Attenuation of pain-related behavior in a rat model of trigeminal neuropathic pain by viral-driven...

ARTICLE doi:10.1016/j.ymthe.2004.12.011

Attenuation of Pain-Related Behavior in a RatModel of Trigeminal Neuropathic Pain byViral-Driven Enkephalin Overproduction in

Trigeminal Ganglion Neurons

Alice Meunier,1,* Alban Latremoliere,2,* Annie Mauborgne,1 Sylvie Bourgoin,2

Valerie Kayser,2 Francois Cesselin,1 Michel Hamon,2 and Michel Pohl1,y

1INSERM U 713, Douleurs et Stress, and 2INSERM U 288, NeuroPsychoPharmacologie Moleculaire, Cellulaire et Fonctionnelle,

CHU Pitie-Salpetriere, 91, Boulevard de l’Hopital, 75013 Paris, France

*These authors contributed equally to this work.

yTo whom correspondence and reprint requests should be addressed at INSERM U 713, Faculte de Medecine Pitie-Salpetriere, 91,

Boulevard de l’Hopital, 75634 Paris Cedex 13, France. Fax: +33 1 40 77 97 90. E-mail: [email protected].

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Trigeminal neuropathic pain represents a real challenge to therapy because commonly used drugsare devoid of real beneficial effect or patients frequently become intolerant or refractory to some ofthese compounds. In a rat model of trigeminal neuropathic pain, which shares numerous similaritieswith human trigeminal neuralgia and trigeminal neuropathic pain, we used a genomic herpessimplex virus-derived vector (HSVLatEnk) to examine the possible effect of a local overproduction ofproenkephalin A (PA) targeted to the trigeminal primary sensory neurons. Unilateral peripheralinoculation of recombinant vectors on the vibrissal pad territory resulted in an about ninefoldincrease in proenkephalin A mRNA levels in trigeminal ganglion ipsilateral to the infected side.Transgene-derived met-enkephalin accumulated in numerous nerve cell bodies of trigeminalganglion and was transported through the sensory nerve fibers located in the infraorbital nerve.Bilateral mechanical hyperresponsiveness, which developed 2 weeks after chronic constrictive injuryof the left infraorbital nerve, was significantly attenuated in animals overproducing PA in thetrigeminal ganglion ipsilateral to the lesioned infraorbital nerve. This antiallodynic effect wasreversed by both the opioid receptor antagonist naloxone and the peripherally acting antagonistnaloxone methiodide. Our data demonstrate that the local overproduction of PA-derived peptides intrigeminal ganglion sensory neurons evoked a potent antiallodynic effect through the stimulation ofmainly peripherally located opioid receptors and suggest that targeted delivery of endogenousopioids may be of interest for the treatment of some severe forms of neuropathic pain.

Key Words: gene therapy, trigeminal neuropathic pain, proenkephalin A, met-enkephalin,herpes simplex vector

INTRODUCTION

Trigeminal neuropathic pain is a frequently occurringpathological condition in humans that differs in nume-rous aspects from neuropathic pain affecting limbs, ismostly resistant to morphine, and represents a realchallenge to therapy [1–3]. Although antidepressantsand anticonvulsants offer some therapeutic benefit intrigeminal neuralgia, an important proportion of patientsbecome refractory to these drugs [4]. A recently deve-

loped rat model of trigeminal neuropathic pain obtainedby chronic constrictive injury of the infraorbital nerve(CCI-ION) shares many characteristics with clinical dis-orders in humans suffering from trigeminal neuralgia ortrigeminal neuropathic pain [5–7]. In particular, pain-related behavioral abnormalities observed in rats withCCI-ION are difficult to treat with tricyclic antidepres-sants and single or repeated administrations of morphineare ineffective on the mechanical hyperresponsiveness of

MOLECULAR THERAPY Vol. 11, No. 4, April 2005

Copyright C The American Society of Gene Therapy

1525-0016/$30.00

IG. 1. Presence of latency-associated transcripts and increases in PA mRNA

vels in left trigeminal ganglion 3 weeks after HSVLatEnk inoculation on left

t vibrissal pad. (A) Ten-micrometer sections of both left and right trigeminal

anglia from HSVLatEnk-infected or uninfected rats were incubated with

igoxigenin-labeled LAT cRNA. LAT expression was detected in numerous

erve cell bodies in the left (ipsilateral to the infection) trigeminal ganglion of

SVLatEnk-infected rats. In contrast, comparable to both ganglia from

ninfected rats (not shown), no LAT-containing neurons were detected in

e right ganglion contralateral to the HSVLatEnk-inoculated vibrissal pad.

he photomicrographs are representative of three animals per group. Scale

ar, 100 Am. (B) Total RNA was extracted from left or right trigeminal

anglion of HSVLatEnk-infected rats (n = 3) and 2 Ag of total RNA was reverse-

anscribed in the presence of six different dilutions of synthetic fragment and

mplified for 30 cycles. Optical density of PCR amplification products of PA

RNA (402 bp) or of the synthetic fragment (241 bp) was used for the plot

rawing [13]. Representative gel analyses of PCR products are shown.

