanical injury model and in DB rats. This distinction may reflect recent findings that the DRG ₂-1 subunit differs in structure from the spinal cord ₂-1 subunit (Luo, 2000) and thus they may be regulated by different mechanisms. Our Western blot studies do not allow us to determine the cell type(s) within the spinal cord in which this induction takes place, and immunocytochemical investigations are clearly required before conclusions regarding the mechanisms of spinal ₂-1 subunit regulation can be formed.

It is now appreciated that neuropathic pain encompasses a complex series of phenomena that are unlikely to be ascribed to a single etiological mechanism. Indeed, our findings suggest that ₂-1 subunit up-regulation is not a molecular change that uniformly characterizes the neuropathic pain states in all the models. There is some precedence for the differential regulation of a given receptor after different nerve injuries. For example, SNL and peripheral axotomy down-regulate µ-opioid receptors in the spinal cord and DRG (Goff et al., 1998; Zhang et al., 1998), whereas CCI causes their up-regulation (Goff et al., 1998). This complexity is further illustrated by findings that susceptibility to spinal nerve ligation-induced tactile allodynia is animal strain-dependent (Okuse et al., 1997; Mogil et al., 1999; Luo et al., 2001). Interestingly, vincristine-induced allodynia in Harlan rats is less sensitive to treatment with gabapentin (Fig. 4E) than that in Holtzman rats to treatment with pregabalin (Nozaki-Taguchi et al., 2001), a similar but more potent antiallodynic drug (Field et al., 1999). It seems that the gabapentin insensitivity in our VIN rats is not due to inadequate dose of the drug because both drugs have similar binding affinities to the ₂-1 subunit (Suman-Chauhan et al., 1993) and the difference in drug doses between our study (up to 300 mg/kg gabapentin i.p.) and another study (80 mg/kg pregabalin i.p.; Nozaki-Taguchi et al., 2001) exceeds the difference in antiallodynic potencies of these drugs in vivo (Field et al., 1999). It is likely that rat strain-related factors may contribute to this discrepancy. This strain-dependent discrepancy in drug efficacy also occurred in cyclooxygenase-inhibitor treatment in rats with inflammatory pain (C. Svensson, manuscript in preparation) and -opioid antagonist treatment in mice with acute, thermal nociception (Mogil et al., 1997).

Even though it was not proven, it was less likely that altered pharmacokinetics of gabapentin accounted for the gabapentin insensitivity in VIN rats because the antiallodynic effects of gabapentin in other models, including the DB model, occurred quickly after intraperitoneal administration. This suggests that the effective plasma concentration of the drug is reached rapidly after intraperitoneal injection, presumably due to the facts that gabapentin is highly soluble, not metabolized, and not bound to plasma proteins (The U.S. Gabapentin Study Group 5, 1993). Thus, we did not anticipate that VIN treatment would diminish the rapid distribution of the drug, especially at a dose that was 6-fold of the effective dose seen in other models. Although increased expression of the ₂-1 subunit is not a common finding to all nerve injury models that exhibit tactile allodynia, it is possible that this induction does contribute to allodynia in the models in which it occurs. This association is supported by our observation that models that showed induction of the ₂-1 subunit in either the DRG or spinal cord also exhibited tactile allodynia that could be alleviated by gabapentin. Gabapentin has been reported to bind to the ₂-1 subunit in vitro and this could represent the antiallodynic mechanism, assuming that increased ₂-1 subunit levels contribute to allodynia and gabapentin also binds to the ₂-1 subunit in vivo. Thus, it is possible that elevated ₂-1 subunit undergoes redistribution to the central axons and/or injured primary afferents and participates in the generation and maintenance of spontaneous ectopic discharge, either through altered calcium channels or an unknown mechanism. This hypothesis is supported by findings that a redistribution of tetrodotoxin-resistant sodium channel PN3 occurs after CCI (Novakovic et al., 1998), and a similar redistribution of calcium channels is implied because application of N-type calcium channel blockers to the site of CCI can suppress mechanical allodynia (Xiao and Bennett, 1995). The involvement of N-type calcium channel in spinal nerve ligation-induced allodynia was demonstrated in a recent study showing that nerve injury-induced allodynia was suppressed in mice lacking the N-type specific, channel forming 1_B subunit (Saegusa et al., 2001).

Alternatively, increased ₂-1 subunit levels could contribute to altered excitability of sensory neurons and other cell types in the sensory pathway that can be stabilized by gabapentin. This is supported by recent findings that gabapentin's antihyperalgesic action depends on the state of target cells. It has been shown that gabapentin inhibits excitatory postsynaptic currents in spinal dorsal horn neurons from hyperalgesic, but not control, animals (Patel et al., 2000). In addition, gabapentin inhibits substance P facilitation in K⁺-evoked release, but not direct K⁺-evoked release, of glutamate from rat caudal trigeminal nucleus (Maneuf et al., 2001). Finally, gabapentin's actions on N-methyl-D-aspartate receptors of spinal dorsal horn neurons require elevated intracellular protein kinase C levels, a state seen in inflamed, but not normal, spinal cord tissue (Gu and Huang, 2001).

In combination with the hypothesis that gabapentin may act on other cellular components in addition to calcium channels (Taylor et al., 1998), our data suggest that distinct neuroplasticities in different neuropathies may underlie the complexity of gabapentin's antiallodynic actions. It is possible that gabapentin may interact with different components that may or may not include calcium channel ₂-1 subunit. In either event, more studies are required to unravel the mechanism of gabapentin's antiallodynia actions in neuropathic pain models.