gabapentin and pregabalin suppress tactile allodynia and potentiate spinal cord stimulation in a...

European Journal of Pain (2002) 6: 261±272doi:10.1053/eujp.2002.0329, available online at http://www.idealibrary.com on 1

Gabapentin and pregabalin suppress tactileallodynia and potentiate spinal cordstimulation in a model of neuropathy

Johan Wallin, Jian-Guo Cui, Vadim Yakhnitsa, GastoÂ n Schechtmann,BjoÈ rn A. Meyerson and Bengt Linderoth

Department of Clinical Neuroscience, Section of Neurosurgery, Karolinska Institutet,Stockholm, Sweden

Spinal cord stimulation (SCS) is an effective tool in alleviating neuropathic pain. However, a number of well-selected patients fail to obtain satisfactory pain relief. Previous studies have demonstrated that i.t. baclofenand/or adenosine can enhance the SCS effect, but this combined therapy has been shown to be useful in lessthan half of the cases and more effective substances are therefore needed. The aim of this experimental studyin rats was to examine whether gabapentin or pregabalin attenuates tactile allodynia following partial sciaticnerve injury and whether subeffective doses of these drugs can potentiate the effects of SCS in rats which donot respond to SCS. Mononeuropathy was produced by a photochemically induced ischaemic lesion of thesciatic nerve. Tactile withdrawal thresholds were assessed with von Frey filaments. Effects of increasing dosesof gabapentin and pregabalin (i.t. and i.v.) on the withdrawal thresholds were analysed. These drugs werefound to reduce tactile allodynia in a dose-dependent manner. In SCS non-responding rats, i.e. where stimula-tion per se failed to suppress allodynia, a combination of SCS and subeffective doses of the drugs markedlyattenuated allodynia. In subsequent acute experiments, extracellular recordings from wide dynamic range neu-rones in the dorsal horn showed prominent hyperexcitability. The combination of SCS and gabapentin, at thesame subeffective dose, clearly enhanced suppression of this hyperexcitability. In conclusion, electrical therapyand pharmacological therapy in neuropathic pain can, when they are inefficient individually, become effectivewhen combined. # 2002 European Federation of Chapters of the International Association for the Studyof Pain. Published by Elsevier Science Ltd. All rights reserved.

KEYWORDS: neuropathy, pain, gabapentin, pregabalin, spinal cord stimulation, rat.

INTRODUCTION

Chronic neuropathic pain caused by peripheralnerve injury is often associated with sensoryabnormalities such as tactile allodynia, which canbe described as pain induced by normally inno-cuous mechanical stimuli applied to the hyper-sensitive area. Such evoked pain is generallydifficult to control. Hypersensitivity to innocuous

Paper received 29 August 2001 and accepted in revisedform 6 November 2001.Correspondence to: Dr Bengt Linderoth, Department ofClinical Neuroscience, Section of Neurosurgery,Karolinska Institutet, S-171 76 Stockholm, Sweden.Tel: �46 8 517 725 92; Fax: �46 8 30 70 91;E-mail: [email protected]

1090-3801/02/040261 + 12 $35.00/0& 2002 European Federation of Chapters of the InternatioPublished by Elsevier Science Ltd. All rights reserved.

stimuli is also present in rats with peripheralnerve lesions (Bennett andXie, 1988; Seltzer et al.,1990) and thus resembles allodynia observed inpatients with neuropathy.

Spinal cord stimulation (SCS) has been usedsince the late 1960s as a powerful tool in alle-viating chronic pain (Meyerson and Linderoth,2000b). Neuropathic pain following peripheralnerve injury is by many considered the best indi-cation for SCS, but about 30% of well-selectedpatients fail to obtain satisfactory pain relief(Simpson, 1994). Despite research in recent years,the underlying mechanisms behind the positiveeffects of SCS are still unclear to a large extent(Linderoth and Foreman, 1999; Meyerson andLinderoth, 2000a).

nal Association for the Study of Pain.