ARTICLEdoi:10.1016/j.ymthe.2004.12.011

animals [7]. Chronic administration of a high dose ofmorphine (5 mg day�1) starting 1 month after the IONligation was shown to reduce mechanical hypersensitiv-ity in CCI-ION. However, this effect rapidly vanished astolerance to morphine developed and was finally com-pletely absent when chronic infusion of morphine startedduring the first 2 weeks following ION ligation [8].Interestingly, by contrast to neuropathic pain-relatedbehavior involving the limbs, mechanical allodyniaassociated with CCI-ION has been recently shown to bereduced by triptans, the potent antimigraine moleculeswith 5-HT1B/1D receptor agonist properties [9]. These laterdata thus further suggest that neuropathic pain in thetrigeminal area presents important differences, comparedwith peripheral pain at the spinal level, which probablyreflect anatomophysiological particularities of the trige-minal sensory system [10,11].

We and others have recently demonstrated that herpessimplex virus type 1 (HSV-1)-mediated transfer andoverexpression of proenkephalin A (PA) in primarysensory neurons at the lumbar level has a potentantihyperalgesic effect in rat models of chronic pain[12–14]. In addition, in a rat model of neuropathic painconsecutive to spinal nerve ligation, Hao and co-workers[15] also reported that overexpression of PA in lumbardorsal root ganglia ameliorated mechanical allodynia.

Here, we examined in this particular model of severetrigeminal neuropathic pain the possible effects oftargeted overproduction of PA-derived opioid peptidesin trigeminal ganglion sensory neurons, with specialattention to the period (2 weeks after infraorbital nerveligation) when mechanical hypersensitivity was fullyestablished but chronic morphine treatment was devoidof any antiallodynic potency.

RESULTS

Peripheral Inoculation of HSVLatEnk on Rat VibrissalPad Results in Robust Met-enkephalin Production inTrigeminal Sensory Ganglia NeuronsThree weeks following unilateral application of anHSVLatEnk suspension on the slightly scarified left ratface, in situ hybridization histochemistry with cRNAprobe of HSV latency-associated transcripts (LATs)showed accumulation of LATs only in the left trigeminalganglion, ipsilateral to the infection. Indeed, in the rightganglion, as in the trigeminal ganglia from uninfectedrats (not shown), we detected no LAT hybridization (Fig.1A). The presence of LATs in numerous nerve soma of theleft ganglion demonstrated the ability of the HSV vectorto penetrate into this structure and to establish a latentinfection of sensory nerves after peripheral infection.

The levels of pEnkA mRNA measured by quantitativeRT-PCR in the left trigeminal sensory ganglion wereabout ninefold higher (P b 0.001; n = 3) than thosefound in the right ganglion (contralateral to the infec-



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tion) (Fig. 1B). Proenkephalin A mRNA concentrations inthe right ganglion of HSVLatEnk-infected rats werecomparable to those determined in both trigeminalganglia of control uninfected animals (not shown).Finally, infection of rats with control HSVLath-gal vector

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did not affect the amount of pEnkA mRNA thatremained, comparable to control values (not shown).

Immunofluorescence investigations showed that no oronly a few neurons containing met-enkephalin (ME)-likeimmunoreactive material (MELM) were present in trige-minal sensory ganglia of uninfected or sham-infected(HSVLath-gal) rats. Infraorbital nerve, which includesperipheral branches of trigeminal sensory neurons, con-tained rare and discrete nerve fibers stained for MELM. Incontrast, in HSVLatEnk-infected rats, numerous neuronalcell bodies moderately or heavily stained for MELM werepresent in the left trigeminal ganglion (Fig. 2A). Numer-ous nerve processes containing dense MELM labelingwere visualized in the left infraorbital nerve (Fig. 2B). Onthe other hand, as in control rats, almost no MELM-containing neurons or nerve processes were present inthe contralateral (right) ganglion or infraorbital nerve inHSVLatEnk-infected rats (Figs. 2C and 2D).

