Development and expression of neuropathic pain in CB1 knockout mice

Neuropharmacology 50 (2006) 111e122

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Development and expression of neuropathic pain inCB1 knockout mice

Anna Castane a,1, Evelyne Celerier a,1, Miquel Martın a, Catherine Ledent b,Marc Parmentier b, Rafael Maldonado a, Olga Valverde a,*

a Laboratori de Neurofarmacologia, Facultat de Ciencies de la Salut i de la Vida, Ciencies Experimentals i de la Salut,

Universitat Pompeu Fabra, C/Dr. Aiguader 80, 08003 Barcelona, Spainb IRIBHM, Universite libre de Bruxelles, N-1070 Bruxelles, Belgium

Received 2 June 2005; received in revised form 29 July 2005; accepted 29 July 2005

Abstract

Neuropathic pain is a clinical manifestation characterized by the presence of spontaneous pain, allodynia and hyperalgesia. Here,we have evaluated the involvement of CB1 cannabinoid receptors in the development and expression of neuropathic pain. For thispurpose, partial ligation of the sciatic nerve was performed in CB1 cannabinoid receptor knockout mice and their wild-type litter-

mates. The development of mechanical and thermal allodynia, and thermal hyperalgesia was evaluated by using the von Frey fila-ments, cold-plate and plantar tests, respectively. Pre-surgical tactile and thermal withdrawal thresholds were similar in bothgenotypes. In wild-type mice, sciatic nerve injury led to a neuropathic pain syndrome characterized by a marked and long-lasting

reduction of the paw withdrawal thresholds to mechanical and thermal stimuli. These manifestations developed similarly in micelacking CB1 cannabinoid receptors. We have also investigated the consequences of gabapentin administration in these animals.Gabapentin (50 mg/kg/day, i.p.) induced a similar suppression of mechanical and thermal allodynia in both wild-type and CB1knockout mice. Mild differences between genotypes were observed concerning the effect of gabapentin in the expression of thermal

hyperalgesia. Taken together, our results indicate that CB1 cannabinoid receptors are not critically implicated in the development ofneuropathic pain nor in the anti-allodynic and anti-hyperalgesic effects of gabapentin in this model.� 2005 Elsevier Ltd. All rights reserved.

Keywords: Allodynia; CB1 cannabinoid receptors; Gabapentin; Hyperalgesia; Neuropathic pain

1. Introduction

Neuropathic pain as a consequence of nerve injury ischaracterized by the presence of exaggerated response topainful stimuli (hyperalgesia), pain response to normallyinnocuous stimuli (allodynia) and spontaneous pain

* Corresponding author. Tel.: C34 93 542 28 30; fax: C34 93 542

28 02.

E-mail address: [email protected] (O. Valverde).1 These authors contributed equally to this work.

0028-3908/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuropharm.2005.07.022

(Bridges et al., 2001a). These pathological pain sensa-tions are associated with various complex physiologicalchanges in the peripheral and central nervous system,such as spontaneous neuron discharging, alteration ofion channel expression, sprouting of primary afferentneurons, peripheral and central sensitisation, spinal reor-ganization and changes in inhibitory pain descendingpathways (Basbaum, 1999; Banos et al., 2003;Wood et al., 2004). Due to the different adaptationsoccurred during neuropathic pain, a great diversity ofpharmacological agents has been used to improve its

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112 A. Castane et al. / Neuropharmacology 50 (2006) 111e122

symptomatology, including tricyclic antidepressants,NMDA receptor antagonists, local anaesthetics, GABAagonists, opioid agonists and anti-epileptic drugs, suchas gabapentin. Actually, gabapentin is one of the mostuseful compounds to treat neuropathic pain in humans(Spina and Perugi, 2004; Maizels and Mc Carberg,2005). However, majority of these treatments have a lim-ited effectiveness or produce undesirable side effects(McQuay et al., 1996; Sindrup and Jensen, 1999; Banoset al., 2003; Foley, 2003; Lim et al., 2003). Recently, can-nabinoids have emerged as new possible candidates forthe treatment of neuropathic pain (Herzberg et al.,1997; Banos et al., 2003; Goya et al., 2003). Indeed, can-nabinoid agonists reduce the allodynia and hyperalgesiathat accompany neuropathies in experimental animals(Herzberg et al., 1997; Bridges et al., 2001b; Fox et al.,2001), although the specific mechanism of action remainsunclear. Both CB1 and CB2 cannabinoid receptors maybe involved in the anti-allodynic and anti-hyperalgesic ef-fects of cannabinoids (Bridges et al., 2001b; Malan et al.,2002, 2003; Goya et al., 2003; Ibrahim et al., 2003; Costaet al., 2004; Scott et al., 2004). The high density of CB1cannabinoid receptors in sensitive fibres of large diameter(Ab and Ad), which are associated to the aberrant paintransmission during neuropathies, could be a neuroana-tomical site of action of cannabinoid agonists (HohmannandHerkenham, 1999; Piomelli et al., 2000; Stander et al.,2005). CB2 receptors are located on non-neuronal cells inthe vicinity of nociceptiveneurons and seemtobe involvedin the modulation of pro-inflammatory agents such asprostaglandin E2 and nitric oxide (Costa et al., 2004).