262 J. WALLIN ET AL.

As knowledge of the neurochemical mechan-isms behind SCS and the pathophysiology ofneuropathic pain has accumulated, it has beensuggested that a combination of SCS and phar-macological agents might be useful to improvepain relief. To date, there are for example robustdata showing that the development of neuro-pathic pain involves an impairment of theGABAergic (GABA, -aminobutyric acid) sys-tem in the spinal dorsal horn (e.g. Castro-Lopeset al., 1993; Cui et al., 1996, 1997b). It has beendemonstrated that SCS induces an increase ofGABA release as well as a decrease in the releaseof glutamate and aspartate in the dorsal horn ofsciatic nerve injured allodynic rats (Stiller et al.,1996; Cui et al., 1997a). In line with these obser-vations, studies in both animal models andpatients show that the GABAB receptor agonistbaclofen, when administered intrathecally, maypotentiate the pain suppressing effect of SCS(Meyerson et al., 1997; Cui et al., 1998). However,this combined therapy is successful in less thanhalf of the patients and there is a need for new andmore effective drugs to be used in combinationwith SCS.

Gabapentin (Neurontin1) and pregabalin aretwo structurally related anticonvulsant drugswhich have been shown to suppress tactile allo-dynia in rats induced by streptozocin (Field et al.,1999b), chronic constriction injury (CCI) as wellas by spinal nerve ligation (Xiao and Bennett,1995; Abdi et al., 1998; Field et al., 1999a).Gabapentin and pregabalin bind specifically, andwith similar affinity, to the same site in centralnervous system neurones (Taylor et al., 1998)identified as the�2� subunit of voltage-dependentCa2� channels (Gee et al., 1996). It can thereforebe assumed that the anti-allodynic effects ofgabapentin and pregabalin involve the samecentral mechanisms. Regarding gabapentin, thereare numerous animal studies suggesting that thisdrug may be useful for controlling acute noci-ceptive, as well as many different types of neu-ropathic, pain (Hunter et al., 1997; Jun andYaksh, 1998; Partridge et al., 1998; Kayser andChristensen, 2000). Results from recent clinicalstudies have suggested that gabapentin is effectivein the treatment of, for example, reflex sympa-thetic dystrophy (Mellick and Mellick, 1997),

European Journal of Pain (2002), 6

pain in diabetic neuropathy (Backonja et al.,1998) and postherpetic neuralgia (Rowbothamet al., 1998).

The present study was undertaken to firstexamine the abilities of i.v. and i.t. gabapentinand pregabalin to suppress tactile allodynia inrats subjected to ischaemic sciatic nerve injury.The principal aim was to analyse the capabilitiesof low or subeffective doses of these drugs topotentiate the SCS effect in rats with mono-neuropathy where the stimulation per se had nosignificant suppressing effect on the allodynia.Furthermore, we recorded neuronal activity ofwide dynamic range (WDR) cells in the spinaldorsal horn following SCS and i.t. gabapentininfusion alone, as well as following SCS in com-bination with gabapentin.

MATERIAL AND METHODS

Animals

Male Sprague-Dawley rats (200±400 g, n� 66,B&KUniversal AB, Sollentuna, Sweden), housedat the local animal department, were used. Theanimals were exposed to a 12 h light±dark cycleand were provided with food and waterad libitum. The experiments were carried outaccording to the recommendations of the Com-mittee for Research and Ethical Issues of theIASP (1983) and were approved by the regionalethical committee for animal research. All effortswere made to minimise animal suffering and toreduce the number of animals used.

All surgical procedures were performed undergeneral halothane anaesthesia and under sterileconditions. Briefly, anaesthesia was induced with4%halothane andmaintainedwith 1±2% in a 1 : 1mixture of air and oxygen delivered at a rate ofapproximately 2 l/min. A heating pad was used tomaintain a constant body temperature of 37 �Cduring surgery.