As a major part of the central branches of thetrigeminal sensory neurons project to the trigeminalspinal nucleus, we examined the presence of MELM inparticular in the caudal part of this nucleus (subnucleuscaudalis), where thermal and pain messages from the faceterritory are conveyed [16,17]. In HSVLatEnk-infectedrats, MELM labeling present in superficial layers of theleft part (ipsilateral to the infection) of nucleus wasunchanged compared with the density and area of MELMlabeling in the contralateral part of this structure (Fig. 3).Immunohistochemical staining for MELM in the trige-minal nucleus of HSVLatEnk-infected rats was consis-tently comparable to that found in control rats (notshown).

FIG. 2. Immunofluorescent detection of met-enke-

phalin-like material in left and right trigeminal ganglia

and infraorbital nerves in HSVLatEnk-infected rats.

Fifteen-micrometer sections were stained with anti-

ME monoclonal antibody (Valbiotech). (A) Numerous

neuronal cell bodies in the left (ipsilateral to the

infection) trigeminal ganglion were heavily (arrows)

or moderately (arrowheads) stained for ME. (B) The

presence of immunoreactive ME-like material was also

visualized in numerous nerve fibers in the left

infraorbital nerve. In contrast, in (C) trigeminal

ganglion and (D) infraorbital nerve dissected out from

the right, uninfected side of the rat head, no, or only

scarce, ME labeling was detected. The photomicro-

graphs are representative of three to five animals per

group. Scale bar, (A, C) 100 Am, (B, D) 50 Am.

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Chronic Constriction Injury of the Infraorbital NerveInduces Severe Mechanical Allodynia That IsAttenuated by Local HSVLatEnk-Mediated MEOverproductionTwo weeks after CCI of the left infraorbital nerve,uninfected or HSVLath-gal-infected rats presented con-siderably decreased response threshold to mechanicalstimulation of the left face (0.20 F 0.07 g, mean F SEM,n = 15; 0.20 F 0.04 g, mean F SEM, n = 6, respectively)compared with values measured in sham-operated rats(12.5 g, i.e., the experimental cut-off value) or withpreligation thresholds of each animal (12.5 g) (Fig. 4A).Hypersensitivity to mechanical stimulation remainedconstant for at least 4 weeks after the infraorbital nerveligation (not shown). Most of the lesioned animals (16 of21) presented a brisk withdrawal to the mechanicalstimulus followed by an attack reaction. As we measuredno difference in mechanical stimulation hypersensitivityafter lesion of the infraorbital nerve in uninfected versusHSVLath-gal-infected rats, we will hereafter refer toanimals of both groups as lesioned control rats. Mechan-ical stimulation of the right face territory innervated bythe uninjured infraorbital nerve also showed a markedlydecreased response threshold (0.26 F 0.07 g vs. 12.5 g insham-operated rats) that was comparable to the valuesmeasured in the territory ipsilateral to the lesioned leftinfraorbital nerve.

By contrast, in rats infected with HSVLatEnk on theleft vibrissal pad 1 week before their left infraorbital nervewas lesioned, hypersensitivity to mechanical stimulationof the left face, developing 2 weeks after infraorbitalnerve CCI, was significantly weaker than that determined



FIG. 3. Photomicrograph reconstitution of MELM

immunolabeling in left and right trigeminal nucleus

in HSVLatEnk-infected rats. Fifteen-micrometer sec-

tions prepared from the brain stem including the

subnucleus caudalis of the trigeminal nucleus were

processed for ME labeling. Analysis of immunolabel-

ing through the whole trigeminal nucleus revealed no

differences in density or distribution of ME staining

between the left, infected side and the right side of

the nucleus. Every fifth section is represented. The

photomicrographs are representative of five animals

per group. Scale bar, 300 Am.


in lesioned control rats (3.25 F 0.87 g, P b 0.005, n = 21).We never observed the abrupt attack reaction aftermechanical stimulation of the left face in HSVLatEnk-infected rats. The reduced mechanical response thresh-old, determined in a group of HSVLatEnk-infected rats 3weeks after infection (i.e., 4 weeks after infraorbital nerveligation), persisted during this observation period. On theother hand, mechanical stimulation of the right face(contralateral to the infection and lesion side) of HSVLa-tEnk-infected rats was unaffected at any time after thetreatment and the mechanical response thresholdremained comparable to the values observed in lesionedcontrol rats (Fig. 4A).

Antiallodynic Potency of Targeted MEOverproduction Is Reversed by a Peripherally ActingOpioid Receptor AntagonistMechanical hypersensitivity successive to the infraorbi-tal nerve CCI was not modified in control or HSVLa-tEnk-infected rats implanted with saline-containingminipumps (not shown). Three days administration ofnaloxone or naloxone methiodide at the dose of 3 mg �kg�1 � day�1 was without any significant effect onbilateral mechanical response threshold in lesionedcontrol rats (Fig. 4B), whereas in HSVLatEnk-infectedrats either naloxone or naloxone methiodide reversedthe antiallodynic effect (P b 0.05, n = 6 and P b 0.001,n = 5, respectively (Fig. 4B).