Several anatomical studies in rats suggest the exis-tence of an endogenous cannabinoid tone controllingthe development of neuropathic pain symptoms. Thus,chronic constriction of the sciatic nerve induced a timedependent up-regulation of spinal CB1 cannabinoid re-ceptors primarily within the ipsilateral superficial spinalcord dorsal horn (Lim et al., 2003). An up-regulation ofCB1 cannabinoid receptors was also observed in thecontralateral thalamic region after unilateral axotomyof the tibial branch of the sciatic nerve in rats (Sieglinget al., 2001). In contrast, pharmacological studies havereported controversial results. Thus, the administrationof the CB1 receptor antagonist, rimonabant, increasedthermal hyperalgesia and mechanical allodynia in ratsafter a chronic constriction of the sciatic nerve (Herzberget al., 1997), but not following L5/L6 spinal nerve liga-tion (Bridges et al., 2001b). The use of knockout micelacking CB1 cannabinoid receptors represents a very use-ful tool to better understand the participation of theendocannabinoid system in the mechanisms underlyingneuropathic pain. In our study, we have first evaluatedthe consequences of CB1 cannabinoid receptor deletionin the development and expression of neuropathic pain.Secondly, we have investigated whether the absence ofCB1 cannabinoid receptors could modify the responses

to one of the most current effective treatments for neuro-pathic pain, the anti-epileptic drug gabapentin, whichmechanism of action for its analgesic effects remainsunclear.

2. Materials and methods

2.1. Animals

Male CB1 knockout mice and wild-type littermatesweighing 26e30 g at the beginning of the experimentswere used. The generation of mice lacking CB1 cannabi-noid receptor was described previously (Ledent et al.,1999). In order to homogenize the genetic backgroundof mice, the first generation heterozygous were bredfor 15 generations on a CD1 background, with selectionfor the mutant CB1 gene at each generation. The CB1receptor knockout mice derived from the backcrossingof chimeric CD1-CB1 receptor knockout mice devel-oped by Ledent et al. (1999) with wild-type CD1 females(Charles River, France). Beginning with the 15th gener-ation backcrossing mice, heterozygoteeheterozygotemating of CB1 knockout mice produced wild-typeand knockout littermates for subsequent experiments.Breeding couples were periodically renovated by crossingheterozygote mice with wild-type CD1 females (CharlesRiver, France) in order to maintain a genetically diverseoutbred background. All animals used in a given exper-iment originated from the same breeding series and werematched for age and weight. Mice were housed 5 per cagein a temperature (21G 1 �C) and humidity (55G 10%)-controlled room with a 12-h lightedark cycle (lightbetween 8 a.m. and 8 p.m.). Food and water were avail-able ad libitum except during behavioural observations.Mice were habituated to their new environment for 1week after arrival before starting the experimental proce-dure. The observer was blind to the treatment, genotypeand surgical procedure of each subject.

All experimental procedures and animal husbandrywere conducted according to standard ethical guidelines(NIH, publication no. 85-23, revised 1985; EuropeanCommunities Council Directive 86/609/EEC) and ap-proved by the Local Ethical Committee (IMAS-IMIM/UPF).

2.2. Drugs

Gabapentin was kindly supplied by Medichem S.A.(Celra, Girona, Spain). Gabapentin was dissolved insaline and administered in a volume of 10 ml/kg byintraperitoneal route (i.p.).

113A. Castane et al. / Neuropharmacology 50 (2006) 111e122

2.3. Surgery

The partial sciatic nerve ligation model was used to in-duce neuropathic pain (Seltzer et al., 1990; Malmbergand Basbaum, 1998). Thismodel consists of partial injuryto the sciatic nerve at mid-thigh level. Briefly, mice wereanaesthetized with halothane (induction: 3%; surgery:1%) and the common sciatic nerve was exposed at thelevel of the mid-thigh of the right hindpaw. At about1 cm proximally to the nerve trifurcation, a tight ligaturewas created around 33e50% of the sciatic nerve using9-0 18$ non-absorbable virgin silk suture (Alcon� surgi-cal, Texas, USA), leaving the rest of the nerve ‘‘unin-jured’’. The muscle was then stitched, and the incisionwas closed with wound clips. Control animals (sham-operated mice) underwent the same surgical procedureexcept that the sciatic nerve was not ligated. Aftersurgery, animals were allowed to recover for 24 h priorto initiation of nociceptive behavioural tests.

2.4. Nociceptive behavioural tests

Hyperalgesia to noxious thermal stimulus and allody-nia to cold and mechanical stimuli were used as outcomemeasures of neuropathic pain by using the following be-havioural models.

Thermal hyperalgesia was assessed as previously re-ported (Hargreaves et al., 1988). Paw withdrawal latencyin response to radiant heat was measured using plantartest apparatus (Ugo Basile, Varese, Italy). Briefly, micewere placed in Plexiglas� boxes (20 cm high, 9 cm diam-eter) positioned on a glass surface. Mice were allowed tohabituate to the environment for 30 min before testing inorder to allow an appropriate behavioural immobility.The heat source was then positioned under the plantarsurface of the hindpaw and activated with a light beamintensity, chosen in preliminary studies to give baselinelatencies from 8 to 10 s in control mice. A cut-off timeof 20 s was used to prevent tissue damage in absence ofresponse. The mean paw withdrawal latencies for theipsilateral and contralateral hindpaws were determinedfrom the average of 3 separate trials, taken at5e10 min intervals to prevent thermal sensitisation andbehavioural disturbances.

Thermal allodynia to cold stimulus was assessed byusing the hot/cold-plate analgesia meter (Columbus,OH, USA), as previously described (Bennett and Xie,1988). A glass cylinder (40 cm high, 20 cm diameter)was used to keep mice on the cold surface of the platewhich was maintained at a temperature of 5G 0.5 �C.The number of elevations of each hindpaw was then re-corded for 5 min.