Induction of nerve injury

A photochemically induced ischaemic nervelesion (Gazelius et al., 1996) was used to producemononeuropathy. The left sciatic nerve was

ANTICONVULSANTS POTENTIATE PAIN SUPPRESSION BY SCS 263

exposed by blunt dissection at the mid-thigh leveland isolated from surrounding tissues. An arrow-shaped strip of aluminium foil was then insertedunder the nerve in order to reflect the laserbeam.Aphotochemically activedye,ErythrosinB(50mg/kg, Sigma-Aldrich, Steinheim, Germany),was administered by i.v. injection in a tail vein.Immediately after the injection, the sciatic nervewas irradiated for 20min by a green low-energylaser (Laser-Compact Company Ltd, Moscow,Russia) with a wavelength of 532 nm and anoutput power of 5mW. The wound was thensutured in layers and the animal was put back inits cage to recover. The choice of this ischaemicmodel of neuropathy for the present study wasbased on previous observations that the incidenceof allodynia is very high and that hypersensitiveanimals exhibit an extremely low response rate toSCS (Cui et al., 1998). In total, 56 rats withischaemic nerve injury were studied.

For comparison, 10 rats subjected to the CCImodel (Bennett and Xie, 1988), where the sciaticnerve was loosely ligated four times approxi-mately 1mm apart with a 4-O catgut suture, werealso included in the study.

Implantation of spinal electrodes

After exposure of the spine, a small laminectomywas carried out at the thoracic vertebra T12. Thecathode (a thin solid silver rectangular plate:3mm� 1.5mm� 0.25mm) of the spinal electrodewas introduced in the dorsal epidural space,whereas the anode (a solid silver disc, 6mm indiameter) was placed subcutaneously on the leftlateral side of the spine.Amicrocontact connectedtothetwopolesviaplaitedinsulatedTeflon-coatedstainless steel wires was then tunnelled subcuta-neously and fixed to the neck skin. To avoiddamage to themicrocontact, the animalwasput ina separate cage after surgery and allowed to re-cover for at least 24 h before further experiments.

Implantation of intrathecal catheters

Under halothane anaesthesia, a PE-10 catheterwas inserted via a 21G needle used for puncturing

the lumbosacral canal and advanced in a caudo-rostral direction up to the lumbar enlargement.The catheter was fixed to the fascia with tissueglue, tunnelled subcutaneously and fixed to theneck skin. In order to verify the position of thecatheter physiologically, 300�g of lidocain(Xylocain, AstraZeneca, SoÈ dertaÈ lje, Sweden) wasinjected, which induces a transient completeparalysis of the hind limbs.

Spinal cord stimulation

The parameters used for SCS in this animalmodel have been chosen to mimic those used inthe clinic. Monopolar electrical stimulation wasapplied with a frequency of 50Hz, a pulse widthof 0.2ms and a stimulation intensity individuallyset to 2/3 of the motor threshold. The parametersused here are the same as in previous studies (e.g.Linderoth et al., 1991). The motor threshold wasrecognized as slight twitching of the lower trunkmuscles. For behavioural testing, SCS wasapplied for 30min and the withdrawal thresholdsto mechanical stimulation were assessed regularlywith von Frey filaments. During the experiments,the animals were allowed to move freely in cir-cular observation cages with wire mesh floors.Stimulation was started 60min after i.v. admin-istration and immediately after i.t. administrationof gabapentin or pregabalin. For the electro-physiological experiments, SCS was applied twicefor 5min each time. First, SCS was given afteridentification of input properties of dorsal hornneurones and then gabapentin was injected i.t.5±20min after cessation of SCS. Secondly, addi-tional SCS was applied 10±20min followingadministration of gabapentin.

Testing of withdrawal response to tactilestimuli

In order to quantify the degree of allodynia,withdrawal thresholds to static tactile stimula-tion were evaluated with von Frey nylonmonofilaments.