DISCUSSION

Trigeminal sensory nerves relay sensory and propriocep-tive information from orofacial regions and meninges.Despite evident analogies with the spinal sensorysystem, accumulating data are in favor of fundamentalanatomical, functional, as well trophic factor require-ment particularities of the trigeminal sensory system[10,18]. Together with the functional complexity oftrigeminal sensory systems, this specificity mightaccount, at least in part, for the real therapeuticchallenge that represents trigeminal neuropathic pain.



Interestingly, the particularity of the trigeminal sensorysystem is, at least in some aspects, also found in the ratmodel of trigeminal pain. Indeed, unlike neuropathicpain, which develops in the limb only on the side of thechronically constricted sciatic nerve [19], a pronouncedbilateral mechanoallodynia results from the unilateralloose ligation of the ION [5], probably reflecting theanatomofunctional organization of the trigeminal com-plex [11,20,21]. Here we report that in this rat model ofsevere trigeminal neuropathic pain, resistant to mor-phine treatment, which thus shares numerous behav-ioral abnormalities with human trigeminal neuralgiaand neuropathic pain, targeted genomic HSV vector-mediated overproduction of pEnkA-derived peptides intrigeminal ganglion sensory neurons evoked a potentantiallodynic effect.

The antinociceptive efficacy of HSV-driven overex-pression of pEnkA was recently reported at the spinallevel [12,14] and, in particular, we demonstrated bothantihyperalgesic and antiinflammatory potency of over-produced enkephalin-derived peptides in lumbar dorsalroot ganglia sensory neurons of rats suffering fromchronic inflammatory pain [13]. Using a similar ap-proach, Hao and co-workers [15] extended these data byshowing that transgene-mediated enkephalin productionin lumbar dorsal root ganglia may also attenuate neuro-pathic pain in the rat induced by lumbar fifth spinalnerve ligation and, contrary to the morphine antiallo-dynic effect, without signs of tolerance.

The HSV-derived vectors bearing the rat pEnkA codingsequence under the control of the LAT-long terminalrepeat (LTR) promoter that we generated were deleted forthe HSV thymidine kinase gene and were thus severelyimpaired for acute replication in neurons. After periph-eral (foot pad) inoculation, these vectors have beenshown to drive robust expression of pEnkA in primarysensory neurons of the lumbar dorsal root ganglia.Indeed, the production of transgene-derived authenticmet-enkephalin rose progressively during the first 3weeks after the infection [22] and then lasted for at least2 months [13]. In this study, peripheral application of

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FIG. 4. Mechanical response thresholds in control rats (preligation) and in rats

with ligation of left infraorbital nerve treated or not with HSVLatEnk.

Sensitivity of rats to mechanical stimulation of the vibrissal pad territory

was evaluated with a graded series of von Frey filaments. (A) Unilateral loose

ligation of left infraorbital nerve (ION) induced 15 days later a strong

mechanical allodynia at both left face (L) and contralateral right (R) face of

animals (n = 22). No difference in mechanical stimulation hypersensitivity was

measured in uninfected versus HSVLath-gal-infected rats and consequently

both groups were termed lesioned control rats. In rats inoculated on their left

vibrissal pad with HSVLatEnk 1 week before their left ION was constricted,

ligation-induced mechanical allodynia was attenuated 3 weeks (3 w) later

(i.e., 2 weeks after ION ligation) only on the left face, ipsilateral to the

HSVLatEnk application (n = 21). Decreased mechanical hypersensitivity in

HSVLatEnk-treated rats persisted 5 weeks (5 w) after left face infection (n = 7).

Data (means F SEM) are expressed in grams. *P b 0.005 for ION ligation

control versus HSVLatEnk-infected rats; **P b 0.001 for preligation versus ION

ligation rats; two-tailed unpaired t test. (B) Animals used for the evaluation of

the mechanical response threshold 3 weeks after they were inoculated with

HSVLatEnk were implanted subcutaneously for 3 days with an Alzet osmotic

minipump delivering 3 mg � kg�1 � day�1 of either naloxone (n = 6) or

naloxone methiodide (n = 5) and mechanical sensitivity in their left and right

(not shown) vibrissal pad territory was assessed once again. *P b 0.05 and

**P b 0.001 for untreated versus naloxone and naloxone methiodide-treated

HSVLatEnk-infected animals, respectively; two-tailed paired t test.