Mechanical allodynia was quantified by measuring thehindpaw withdrawal response to von Frey filament stim-ulation (Chaplan et al., 1994). In brief, animals wereplaced in a Plexiglas� box (20 cm high, 9 cm diameter)

with a wire grid bottom through which the von Frey fil-aments (bending force range from 0.008 to 2 g) (NorthCoast Medical, Inc., San Jose, CA, USA) were appliedby using the upedown paradigm, as previously reported(Chaplan et al., 1994). The filament of 0.4 g was firstused. Then, the strength of the next filament was de-creased when the animal responds or increased whenanimal does not respond. This upedown procedurewas stopped 4 measures after the first change in animalresponding (i.e. from response to no response or fromno response to response). The threshold of responsewas then calculated by using the upedown excel pro-gram provided by the Dr. A. Basbaum’s laboratory(UCSF, San Francisco, USA). Animals were allowedto habituate for 1e2 h before testing in order to allowan appropriate behavioural immobility. Clear paw with-drawal, shaking or licking was considered as nocicep-tive-like response. Both ipsilateral and contralateralhindpaws were tested.

2.5. Experimental protocol

In a first set of experiments we investigated the influ-ence of CB1 receptor deletion on the development ofneuropathic pain. Animals were habituated for 1 h tothe environment of the different experimental testsduring 4 days. After the habituation period, baseline re-sponses were established during 2 consecutive days inthe following sequence: von Frey, plantar and cold-platetests. One day after baseline measurements, sciatic nerveinjury was induced as previously described. CB1 canna-binoid receptor knockout mice and wild-type littermateswere tested in each paradigm on days 1, 3, 6, 8, 10, and15 after the surgical procedure using the same sequenceas for baseline responses.

In a second set of experiments, the effectiveness of ga-bapentin on the expression of neuropathic pain was in-vestigated in both CB1 cannabinoid receptor knockoutmice and wild-type littermates. Animals received gaba-pentin (50 mg/kg/day, i.p.) from day 8 to day 14 andthey were tested on days 1, 3, 6, 8, 10, 13 and 15 afterthe surgical procedure using the same sequence as forbaseline responses. The behavioural testing started45 min after gabapentin administration.

2.6. Statistical analysis

Data obtained in the plantar test, cold-plate test andvon Frey filament stimulation model were compared oneach experimental day by using a two-way ANOVA re-peated measures on the factor paw and genotype as be-tween factor of variation followed by correspondingFisher’s test to compare between paws and genotypeswhen appropriate. The effects of gabapentin treat-ment were also compared by using one-way ANOVA(day as within group factor) and post hoc analysis


(NewmaneKeuls) when required. Differences were con-sidered significant if the probability of error was lessthan 5%.

3. Results

3.1. Development of neuropathic pain in CB1cannabinoid receptor knockout mice andwild-type littermates

3.1.1. Plantar test (thermal hyperalgesia)Sciatic nerve ligature similarly decreased withdrawal

latency of the ipsilateral hindpaw to a thermal stimulusin both CB1 knockout and wild-type mice (Fig. 1A, seeTable 1 for two-way ANOVA). Baseline values weresimilar in both genotypes. Sham operation did notproduce any modification of nociceptive response inwild-type and knockout mice. A marked and long-last-ing decrease of paw withdrawal latency was observedin the ipsilateral paw of wild-type mice exposed to par-tial sciatic nerve injury on day 1 (P! 0.01), day 3(P! 0.05), day 6 (P! 0.01), day 8 (P! 0.01), day 10(P! 0.001) and day 15 (P! 0.01) after surgery (ipsilat-eral vs contralateral paw). The same degree of thermalhyperalgesia was developed in CB1 receptor knockoutmice. Thus, a significant decrease of the ipsilateral pawwithdrawal latency was observed on day 3 (P! 0.001),day 6 (P! 0.001), day 8 (P! 0.01), day 10 (P! 0.01),and day 15 (P! 0.001) after surgery (ipsilateral vs con-tralateral paw). Genotype differences were only observedon day 6, whenCB1 knockoutmice showed a significantlylower withdrawal latency of the ipsilateral paw thanwild-type littermates (P! 0.01) (Fig. 1A).

3.1.2. von Frey filament stimulation(mechanical allodynia)

Sciatic nerve ligature led to a profound decrease ofthe threshold for evoking withdrawal of the ipsilateralhindpaw to a mechanical stimulus in both CB1 knock-out and wild-type mice (Fig. 1B, see Table 1 for two-way ANOVA). Baseline values were similar in bothgenotypes. Sham-operated CB1 knockout mice showeda significant decrease of the threshold required to induceipsilateral paw withdrawal when compared to thecontralateral paw on day 3 (P! 0.01) and day 6(P! 0.05). Significant differences were also observedbetween sham-operated CB1 knockout mice and wild-type littermates when compared the responses of theipsilateral paw on day 6 (P! 0.01). Partial sciatic nerveinjury in wild-type mice led to a significant decrease ofthe threshold for evoking withdrawal of the ipsilateralhindpaw to mechanical stimulation from the 1st dayafter surgery and persisting during the whole durationof the experiment. Indeed, a significant effect of nerveinjury was observed on day 1 (P! 0.01), day 3(P! 0.001), day 6 (P! 0.001), day 8 (P! 0.01), day

10 (P! 0.001) and day 15 (P! 0.001) after surgery (ip-silateral vs contralateral paw). CB1 receptor knockoutmice exposed to sciatic nerve injury showed similar allo-dynic responses than wild-type mice on day 1 (P! 0.01),day 3 (P! 0.01), day 6 (P! 0.001), day 8 (P! 0.001),day 10 (P! 0.001) and day 15 (P! 0.001) after surgery(ipsilateral vs contralateral paw).