The animal was put in a Plexiglass cage with ametal mesh floor and allowed to adapt for at least



10min before testing. The filaments were appliedthrough the floor to the mid-plantar surface ofthe paw, so that the filament bent gently. VonFrey filaments with calibrated stiffnesses corre-sponding to 1, 2, 4, 5, 8, 11, 12, 17, 19 and 30 gwere used. Testing was started with the softestfilament and continued in ascending order ofstiffness. The filament corresponding to 30 g wasselected as cut-off. The withdrawal thresholdswere compared for the same parts of the hindpaw of the intact and the nerve-injured paw.Only animals that had developed tactile allo-dynia, defined as withdrawal from at least 5out of ten applications of a filament corre-sponding to 8 g or less, were included in theexperiment. All animals were subjected to vonFrey testing every 30min when given a drug i.v.and every 10min when given a drug by i.t.administration.

Electrophysiological study

Subsequent to the behavioural experiments, acuteelectrophysiological experiments were conductedwith SCS in combination with i.t. gabapentin inthe same rats and the same subeffective doses thatwere used in the behavioural tests. These experi-ments were carried out under halothane anaes-thesia and the heart rate was monitored throughelectrocardiogram electrodes implanted sub-cutaneously into the forepaws.

The spinal cord was exposed by a laminectomyof T11±L1. In order to avoid interference frombreathing movements, the vertebral column wasrigidly fixed in the horizontal position by clampsholding the spinal processes and lifting theanimal, so that it was hanging in the experimentalframe. The dura mater was then incised andreflected, and a pool using the skin flaps aroundthe exposed spinal cord was created and filledwith preheated (37 �C) paraffin oil.

Recordings were made with tungsten micro-electrodes (impedance at 1000Hz was 4±5M)(Frederick Haer, Brunswick, GA, USA) drivenby an electronically controlled motor unit in stepsof 2 mm (SCAT-01, Digitimer, Welwyn, GardenCity, UK). Extracellular single-unit activity wasrecorded from WDR neurones in the spinal


dorsal horn ipsilaterally to the nerve injury insegments L3±L5 medially to the dorsal root entryzone. Unitary activity was amplified and led toan analogue±digital system (Neurolog System,Digitimer Ltd, Welwyn Garden City, UK, andMacLab/4, Castle Hill, Australia) via a windowdiscriminator for recording neuronal responsesand construction of peristimulus time histo-grams. The distance between stimulating andrecording sites varied between 5 and 10mmdepending on the position of the recordingelectrode.

Identification of a WDR neurone was carriedout by testing its ability to respond, in a gradualmanner, to various stimuli (brush< press<pinch) applied to the plantar surface of the nerve-injured paw (Yakhnitsa et al., 1999). After iden-tification, the cell was tested with innocuousmechanical stimuli which consisted of gentle pawpressure with a flat-tipped forceps. Mechanicalstimuli were applied for 10 s at least 60 s apart.

Drug administration

Gabapentin (Neurontin1) and pregabalin (CI-1008, S-(�)-3-isobutylGABA) were obtainedfrom Parke-Davis, division of Warner-LambertCo, Ann Arbor, MI, USA (now Pfizer Inc).Drugs were administered at an average volumeof 2ml/kg for i.v. injection and a volume of 10 mlfor i.t. injection. In the first part of the experi-ment, both dose-response and time-responsecharacteristics for the drugs were studied. Dosesof 10±100mg/kg and 5±50mg/kg were used fori.v. injection of gabapentin and pregabalinrespectively and the animals were tested for 5 h.For i.t. administration, doses of 25±200mg wereused for gabapentin and 1.8±60mg for pregabalin,and the animals were tested for 3 h. In the secondpart of the experiment, individually screenedsubeffective doses of the drugs were given (bothi.v. and i.t.) in combination with SCS. The sub-effective dose for each animal was defined as thehighest dose tested that did not suppress tac-tile allodynia (see definition above in `Testingof withdrawal response to tactile stimuli'). Inthe third part of the experiment, activity inWDR neurones was recorded before and after


administration of the subeffective doses of i.t.gabapentin used in the behavioural tests. Gaba-pentin was delivered through the same spinalcatheter as was used in the behavioural experi-ments, both alone and in combination with SCS.In general, the electrophysiological experimentswere performed 1±3 days after the collection ofbehavioural data was completed.