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HSVLatEnk on depilated and slightly scarified rat vibrissalpad led 3 weeks later to an important increase intransgene mRNA levels and the appearance of numerouscell bodies synthesizing and accumulating MELM in thetrigeminal ganglion ipsilateral to the infection. Similar toour observation in dorsal root ganglia [13], HSVLatEnkinfection, revealed by both LAT and transgene-derivedpEnkA RNA accumulation, was restricted to the sensorytrigeminal ganglion without any sign of cell damage.Numerous nerve fibers containing MELM were visualizedin the left infraorbital nerve (innervating the infectedface) of HSVLatEnk-infected rats, suggesting effectivetransport of MELM from nerve cell bodies to theperipheral terminals of infected sensory neurons. Inaddition, in infected animals no difference in theintensity or area of MELM staining was apparent throughthe subnucleus caudalis of the trigeminal spinal nucleusin the infected side where most of the central projectionsof sensory neurons innervating the rat face terminate[16,17]. Consistent with our previous observations at thespinal level, showing mainly peripherally oriented trans-port and release of overproduced MELM [13,23], thesedata suggest that the major part of viral vector-derivedMELM was transported to the peripheral processes oftrigeminal primary sensory neurons. However, the exis-tence of a limited contingent of sensory neurons trans-porting overproduced MELM also to their centralprojections could not be excluded. Indeed, at the spinallevel we showed that few MELM-containing nerveprocesses projected also to the spinal cord and, using asimilar approach, Goss et al. [14] reported that theantinociceptive effect of enkephalin overproduced inthe dorsal root ganglia could be reversed after intrathecaladministration of an opioid receptor antagonist.

Peripheral inoculation of HSVLatEnk and subsequentoverproduction of pEnkA-derived peptides in trigeminalsensory ganglia attenuated the marked mechanicalhypersensitivity of rats developing after the nerve injury.Although constriction of ION induces bilateral mechan-ical allodynia, an antiallodynic effect was observed onlyat the side infected with HSVLatEnk, and the mechanicalstimulation of the contralateral (right) face resulted inhypersensitivity comparable to that measured in controlrats with CCI-ION. In addition to the significantlyincreased mechanical response threshold, the HSVLa-tEnk-infected animals did not present the attack reactionafter the mechanical stimulation of the left face, fre-quently observed in lesioned control rats, suggesting thatthe paroxysmal stabbing pain (experienced in human asan explosive-like sensation) was absent. The decrease inmechanical hypersensitivity in CCI-ION rats was com-parable at 3 and 5 weeks after peripheral inoculation ofHSVLatEnk, suggesting a prolonged antiallodynic effectof overproduced enkephalins. If applicable in human, thechronic character of trigeminal pain should clearly needthe long-lasting efficacy of viral vector-driven enkephalin




production. Long-term activity of promoters based onLAT-derived elements combined with the Moloney mur-ine leukemia virus LTR, such as that used in ourrecombinant vector to drive pEnkA synthesis, has beenclearly documented [24,25]. Apart from the efficacy ofthe vector promoter and the active production of trans-gene-derived pEnkA, progressive development of toler-ance to continuously delivered enkephalins mightrepresent a possible limitation of such a treatment.Experiments of longer duration than those reported hereshould thus be conducted in this model of trigeminalneuropathic pain to ensure the persistence of the anti-allodynic efficacy of this treatment. Nevertheless, severalelements are in favor of a sustained antinociceptiveactivity of HSV-vector-mediated enkephalin production.Indeed, in a rat model of polyarthritis we have previouslyshown that the antihyperalgesic effect of HSVLatEnkpersisted with the same magnitude at least for 8 weekswithout any signs of tolerance [13]. The absence oftolerance development in response to continuous localproduction of pEnkA-derived peptides is further sup-ported by the data of Hao et al. [15]. In a model ofneuropathic pain at the spinal level, these authorsdemonstrated that, in contrast to rapidly developingtolerance to repeated administration of morphine, enke-phalins produced locally from very similar vectors main-tained a stable antiallodynic effect for 4 weeks. Theapparent lack of tolerance development might also reflectthe conditions of the release of overproduced enkepha-lins from nerve terminals, apparently similar to theinducible, physiological release of endogenous opioidpeptides. We, and others, have previously demonstratedthat viral vector-mediated, forced production of enke-phalins in control healthy rats does not induce any basalanalgesic effect [12,13]. The antinociceptive potency ofthis treatment is buncoveredQ under conditions of pro-longed or chronic pain, associated with continuousstimulation and sensitization of primary sensory neu-rons, presumably leading to the release of overproducedenkephalins from vector-infected neurons. Finally, ourobservation that, in primary sensory neurons at thelumbar level, overproduced enkephalins are stored andtransported in vesicular form and released after electricalstimulation of these neurons further supports the idea ofan inducible rather than constitutive release of viralvector-overproduced pEnkA-derived peptides [23].