3.1.3. Cold-plate test (thermal allodynia)Sciatic nerve ligature enhanced ipsilateral paw eleva-

tions in both CB1 knockout and wild-type mice(Fig. 1C, see Table 1 for two-way ANOVA). No signif-icant differences between genotypes were observed in thebaseline values. However, the baseline number of pawelevations was lower in all the CB1 knockout groupscompared to the wild-type animals. Sham operationdid not produce any modification of the nociceptive re-sponse in wild-type and CB1 knockout mice. However,a significant decrease of ipsilateral and contralateralhindpaw elevations was observed in the mutants follow-ing the consecutive exposure to the test. Indeed, signifi-cant differences between wild-type and CB1 knockoutmice were found on day 8 (P! 0.01), day 10(P! 0.05) and day 15 (P! 0.05) for the ipsilateralpaws, and on day 8 (P! 0.01) and day 10 (P! 0.05)for the contralateral paws. Wild-type and CB1 knockoutmice exposed to partial sciatic nerve injury significantlyincreased the number of ipsilateral paw elevations aftersurgery revealing the development of thermal allodynia.Thus, a significant effect of nerve injury was observed onday 1 (P! 0.01), day 3 (P! 0.05), day 6 (P! 0.01),day 10 (P! 0.05) and day 15 (P! 0.05) after surgery(ipsilateral vs contralateral paw) in wild-type mice, andon days 1, 3, 6, 8 and 15 (P! 0.05 in all the cases) aftersurgery in mice lacking CB1 cannabinoid receptors.

3.2. Effects of gabapentin in the expression ofneuropathic pain in CB1 cannabinoid receptorknockout mice and wild-type littermates

In a preliminary experiment we evaluated the loco-motor effects of acute gabapentin administration inwild-type mice (data not shown). Horizontal activitywas measured during 1 h after gabapentin (50 mg/kg,i.p.) or saline administration, as previously described(Castane et al., 2002). Locomotor activity was similarin saline (mean number of activity counts, 1006G 153)and gabapentin (mean number of activity counts,915G 95) groups.

The administration of gabapentin (50 mg/kg/day) sig-nificantly reversed the behavioural manifestations ofneuropathic pain in wild-type and CB1 cannabinoid re-ceptor knockout mice. Thus, gabapentin attenuatedthermal hyperalgesia and thermal and mechanical allo-dynia. However, these analgesic effects disappeared inboth genotypes as soon as the gabapentin treatment


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DAYS

(A) Plantar test

(B) Von Frey model

(C) Cold-plate test

Ipsilateral WT Ipsilateral KO

Contralateral WT Contralateral KO

Fig. 1. Development of neuropathic pain in CB1 cannabinoid receptor knockout mice (KO) and wild-type littermates (WT). The behavioural man-

ifestations of neuropathic pain were evaluated by using the plantar test (A), the von Frey model (B) and the cold-plate test (C). Data are expressed as

meanG SEM in WT (circles) and CB1 KO (triangles) mice. +P! 0.05, ++P ! 0.01, +++P ! 0.001 (Fisher’s test ipsilateral vs contralateral paw),*P ! 0.05, **P ! 0.01 (Fisher’s test knockout vs wild-type).

was stopped (Fig. 2, see Table 2 for two-way ANOVA).Conversely, gabapentin only induced minor effects insham-operated mice (Tables 2 and 3).

In the plantar test, gabapentin reversed the decreaseof ipsilateral paw withdrawal latency in wild-type and

CB1 knockout mice induced by partial sciatic nerve in-jury (Fig. 2A). In wild-type mice, ipsilateral vs contralat-eral paw comparisons showed a significant hyperalgesiainduced by nerve injury before gabapentin treatmenton day 6 after surgery (P! 0.05). On the contrary,

Table 1

Development

y 8 Day 10 Day 15

P-value F P-value F P-value

Sham-operate

Plantar test

Paw .649 N.S. 0.026 N.S. 0.341 N.S.

Genotype .482 N.S. 0.074 N.S. 2.043 N.S.

Interaction .210 N.S. 0.315 N.S. 0.069 N.S.

von Frey mod

Paw .288 N.S. 2.326 N.S. 0.677 N.S.

Genotype .784 N.S. 2.548 N.S. 0.929 N.S.


Cold-plate tes

Paw .488 N.S. 0.670 N.S. 0.125 N.S.

Genotype .336 !0.01 6.487 !0.05 5.137 !0.05


Mice exposed

Plantar test

Paw .409 !0.001 35.792 !0.001 39.483 !0.001

Genotype .816 N.S. 0.302 N.S. 1.081 N.S.


von Frey mod

Paw .611 !0.001 61.532 !0.001 85.091 !0.001

Genotype .265 N.S. 1.324 N.S. 0.804 N.S.


Cold-plate tes

Paw .345 !0.01 9.942 !0.01 12.953 !0.01

Genotype .834 N.S. 0.740 N.S. 0.344 N.S.