Data analysis

The Friedman non-parametric analysis of var-iance followed byDunn's test was used to analysechanges in withdrawal thresholds in grams fromthe basal levels within the same group overtime. For comparison of withdrawal thresholdsbetween different unrelated groups at the sametime points, the Mann±Whitney U test was used.

Neuronal spontaneous discharges were aver-aged over a 60 s period. Evoked responses werequantified as the mean firing rates during theapplication of stimuli after subtracting thebackground spontaneous discharge. Afterdis-charges were evaluated when the cells continuedfiring above background level, and themagnitudeof afterdischarge was calculated as the meandischarge frequency after cessation of mechanicalstimuli, the spontaneous discharge being sub-tracted. Student's paired t test was used for theevaluation of differences between neuronalresponses.

All data are presented as mean� SEM andp< 0.05 was considered significant in all statis-tical tests. Graphics and calculations were per-formed using GraphPad PRISM version 2.01(GraphPad, SanDiego, CA,USA) andMicrosoftExcel version 5.0 (Microsoft Corporation,Redmond, WA, USA).

RESULTS

Behavioural experiments

Tactile allodynia could be observed after between1 and 7 days following induction of the photo-chemically induced ischaemic sciatic nerve lesionand was displayed by a majority of the operated

animals (approximately 80%). The incidence ofallodynia in rats subjected to CCI was about50%. The withdrawal thresholds to von Freyfilaments in the allodynic animals were between1 and 6 g and the hypersensitivity persisted forabout 1±2 months. There were no significantdifferences in threshold magnitudes between thetwo models of mononeuropathy.

Effects of gabapentin and pregabalin onmechanical allodynia

I.v. and i.t. administration of gabapentin (10±100mg/kg, i.v.; 25±200 mg, i.t.) and pregabalin (5±50mg/kg, i.v.; 3.8±60mg, i.t.) resulted in a sig-nificant suppression of tactile allodynia in a dose-dependent manner (Fig. 1). CCI rats were onlytested with i.v. gabapentin (data not shown), andthe responses in this group did not seem to differfrom the corresponding group of rats with thephotochemical nerve lesion (cf. Kayser andChristensen, 2000). The maximum suppressiveeffects on allodynia were reached at 2±3 h afteri.v. injection and at 60±80min following i.t.injection. There were no significant differencesbetween the drugs regarding maximum effects orlatencies, although pregabalin appeared to bemore potent and a lower dose sufficed to reachthe peak effect. The attenuation of allodynia wasstronger for both gabapentin and pregabalinwhen the drugs were given by i.t. administration.It was obvious that the doses of i.v. gabapentinand pregabalin needed to induce a maximalanti-allodynic effect also produced clear signs ofsedation.

Effects of subeffective doses of gabapentinand pregabalin in combination with SCSon mechanical allodynia

Only animals subjected to the photochemicallyinduced ischaemic nerve injury which did notrespond to SCS with a suppression of allodynia(i.e. a decrease in withdrawal threshold to 8 g orless) were included in these experiments. SCS wasapplied for 30min together with subeffectivedoses of gabapentin (10±30mg/kg, i.v.; 12.5±100 mg, i.t.) or pregabalin (5±10mg/kg, i.v.; 3.5±7.5mg, i.t.) (doses were determined in precedingexperiments for each animal individually). Only


FIG. 1. Dose-response effects of i.t. and i.v. gabapentin ((A) 25±200 mg; (B) 10±100 mg/kg) and i.t. and i.v. preg-abalin ((C) 3.8±60 mg; (D) 5±50 mg/kg) (drugs injected at t� 0 min) on tactile allodynia, evaluated with von Freyfilaments in rats with photochemical nerve injury (6±8 trials per dose group). Both drugs produced a significantsuppression of tactile allodynia in a dose-dependent manner. The maximum effects were reached at 2±3 h fol-lowing i.v. injection and at 60±80 min following i.t. injection. Data are presented as mean withdrawal threshold ing�SEM: *p< 0.05, **p< 0.01 and ***p< 0.001.