The finding that the antiallodynic effect that followedthe enkephalin overproduction in trigeminal ganglionmay be reversed by the opioid receptor antagonistnaloxone suggests that PA-derived opioid peptides evokedlocal antinociceptive effects through the stimulation ofopioid receptors. The spinal trigeminal nucleus caudalis,which represents the major input of sensory neurons fromthe orofacial region, contains a high density of opiatereceptors and both in vitro and in vivo experimentsdemonstrated direct inhibitory activity of morphine and



of A- or y-opioid receptor agonists to inhibit sensoryneurons in this structure [26–28]. Although activation ofthese receptors might account for the reduced mechanicalhypersensitivity in HSVLatEnk-treated CCI-ION rats, ourdata rather support the idea that the major part of theantiallodynic potency of this treatment was mediatedthrough the activation of peripherally located opioidreceptors. Indeed, immunohistochemical experimentsshowed that, in line with our previous observations atthe lumbar level, the major part of the transgene-derivedMELM was present in the peripheral part of sensory nerves.Moreover, the finding that naloxone methiodide, anopioid receptor antagonist acting only at the peripherallevel, fully reversed the antiallodynic effect observed inHSVLatEnk-treated CCI-ION rats strongly supports thehypothesis that peripherally located opioid receptors areinvolved in the attenuation of trigeminal neuropathicpain in this model.

Abundant literature supports the reduced responsive-ness of neuropathic pain to systemic morphine (forreview, see Dellemijn [29]). Although some efficacy ofmorphine is occasionally found in clinical managementof neuropathic pain of other origins [30], facial pain [31]and trigeminal neuralgia [32] appear particularly resistantto opioid therapy. In the rat model of trigeminal neuro-pathic pain induced by CCI-ION, variable results werereported regarding morphine efficacy. While Deseure etal. [33] reported that a high (and, actually, sedative) doseof morphine (10 mg � kg�1 ip) could reverse mechanicalallodynia in this model, single or repeated iv injections ofmorphine at a dose 10-fold higher (1 mg � kg�1) thanthat sufficient to reduce mechanical allodynia successiveto CCI of the sciatic nerve [34] were without any effect onthe mechanical response threshold in CCI-ION rats[7,35].

By contrast, the present data demonstrated thattargeted HSV-mediated overproduction of pEnkA-derivedopioid peptides in the primary sensory neurons of thetrigeminal area evoked an antiallodynic effect in thismodel. Although sustained attenuation of hypersensitiv-ity of animals against mechanical stimulation was with-out apparent signs of tolerance or undesirable side effectsduring the observation period, long-term experimentsshould nevertheless be performed to demonstrate a realpotential for such approaches in the management ofsome extreme forms of chronic pain.

MATERIALS AND METHODS

HSV-Derived Vector Construction

The recombinant HSV-derived vectors impaired for replication were

constructed and produced as described previously [13]. Briefly, a rat

PA-encoding sequence under the control of the LAT-LTR promoter [24]

was inserted into the gC locus of HSV-1 genomic DNA deleted for the

thymidine kinase gene (HSVLatEnk). The modified HSV LAT promoter

allowed long-term in vivo production of transgene in rat primary

sensory neurons [13]. Control vectors were the same, except that the

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transcriptional unit inserted into the gC locus contained the Escherichia

coli h-galactosidase (LacZ) coding sequence (HSVLath-gal). Recombinant

vectors were isolated by PCR analysis and purified by successive

limiting dilutions. Single-plaque-isolated recombinants were then

amplified on Vero cells and purified at about 106 plaque-forming units

(pfu) � Al�1. Viral stock was saved at �808C in saline containing 10%

sucrose.

Animals and Treatments

All experiments were performed in conformity with the institutional

guidelines, which are in compliance with national and international

laws and policies for use of animals in neuroscience research (European

Communities Council Directive No. 87848, October 1987, Ministere de

l’Agriculture et de la Foret, Service Veterinaire de la Sante et de la

Protection Animale; Permissions No. 6186 to M.P.). Male Sprague–

Dawley rats (Centre d’Elevage R. Janvier, Le Genest-St Isle, France),

weighing 175–200 g on arrival, were used. All animals were maintained

under the same conditions (22 F 18C, 60 F 10% relative humidity, 12 h/

12 h light/dark cycle, food and water ad libitum). The animals were

accustomed to the housing facilities for at least 1 week before any

treatment.