Two-way AN asal) and days 1, 3, 6, 8, 10 and 15 after the surgical procedure in

each nocicept

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of neuropathic pain in CB1 cannabinoid receptor knockout mice and wild-type littermates (two-way ANOVA)

Basal Day 1 Day 3 Day 6 Da

F P-value F P-value F P-value F P-value F

d mice

F(1,26) 0.826 N.S. 0.008 N.S. 0.815 N.S. 0.033 N.S. 1

F(1,26) 0.482 N.S. 0.012 N.S. 1.126 N.S. 1.466 N.S. 1

F(1,26) 1.450 N.S. 1.204 N.S. 0.032 N.S. 0.055 N.S. 2

el

F(1,26) 0.018 N.S. 5.505 !0.05 12.571 !0.01 1.071 N.S. 0

F(1,26) 0.172 N.S. 2.523 N.S. 1.450 N.S. 7.304 !0.05 2

F(1,26) 0.485 N.S. 1.005 N.S. 0.846 N.S. 10.364 !0.01 0

t

F(1,22) 3.244 N.S. 0.927 N.S. 3 891 N.S. 1.449 N.S. 0

F(1,22) 1.650 N.S. 1.701 N.S. 3.356 N.S. 4.206 N.S. 10

F(1,22) 0.273 N.S. 0.000 N.S. 1.201 N.S. 0.161 N.S. 0

to sciatic nerve injury

F(1,26) 0.044 N.S. 14.218 !0.001 29.681 !0.001 34.650 !0.001 35

F(1,26) 0.002 N.S. 0.026 N.S. 0.366 N.S. 9.370 !0.01 2

F(1,26) 1.597 N.S. 0.816 N.S. 0.760 N.S. 0.425 N.S. 0

el

F(1,26) 0.065 N.S. 23.350 !0.001 70.217 !0.001 72.213 !0.001 44

F(1,26) 0.090 N.S. 0.789 N.S. 0.054 N.S. 0.083 N.S. 0

F(1,26) 0.031 N.S. 0.002 N.S. 1.398 N.S. 2.147 N.S. 1

t

F(1,19) 0.193 N.S. 16.038 !0.001 12.893 !0.01 17.803 !0.001 10

F(1,19) 0.470 N.S. 0.646 N.S. 0.419 N.S. 0.168 N.S. 0

F(1,19) 0.193 N.S. 0.004 N.S. 0.376 N.S. 0.134 N.S. 0

OVA with paw as within factor and genotype as between factor of variation performed for the pre-surgical values (B

ive behavioural paradigm.


Ipsilateral WT Ipsilateral KO

Contralateral WT Contralateral KO

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MICE EXPOSED TO SCIATIC NERVEINJURY

# # #

Fig. 2. Effect of gabapentin in the expression of neuropathic pain in CB1 cannabinoid receptor knockout mice (KO) and wild-type littermates (WT).

The behavioural consequences of gabapentin administration in the expression of neuropathic pain were evaluated by using the plantar test (A), von

Frey model (B) and cold-plate test (C). Gabapentin reversed the behavioural manifestations of neuropathic pain in both WT and CB1 KO mice

exposed to sciatic nerve injury. Mice received gabapentin (50 mg/kg/day) from day 8 to day 14 after surgery (see Section 2 for details). Data are

expressed as meanG SEM. +P ! 0.05, ++P! 0.01, +++P! 0.001 (Fisher’s test ipsilateral vs contralateral paw), #P! 0.05 (NewmaneKeuls post

hoc vs day 6).

no hyperalgesia was observed during gabapentin treat-ment on days 8, 10 and 13. Once gabapentin treatmentwas stopped, the thermal hyperalgesia returned on day15 (P! 0.05). The effects of gabapentin administrationon sciatic nerve injury induced hyperalgesia were de-layed in mice lacking CB1 cannabinoid receptors. Thus,

ipsilateral vs contralateral paw comparisons showeda significant hyperalgesia on day 6 (P! 0.001) andday 15 (P! 0.01), as expected, but also on day 8(P! 0.001) when starting the gabapentin treatment.Then, hyperalgesia disappeared on days 10 and 13 duringgabapentin administration.


Table 2

Effects of gabapentin in the expression of neuropathic pain in CB1 cannabinoid receptor knockout mice and wild-type littermates (two-way

ANOVA)

Gabapentin (50 mg/kg/day)

Day 6 Day 8 Day 10 Day 13 Day 15

F P-value F P-value F P-value F P-value F P-value

Sham-operated mice

Plantar test

Paw F(1,13) 0.030 N.S. 0.902 N.S. 2.440 N.S. 1.659 N.S. 12.508 !0.01

Genotype F(1,13) 0.193 N.S. 0.454 N.S. 0.003 N.S. 0.030 N.S. 0.333 N.S.

Interaction F(1,13) 0.128 N.S. 2.301 N.S. 1.448 N.S. 0.001 N.S. 1.271 N.S.

Von Frey model

Paw F(1,13) 0.362 N.S. 1.639 N.S. 0.574 N.S. 1.787 N.S. 3.623 N.S.

Genotype F(1,13) 0.027 N.S. 1.135 N.S. 1.363 N.S. 0.058 N.S. 6.705 !0.05


Cold-plate test

Paw F(1,13) 0.578 N.S. 0.059 N.S. 10.920 !0.01 0.000 N.S. 0.040 N.S.

Genotype F(1,13) 3.433 N.S. 1.875 N.S. 5.393 !0.05 2.022 N.S. 4.653 !0.05

Interaction F(1,13) 0.578 N.S. 0.059 N.S. 10.920 !0.01 0.000 N.S. 0.040 N.S.

Mice exposed to sciatic nerve injury

Plantar test

Paw F(1,18) 31.828 !0.001 17.732 !0.001 1.285 N.S. 3.118 N.S. 18.602 !0.001


Interaction F(1,18) 0.002 N.S. 4.717 !0.05 1.894 N.S. 0.933 N.S. 0.471 N.S.