two animals out of 36 responded to SCS alone(�6%) with a significant suppression of allo-dynia, and those were excluded from furtherexperiments. The combined effects of SCS andgabapentin and of SCS and pregabalin were notsignificantly different with regard to maximumanti-allodynic effect. However, there were sig-nificant differences, concerning both latency andduration, between gabapentin and pregabalinwhen given i.t. in combination with SCS(Fig. 2A). A subeffective dose of i.t. gabapentintogether with SCS reached a maximal effect att� 30min, whereas the effect of the combinationof i.t. pregabalin and SCS peaked at t� 50min.On the other hand, the duration of the pregaba-lin±SCS effect (�120min) was almost doublethat of gabapentin in combination with SCS(�70min). Those time differences were not


observed when the drugs were given i.v. togetherwith SCS (Fig. 2B).

Electrophysiological experiments

Neuronal activity was recorded from the dorsalhorn at an average depth of 655.6� 36.6mmbeneath the surface of the spinal cord. Gaba-pentin was in all experiments administered i.t. in adose that in preceding behavioural trials had beenfound to be ineffective.

Effects of SCS and gabapentin onspontaneous discharge

A total of 17 neurones, including 12 WDRcells, one low-threshold cell and four cells with

FIG. 2. Effects on tactile allodynia of SCS per se and in combination with subeffective doses of gabapentin andpregabalin (A) i.t. (12.5±100mg, gabapentin; 3.5±7.5mg, pregabalin) and (B) i.v. (10±30 mg/kg, gabapentin; 5±10 mg/kg, pregabalin) (drugs injected at t� 0 min). Doses were determined in preceding experiments for each animal.SCS was applied for 30 min beginning at t� 0 min for i.t. injection and t� 60 min for i.v. injection. Application ofSCS in combination with gabapentin or pregabalin clearly suppressed tactile allodynia. As indicated by asterisks,there were significant differences in both latency and duration between gabapentin and pregabalin when given i.t.in combination with SCS. These differences were not observed when the drugs were given i.v. together with SCS.Data are presented as mean withdrawal threshold in g�SEM: *p< 0.05, **p< 0.01 and ***p< 0.001.


undetectable receptive fields, displayed sponta-neous discharge. The average spontaneous dis-charge frequency for all cells was 9.6� 3.7spikes/s in the control situation, 9.5� 3.7 spikes/safter SCS per se, 6.8� 2.5 spikes/s after admin-istration of gabapentin only and 7.0� 2.5 spikes/sfollowing SCS in combination with gabapentin.In eight WDR neurones, SCS in combinationwith gabapentin induced a significant suppres-sion of the discharge from 5.3� 1.9 to 1.4� 0.5spikes/s ( p< 0.05), lasting for approximately20min (19.0� 2.5min) after cessation of SCS. Inthe remaining nine neurones, no significantchange of the discharge frequency was observedafter application of gabapentin, SCS or a com-bination of the two.

Effects of SCS and gabapentin onpress-evoked discharge

Examination of press-evoked neuronal activity,after SCS per se, after administration of gaba-pentin and following SCS in combinationwith gabapentin, was performed in 12 WDRneurones.

The mean control discharge frequency ofthe principal response to application ofinnocuous pressure was 62.5� 9.1 spikes/s. After

SCS, the discharge decreased significantly( p< 0.05) to 42.1� 8.6 spikes/s, although thesuppressive effect was short-lasting (1±10min).Following administration of a low dose ofgabapentin alone, the discharge frequency wasnot significantly altered from the control value.However, SCS in combination with gaba-pentin induced a highly significant decrease indischarge frequency to 34.8� 5.9 spikes/s( p< 0.01). This effect lasted for a mean of10.0� 2.9min and in five cells it persisted from20 to 45min after SCS was terminated.