Infection with recombinant vectors. Rats were profoundly anesthetized

(Nembutal, 50 mg � kg�1 ip) and their left side vibrissal pad territory

was depilated using a razor blade and slightly scarified. Five microliters

of viral suspension (~5 � 106 pfu) of either HSVLatEnk or HSVLath-gal

was applied and spread using glass tips. Treatment of sham-infected

rats consisted of vehicle (5 Al of 10% sucrose in 0.9% NaCl) application

onto slightly scarified vibrissal pad. Animals were treated 1 week before

induction of trigeminal neuropathic pain. This experimental protocol

was chosen because previous studies showed that the increase in the

concentrations of MELM in rat sensory ganglia following peripheral

application of HSVLatEnk was maximum 3 weeks after infection [22],

thus coinciding with the hyperresponsiveness to mechanical stimula-

tion, which is fully developed 2 weeks after the nerve lesion [5,6]. In

addition, 3 weeks after the infection, the rats’ vibrissae were grown

again and the animals’ faces were completely healed, allowing the

evaluation of mechanical allodynia with von Frey filaments. A group

of control rats, unilaterally infected with either HSVLatEnk or

HSVLath-gal but in which the infraorbital nerve was not constricted,

was also prepared.

Surgery. Rats were anesthetized with an intraperitoneal injection of

sodium pentobarbital (Nembutal, 50 mg � kg�1) and the head of the rat

was fixed in a Horsley–Clark stereotaxic frame. The unilateral chronic

constriction injury to the left infraorbital nerve was performed under

direct visual control using a Zeiss operation microscope (10–25�). A

midline scalp incision was made, exposing skull and nasal bone. The

infraorbital part of the left infraorbital nerve was exposed using a

surgical procedure adapted from Gregg [36] and Jacquin and Zeigler [37].

The edge of the orbit, formed by the maxillary, frontal, lacrimal, and

zygomatic bones, was dissected free. To give access to the infraorbital

nerve, the orbital contents were gently deflected with a cotton-tipped

wooden rod. The infraorbital nerve was dissected free at its most rostral

extent on the orbital cavity, just caudal to the infraorbital foramen. Two

chromic catgut (5-O) ligations were tied loosely (with about 2 mm

spacing) around the nerve. To obtain the desired degree of constriction,

a criterion formulated by Bennett and Xie [19] was used: the ligations

reduced the diameter of the nerve by a just noticeable amount and

retarded, but did not interrupt the epineural circulation. Blood

circulation through epineural vessels was checked under direct visual

control using the Zeiss operation microscope. The scalp incision was

closed using silk sutures (5-O).

Nociceptive test procedures. Animals were placed individually in plastic

cages and allowed to adapt to the testing environment as described

previously [9]. Mechanical sensitivity was determined with a graded

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series of 10 von Frey filaments (Semmes–Weinstein monofilaments;

Stoelting, Wood Dale, IL, USA), producing a bending force of 0.217,

0.445, 0.745, 0.976, 2.35, 4.19, 4.64, 6.00, 7.37, and 12.5 g. The 12.5-g

filament, the bending force of which already turned the head of the rat,

was chosen as the cutoff. Stimuli were applied within the infraorbital

nerve territory, near the center of the vibrissal pad, on the hairy skin

surrounding the mystacial vibrissae. These areas were stimulated on

both sides of the face, ipsilateral and contralateral to the nerve

constriction and/or infection. Each stimulation consisted of three

consecutive applications (1 s apart) of the filament, beginning with

the filament producing the lowest force. A complete series of von Frey

filaments was applied following an increasing force order, until a well-

defined behavioral response was triggered. As previously described [9],

this response consisted of a brisk withdrawal of the head and/or an

attack/escape reaction. The minimal force applied through von Frey

filaments to trigger at least one of these behaviors was considered the

mechanical response threshold. Thresholds to stimulation of the face by

von Frey filaments were determined 2 days before and 3 and 5 weeks

after infection (i.e., 2 and 4 weeks after surgery).

Pharmacological treatments. Mechanical hypersensitivity of the differ-

ent groups of CCI-ION or infected control rats was first assessed 3 weeks

after infection. The next day, animals were lightly anesthetized

(Nembutal, 30 mg � kg�1 ip). The skin was incised at the level of the

scapula, an Alzet osmotic minipump (Model 1007D; delivery rate 0.5 Al �h�1) was implanted subcutaneously, and the incision was then sutured.

Following the manufacturer’s instructions osmotic minipumps were

filled with either naloxone or naloxone methiodide to administer each

of these drugs at the dose of 3 mg � kg�1 � day�1. bShamQ-treated

animals were implanted with saline-delivering minipumps. Behavioral

studies were performed on the third day after minipump implantation.