Von Frey model

Paw F(1,18) 94.851 !0.001 2.547 N.S. 1.329 N.S. 0.358 N.S. 26.448 !0.001



Cold-plate test

Paw F(1,18) 8.469 !0.01 7.442 !0.05 6.047 !0.05 4.119 N.S. 30.261 !0.001



Two-way ANOVA with paw as within factor and genotype as between factor of variation performed on days 6, 8, 10, 13 and 15 after the

surgical procedure in each nociceptive behavioural paradigm.Mice received gabapentin (50 mg/kg/day) from day 8 to day 14 (see Section 2 for details).

In the von Frey filament stimulation model, gabapentinprevented the decrease induced by partial sciatic nerveinjury on the threshold for evoking withdrawal of theipsilateral hindpaw to mechanical stimulation in bothwild-type and CB1 knockout mice (Fig. 2B). In wild-type mice, ipsilateral vs contralateral paw comparisonsshowed a significant allodynia induced by nerve injuryon day 6 after surgery (P! 0.001). This allodynia wasnot observed during gabapentin treatment on days 8,10 and 13. Once gabapentin treatment was stopped,the mechanical allodynia returned on day 15(P! 0.01). In CB1 receptor knockout mice, ipsilateralvs contralateral paw comparisons also revealed a signifi-cant allodynia on day 6 (P! 0.001) and day 15(P! 0.01) after surgery. However, this allodynia disap-peared during gabapentin treatment on days 8, 10 and13 after surgery. In addition to reversing nerve injuryinduced mechanical allodynia, gabapentin significantlyprolonged withdrawal latencies of the contralateralpaw to a mechanical stimulus in nerve-injured[F(4,32)Z 4.462, P! 0.01] and sham-operated

[F(4,28)Z 5.980, P! 0.01] wild-type mice (one-wayANOVA repeated measures). Thus, post hoc analysis(NeumaneKeuls) showed that paw withdrawal latenciesof the contralateral paw were significantly increased ondays 8, 10 and 13 compared to day 6 in wild-type micewith partial sciatic nerve injury, as well as on day 8compared to day 6 in sham-operated wild-type mice(P! 0.05 in all the cases) (Fig. 2B, Table 3).

In the cold-plate test, the administration of gabapen-tin reversed the increased number of ipsilateral paw ele-vations observed in wild-type and CB1 knockout miceafter sciatic nerve injury (Fig. 2C). In wild-type mice,ipsilateral vs contralateral paw comparisons showeda significant allodynia induced by the partial sciaticnerve injury on day 6 after surgery (P! 0.05). Thisallodynia was not observed during gabapentin treatmenton days 8, 10 and 13. Once gabapentin treatment wasstopped, the thermal allodynia returned on day 15(P! 0.01). In CB1 receptor knockout mice, ipsilateralvs contralateral paw comparisons also showed a signifi-cant allodynia on day 6 (P! 0.01) and day 15


Table

3

Effects

ofgabapentinin

sham-operatedwild-typeandCB1cannabinoid

receptorknockoutmice

Gabapentin(50mg/kg/day)

Day6

Day8

Day10

Day13

Day15

Contralateral

Ipsilateral

Contralateral

Ipsilateral

Contralateral

Ipsilateral

Contralateral

Ipsilateral

Contralateral

Ipsilateral

Plantartest

WT

6.74G

0.58

7.08G

0.61

7.77G

0.77

6.49G

0.43

6.87G

0.59

7.84G

0.83

7.38G

0.50

6.96G

0.46

6.83G

0.60

8.35G

0.42*

KO

7.30G

0.84

7.18G

0.78

7.54G

0.66

7.83G

0.84

7.35G

0.84

7.48G

0.77

7.54G

0.91

7.10G

0.85

7.71G

0.84

8.49G

0.82

vonFreymodel

WT

1.05G

0.13

1.24G

0.12

1.51G

0.00#

1.43G

0.08

1.42G

0.07

1.38G

0.12

1.26G

0.11

1.29G

0.10

0.87G

0.14

0.75G

0.11

KO

1.18G

0.14

1.15G

0.21

1.41G

0.05

1.37G

0.08

1.34G

0.11

1.19G

0.19

1.17G

0.13

1.31G

0.12

1.27G

0.11**

1.17G

0.11**

Cold-plate

test

WT

2.63G

1.35

3.63G

1.84

1.00G

0.87

1.13G

0.64

1.75G

0.59

0.25G

0.25***

0.13G

0.13

0.13G

0.13

1.25G

0.73

1.38G

0.53

KO

0.14G

0.14

0.14G

0.14

0.00G

0.00

0.00G

0.00

0.00G

0.00**

0.00G

0.00

0.00G

0.00

0.00G

0.00

0.00G

0.00

0.00G

0.00**

Data

are

expressed

asthemeanG

SEM

ofthepawwithdrawallatency

(s)in

theplantartest,filamentstrength

(g)in

thevonFreymodelandnumber

ofpawelevationsin

thecold-plate

testforboth

contralateralandipsilateralpawsin

wild-type(W

T)andCBIknockout(K

O)mice.*P!

0.05,**P!

0.01(Fisher’stestknockoutvswild-type),***P!

0.01(Fisher’stestipsilateralvscontralateral

paw),#P!

0.05(N

ewmaneKeulspost

hocvsday6).