The mean afterdischarge frequency follow-ing press stimuli was 5.8� 1.2 spikes/s in thecontrol situation, 2.7� 0.9 spikes/s after SCSper se, 5.4� 1.1 spikes/s after administrationof gabapentin only and 3.1� 0.9 spikes/sfollowing SCS and gabapentin combined. Thus,both SCS per se ( p< 0.05) and SCS in combi-nation with gabapentin ( p< 0.05) induced asignificant suppression of afterdischarge fre-quencies. However, the duration of suppres-sion was 3.9� 2.3min after SCS alone, whereasthe effect of SCS together with gabapentinlasted for 12.4� 4.1min (significant differ-ence; p< 0.05). Some neurones were inhibitedfor even longer periods, up to 25±45min(Fig. 3).


FIG. 3. Effects of SCS per se and in combination with subeffective doses of i.t. gabapentin on the press-evokedactivity of WDR neurone in a rat with tactile allodynia. Neuronal activity was evoked by gentle pressure applied tothe nerve-injured paw. The time scale of the experiment is shown by the axis to the right of the histograms. Controlresponses are illustrated by the bottom histogram. As indicated (broken bars to the right), SCS was applied twicefor 5 min each time. Bars under the histograms indicate the duration of innocuous press stimulus applied to thepaw. Onset of i.t. gabapentin injection is indicated by an arrow. Application of SCS in combination with gaba-pentin markedly suppressed evoked neuronal discharges up to 55 min after cessation of the stimulation.


DISCUSSION

In the present study, it is demonstrated that thetwo anticonvulsants gabapentin and pregabalin


suppress tactile allodynia in a dose±responsefashion in a rats with mononeuropathy. More-over, our results indicate that low doses of thesedrugs act synergistically with SCS, while each


therapy per se has no significant effect on allo-dynia. It is also shown that SCS in combinationwith subeffective i.t. gabapentin induces powerfuland long-lasting depression of press-evoked, aswell as of spontaneous, discharge in dorsal hornWDR neurones.

The majority of the animals subjected to theischaemic nerve injury in this study displayedtypical signs of neuropathy (in terms of hyper-sensitivity to tactile stimulation or `allodynia'),but less than 6% responded to SCS with anincrease of withdrawal threshold. This lownumber of SCS responders is in line with ourprevious results (Cui et al., 1998) but in contrastto what has been observed in other nerve injurymodels (e.g. Stiller et al., 1996).

There is both neurochemical and electro-physiological evidence suggesting that theeffects of SCS are mediated at the level of thespinal cord by antidromic activation of low-threshold fibres in the dorsal columns (Linderothet al., 1991; Meyerson et al., 1995), therebyaltering the release of neurotransmitters (forreview see Linderoth et al., 1993) and suppress-ing the hyperexcitability of WDR neurones(Yakhnitsa et al., 1999). Previous microdialysisexperiments have shown that the levels of exci-tatory amino acids are decreased, while theGABA release is increased in the spinal dorsalhorn of allodynic rats following SCS (Stiller et al.,1996; Cui et al., 1997a). However, the mechan-isms behind the beneficial effects of SCS as atreatment of neuropathic pain are not yet fullyunderstood.

Despite the fact that gabapentin is structurallyrelated to GABA, it does not activate eitherGABAB or GABAA receptors (Taylor et al.,1998). However, it has been reported to activatethe enzyme catalysing the conversion of gluta-mate into GABA, glutamic acid decarboxylase inbrain tissues, suggesting an increase of GABAsynthesis as a possible mechanism (Loscher et al.,1991; Taylor et al., 1998). Moreover, gabapentinhas been reported to bind to the auxiliary �2�subunit of voltage-dependent Ca2� channels inbrain tissue (Gee et al., 1996) and skeletal muscle(Hamilton et al., 1989). These binding sites arealso present in high density in superficial layersof the spinal dorsal horn (Field et al., 1997),