Observers were blinded to the groups of CCI-ION rats (sham-, HSVLath-

gal-, or HSVLatEnk-infected) and to the drug delivered. Behavioral

experiments did not reveal any differences between sham-infected and

HSVLath-gal-infected controls on the one hand and CCI-ION rats on the

other hand.

Quantitative Reverse Transcription-PCR

Animals used for RT-PCR, immunohistochemical, or in situ hybrid-

ization procedures were killed by decapitation 3 weeks after infection,

i.e., 2 weeks after infraorbital nerve constriction. Trigeminal ganglia

were dissected at 0–48C. Tissue pieces for RNA analyses were frozen in

liquid nitrogen and stored at �808C. Total RNA, extracted using the

NucleoSpin RNA II extraction kit (Macherey-Nagel, Hoerdt, France),

was quantified using as reference a scale of total RNA prepared on a

cesium chloride gradient and estimated from the optical density at 260

nm. RT-PCR was performed, as previously described [22], with 2 Ag of

each RNA sample in the presence of various amounts (0.1–80 fg) of

internal synthetic standard prepared according to the PCR MIMIC

construction kit (Clontech). This 241-base standard fragment, flanked

with PA sequences (21 bases), was amplified with the same set of rat

PA-specific primers as the cDNA. Reverse-transcribed RNA was ampli-

fied with 30 cycles (96, 58, and 728C; 1 min each) according to the

access RT-PCR system instructions (Promega, Madison, WI, USA) using

40 pmol of primers in a mixture containing 10 mm each dNTP, 25

mM MgSO4, 2.5 u of AMV reverse transcriptase, 2.5 u of Tfl DNA

polymerase, 6.5 u of RNase inhibitor (RNasin) in 1� reaction buffer.

The RT-PCR products were electrophoresed on 1.2% ethidium bromide-

stained agarose gel and quantified with the gel analyzer GDS 5000

(UVP, Cambridge, UK).

Immunohistochemistry

Deeply anesthetized animals (Nembutal, 50 mg kg�1 ip) were perfused

transcardially with 100 ml of saline (0.9% NaCl) supplemented with

0.1% sodium nitrite, followed by 600 ml of 4% paraformaldehyde in

PBS, at room temperature. Segments of infraorbital nerves and

trigeminal ganglia were dissected out and cryoprotected in 10% sucrose




(24 h, 48C). Fifteen-micrometer cryostat sections were preincubated (30

min, room temperature) in PBS containing 0.3% Triton X-100 and 3%

normal donkey serum (Jackson Immunoresearch, USA) and then

incubated overnight at 48C in the same buffer supplemented with a

monoclonal anti-ME antibody (1:1000; Valbiotech, France). After being

washed in PBS, sections were incubated for 1 h with rhodamine (Cy3)-

conjugated anti-mouse immunoglobulin (1:800; Interchim, France),

rinsed in PBS, mounted in Fluoromount-G (Clinisciences, France),

and examined using a Leica confocal microscope.

In Situ Hybridization

Animals were anesthetized and perfused as described in the protocol

for immunohistochemistry. Trigeminal sensory ganglia were dissected,

postfixed for 2 h in the same solution at 48C, cryoprotected in 10%

sucrose–PBS, frozen, and stored at �808C until used. Ten-micrometer

cryostat sections were mounted on slides, dehydrated through a

graded series of ethanol concentrations (30–100%), and incubated in

the presence of a cRNA probe for LATs labeled with digoxigenin-11–

UTP according to the instructions of the manufacturer (Promega).

Hybridization was performed overnight at 658C in 1� SSC, 50%

formamide, 10% dextran sulfate, 1 mg/ml rRNA, and 1� Denhardt’s

solution. On the following day, sections were washed twice in 1� SSC,

50% formamide, and 0.1% Tween 20, at 658C, and twice with 100

mM maleic acid, 150 mM NaCl, and 1% Tween 20, at room

temperature. The digoxigenin-labeled hybrids were detected with

alkaline phosphatase-conjugated anti-digoxigenin antibody following

the instructions of the manufacturer (Roche Products, Hertfordshire,

UK).

Statistical Analyses

Data presented as means F SEM were subjected to the unpaired Student t

test. Mechanical response thresholds after treatment versus before treat-

ment with opioid receptor antagonists were compared using the paired

Student t test. When P N 0.05, the corresponding difference was

considered to be not significant.

ACKNOWLEDGMENTS

We are grateful to Andre Bogdan (INSERM U 713) for critical reading of the

manuscript and helpful discussions. This work was supported by grants from

INSERM, Bristol–Myers Squibb Foundation (Unrestricted Biomedical Research

Grant), and Institut UPSA de la Douleur.

RECEIVED FOR PUBLICATION OCTOBER 7, 2004; ACCEPTED DECEMBER 17,

2004.

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