(P! 0.01) after surgery. This allodynia disappearedduring gabapentin treatment on days 8, 10 and 13 aftersurgery.

4. Discussion

In this study, we have investigated the role played byCB1 cannabinoid receptors in the development and ex-pression of neuropathic pain in mice after partial sciaticnerve ligation. It is well established that injury to a pe-ripheral nerve can lead to hyperalgesia to noxious ther-mal stimuli and allodynia to cold and mechanical stimuliin rodents, two features commonly observed in humanneuropathies (Rowbotham, 1995). Partial ligation ofthe sciatic nerve in rodents presents the advantage ofa reliable measurement of these two pain manifestationswithout a relevant mirror image in the intact limb(Malmberg and Basbaum, 1998), and is associated witha poor inflammatory component compared to otherrodent neuropathic pain models (Kim et al., 1997;Bridges et al., 2001a).

The endogenous cannabinoid system plays an impor-tant role in the control of pain processes (Cravatt andLichtman, 2004). Cannabinoid agonists are effective ina wide range of acute, inflammatory and chronic noci-ceptive models including neuropathic pain (Pertwee,2001). In addition, endocannabinoids mediate a protec-tive role during visceral inflammation through the acti-vation of CB1 cannabinoid receptors (Massa et al.,2004). Thus, CB1 knockout and rimonabant-treatedmice exposed to an experimental colitis exhibiteda higher sensibility to chemical-induced visceral inflam-mation. Conversely, FAAH-deficient mice showed pro-tection against colonic inflammation (Massa et al.,2004). The endogenous cannabinoid system may alsoplay a protective role in neuropathic pain states. Thus,anatomical studies have demonstrated an up-regulationof CB1 and CB2 cannabinoid receptors followingperipheral nerve injury (Siegling et al., 2001; Limet al., 2003; Zhang et al., 2003), and pharmacologicalstudies have shown analgesic effects of both CB1 (Maoet al., 2000; Bridges et al., 2001b; Fox et al., 2001) andCB2 (Ibrahim et al., 2003) cannabinoid agonists onexperimental models of neuropathic pain.

In our study on genetically modified mice with CB1receptor deletion, similar baseline withdrawal thresholdsto thermal and mechanical stimuli were found in wild-type and mutant mice, suggesting that CB1 cannabinoidreceptors do not seem to tonically modulate thermal andmechanical nociceptive sensitivity (Scott et al., 2004).Previous studies have evaluated the nociceptive thresh-old of CB1 knockout mice after the application of differ-ent nociceptive stimuli. In agreement with this currentwork, no significant differences were observed in thesame line of CB1 mutants after the application of acute


thermal (hot-plate and tail-immersion) and mechanical(tail-pressure) nociceptive stimuli (Ledent et al., 1999;Valverde et al., 2000). However, Zimmer et al. (1999) re-ported a thermal hypoalgesic phenotype of CB1 knock-out mice in the hot-plate test, without changes in thetail-flick test. Furthermore, a recent study in CB1knockout mice reported normal acute thermal nocicep-tive sensitivity, but increased tactile sensitivity in themutant animals (Ibrahim et al., 2003). A possible expla-nation for these discrepancies would be the use of micewith distinct genetic background (CD1, C57Bl/6, and129/SvJ), and the different experimental conditions forthe nociceptive tests in each previous study.

In the present study, sham operation did not producemajor consequences neither in wild-type nor in CB1knockout mice. Nevertheless, a significant decrease inthe number of ipsilateral and contralateral hindpaw ele-vations was observed in sham-operated mutants com-pared to wild-type mice following the consecutiveexposure to the cold-plate test. This result may suggesta different motor reactivity of CB1 knockout mice inthese specific experimental conditions. However, wecannot exclude that a faster desensitization to the coldstimuli occurs in the mutant mice. No other behaviouralmodification was observed in sham-operated CB1knockout mice during the repeated exposure to the dif-ferent nociceptive models.

Our results reveal that CB1 cannabinoid receptorknockout mice do not show altered pain responses afterthe partial sciatic nerve ligation. A similar and persistentincrease of thermal hyperalgesia as well as thermal andmechanical allodynia was observed in wild-type and mu-tant mice, suggesting that CB1 cannabinoid receptorsare not critically involved in the development and ex-pression of neuropathic pain. The overall developmentof neuropathic pain was studied along 15 days, and onlya significant difference between wild-type and mutantmice was observed on day 6 after nerve injury in the ex-pression of thermal hyperalgesia. Our results are consis-tent with previous studies reporting no genotypedifferences in neuropathic pain-induced thermal hyper-algesia evaluated 10 days after L5/L6 spinal nerve injury(Ibrahim et al., 2003) and 7 or 14 days after chronic con-striction of the sciatic nerve (Costa et al., 2005) in differ-ent strains of CB1 mutants. Additionally, neuropathicpain was not altered in FAAH-deficient mice afterchronic sciatic nerve constriction (Lichtman et al.,2004). Taken together, all these results suggest that theendogenous cannabinoid system does not seem to playa critical role in the regulation of persistent neuropathicpain states.

In contrast to data obtained on genetically modifiedmice, pharmacological studies in rodents have providedevidence that activation of CB1 receptors by exogenousadministration of cannabinoid agonists reduces painsensitivity in a variety of neuropathic pain models.