which implies that gabapentin may interact withnociceptive transmission. The �2� subunit isconsidered to be present in all types of voltage-dependent Ca2� channels, including the N type(for review see Varadi et al., 1995), which hasbeen shown to be one of the ion channelsresponsible for the triggering of neurotransmitterrelease from presynaptic terminals (Takahashiand Momiyama, 1993). Although it is difficult tosay whether the effect of gabapentin is mediatedat a pre- or postsynaptic level, it is likely thatCa2� currents are in some way involved. A recentpatch clamp study by Shimoyama et al. (2000)demonstrated that gabapentin exerts an inhibi-tory effect on bothN-methyl-D-asparte (NMDA)and non-NMDA receptor currents in superficialdorsal horn neurones, suggesting that the gluta-minergic transmission is attenuated. This findingfurther indicates that gabapentin may have sup-pressive effects on central sensitization. Takentogether, it cannot be excluded that gabapentininteracts with the GABA system, in a direct orindirect way, even though an actual GABArelease induced by gabapentin has been demon-strated only at a supraspinal level (GoÈ tz et al.,1993; Petroff et al., 1996). Nevertheless, themechanisms of action of gabapentin and pre-gabalin are still under intensive investigationand it can be assumed that they are pharmaco-logically manifold (for reviews see Novelli andTrovati, 1998; Nicholson, 2000).

Electrophysiological studies have previouslydemonstrated that WDR neurones in animalmodels of mononeuropathy are hyperexcitableand display enhanced stimulus-evoked dischargeas well as spontaneous activity (Sotgiu et al.,1992; Pertovaara et al., 1997). Recently, it wasreported that SCS may exert suppressive effectson the dorsal horn neuronal hyperexcitabilityfollowing peripheral nerve injury (Yakhnitsaet al., 1999). Although gabapentin has beenimplied to operate at peripheral (Carltonand Zhou, 1998; Pan et al., 1999) and spinal(Chapman et al., 1998; Field et al., 1999b), as wellas supraspinal (Kayser and Christensen, 2000),levels, our data support the notion that its anti-allodynic effect is mediated mainly via a spinalmechanism.However, peripheral and supraspinalsites of action of gabapentin following systemic



administration (i.v.) may constitute supplemen-tary mechanisms that can also account for theobserved anti-allodynic effects.

The relatively large systemic doses of bothgabapentin and pregabalin which were needed toinduce a marked attenuation of allodynia whendrugs were administered alone often producedsigns of sedation in the animals. In line with thisobservation, somnolence has been reported to beone of themost commonly reported side-effects inclinical trials of gabapentin, observed in about20%of patients (Ramsay, 1994). In contrast, SCShas very few side-effects, although this procedureis resource demanding and, like all surgicaltherapies, carries some risk for complications(Meyerson and Linderoth, 2000b). The low`subeffective' doses (both i.t. and i.v.) of gaba-pentin and pregabalin used in this study in com-bination with SCS did not result in anyobservable sedation. Thus, the combination ofneurostimulation and low doses of a pharmaco-logical agent, such as gabapentin or pregabalin,may provide a useful strategy for the treatmentof neuropathic pain. Results similar to thosereported here, where the pain suppressing effectsof SCS can be potentiated when stimulation iscombined with pharmacotherapy, have pre-viously been demonstrated for R-PIA, a selectiveadenosine A1 receptor agonist, and for theGABAB receptor agonist baclofen in rat modelsof neuropathy (Cui et al., 1997a, 1998). Further-more, clinical trials have demonstrated that i.t.baclofen and/or adenosine can enhance painrelief when combined with SCS in some patientssuffering from neuropathic pain of peripheralorigin (Meyerson et al., 1997).

In conclusion, the present results suggest thata combination of SCS and low doses of eithergabapentin or pregabalin effectively suppresstactile allodynia in a rat model of mono-neuropathy. Future clinical studies in this areacould contribute to the development of newcombined therapeutic strategies with lowerfrequency of side-effects in the treatment ofneuropathic pain. Thus, two different ways oftreating neuropathic pain, each of which is inef-ficient on its own, can become effective whencombined.


ACKNOWLEDGEMENTS

The authors are indebted to GoÈ te HammarstroÈ mfor excellent technical assistance. This study wassupported by the Swedish Medical ResearchCouncil (MFR K99-04x-12210-03A) and Med-tronic Europe S.A. Parke-Davis AB (Pfizer Inc.)kindly supplied gabapentin and pregabalin.

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