THC and synthetic cannabinoids, such as WIN55,212-2, CP-55,940 and HU-210, alleviated thermaland mechanical hyperalgesia and allodynia associatedwith chronic constriction injury of the sciatic nerve(Herzberg et al., 1997; Mao et al., 2000; Costa et al.,2004), L5 spinal nerve ligation (Bridges et al., 2001b)and partial sciatic nerve ligation (Fox et al., 2001). Theseresponses were selectively blocked by the administrationof the CB1 receptor antagonist rimonabant (Mao et al.,2000; Bridges et al., 2001b; Fox et al., 2001). Recently, ithas been described that chronic administration of rimo-nabant induces pain relief in rats and mice with chronicconstriction of the sciatic nerve, through a CB1 receptormechanism (Costa et al., 2005). This effect does notseem to be related to a direct antinociceptive action ofrimonabant on the pain signalling system, but to beassociated with a reduction of pro-inflammatory andpro-nociceptive mediators, as well as an enhancementof nerve regeneration (Costa et al., 2005). Additionally,an overactivity of CB1 cannabinoid receptors has beenpreviously reported in response to nerve injury (Sieglinget al., 2001; Lim et al., 2003). Thus, CB1 cannabinoid re-ceptors were up-regulated in the thalamus (Sieglinget al., 2001) and the spinal cord (Lim et al., 2003) of ratswith peripheral neuropathy. In the later study, the intra-thecal administration of the ERKeMAP kinase inhibi-tor, PD98059, blocked both CB1 up-regulation andthe enhanced efficacy of CB1 agonists to reduce hyperal-gesia, suggesting that such an up-regulation contributesto the therapeutic effects of exogenous applied cannabi-noids (Lim et al., 2003). Together, these studies using ge-netic and pharmacological approaches suggest that theup-regulation of CB1 cannabinoid receptors appearsto be important for the pharmacological efficacy of can-nabinoid agonists on neuropathic pain, but does notseem to be sufficient to suppress the development of sucha pathological pain.

To date, neuropathic pain is not adequately managedwith the available pharmacological tools and representsa substantial unmet medical need. Different pharmaco-logical treatments have been used to alleviate neuro-pathic pain in humans (Banos et al., 2003; Foley,2003). Among these treatments, the anti-epileptic drug,gabapentin, seems to be particularly efficient in animalmodels of neuropathic pain, as well as in clinical trialsand human therapy (Backonja and Glanzman, 2003).Gabapentin is a lipophilic 3-cyclohexyl analogue of GA-BA that binds to the a2d subunit of voltage-gated calci-um channels (L, R, P/Q, I, N-type calcium channels)and blocks high voltage calcium currents (Gee et al.,1996; Rogawski and Loscher, 2004). This compound al-so increases GABA synthesis and turnover (Loscheret al., 1991). Nevertheless, the mechanism of action ofgabapentin in alleviating neuropathic pain symptomshas not been fully identified. Considering the pharmaco-logical relevance of CB1 cannabinoid receptors for the


treatment of neuropathic pain, we have evaluated if thepharmacological efficacy of gabapentin is modified inCB1 knockout mice. Gabapentin reversed thermal hy-peralgesia as well as thermal and mechanical allodyniain wild-type mice from the first administration, demon-strating its analgesic effects on the main manifestationsof neuropathic pain in this animal model, and in agree-ment with human clinical conditions (Backonja andGlanzman, 2003). The analgesic effects of gabapentinwere maintained during the whole chronic treatment in-dicating the lack of tolerance over this time course.However, the neuropathic pain manifestations returnedas soon as the treatment was stopped. Previous studieshave also shown that chronic administration of gaba-pentin inhibits the hypersensitivity to mechanical stimu-lation induced by partial ligature of the sciatic nerve inrodents (Patel et al., 2001; Bortalanza et al., 2002). Thus,chronic treatment with gabapentin (70 mg/kg/day, p.o.)attenuated mechanical allodynia in mice with partial sci-atic nerve injury, an effect that lasted up to 3 days afterstopping the treatment (Bortalanza et al., 2002). Similar-ly, repeated administration of gabapentin (100 and250 mg/kg, s.c.) reversed mechanical hyperalgesia in ratswith partial sciatic nerve ligation (Patel et al., 2001). Inthe present study, the analgesic effects of gabapentin onneuropathic pain were maintained in mice lacking CB1cannabinoid receptors. Only a slight delay in the effectsof gabapentin on thermal hyperalgesia was observed inCB1 knockout mice in comparison with wild-type litter-mates. Interestingly, CB1 knockout animals also showedin this study a higher hyperalgesic response on day 6 af-ter nerve injury compared to wild-type mice. Therefore,this particular component of the neuropathic pain seemsto be especially sensitive to the consequences of a geneticdisruption of CB1 cannabinoid receptors.

In conclusion, the present study demonstrates thatthe deletion of CB1 cannabinoid receptors does not sig-nificantly alter the development of neuropathic pain af-ter partial sciatic nerve ligation. The analgesic effects ofgabapentin on neuropathic pain, here revealed in boththermal and mechanical allodynia and thermal hyperal-gesia, were also preserved in these mutant mice. Onlyminor changes on neuropathic pain-induced thermal hy-peralgesia and the effects of gabapentin on such a re-sponse were observed after the deletion of CB1cannabinoid receptors.

Acknowledgements

This work was supported by grants from the SpanishMinisterio de Ciencia y Tecnologıa (SAF 2004/0568 andGEN 2003-20651-C06-04), Redes del Instituto CarlosIII (C 03/06 and G 03/005), and European Communities(QLRT 2001-01691 and NEWMOOD# LSHM-CT-

2003-503474). AC is a fellowship from DURSI (Gener-alitat de Catalunya).

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Development and expression of neuropathic pain in CB1 knockout mice

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