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Research Report

Botulinum neurotoxins and formalin-induced pain: Central vs.peripheral effects in mice

Siro Luvisettoa, Sara Marinellia, Francesco Lucchettia, Fabrizio Marchia, Stefano Cobianchia,Ornella Rossettob, Cesare Montecuccob, Flaminia Pavonea,⁎aIstituto di Neuroscienze del CNR, Psicobiologia e Psicofarmacologia, Centro Europeo di Ricerca sul Cervello, Via del Fosso di Fiorano 64,00143-Roma, ItalybDipartimento di Scienze Biomediche Sperimentali, Università di Padova, V.le G. Colombo 3. 35121-Padova, Italy

A R T I C L E I N F O

⁎ Corresponding author. Fax: +39 06 50170330E-mail address: f.pavone@ipsifar.rm.cnr.it

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.01.117

A B S T R A C T

Article history:Accepted 28 January 2006Available online 9 March 2006

Neurotoxins affecting neuroexocytosis can represent an innovative pharmacologicalapproach to the investigation of neural mechanisms of pain. Our interest has beenfocused on the use of botulinum neurotoxins (BoNTs), whose peripheral effects areextensively documented, while the effects on the central nervous system are much lessclear. We have investigated both peripheral (sc into the hindpaw) and central (icv) effects oftwo BoNTs isoforms, BoNT/A and BoNT/B, on inflammatory pain. BoNT/A (sc: 0.937–15; icv:0.937–3.75 pgtox/mouse) and BoNT/B (sc: 3.75, 7.5; icv: 1.875, 3.75 pgtox/mouse) were injectedin CD1 mice and tested in the formalin test 3 days later. Licking response, as index of pain,and behavioral parameters, such as general activity and grooming, were recorded for 40minduring the test. BoNT/A partially affects the licking response in the second phase of formalintest in a similar magnitude of attenuation whether peripherally or centrally administered.BoNT/A does not significantly affect licking behavior during the first phase of the test.Peripheral administration of BoNT/B attenuates the licking response during the first phasenot modifying the second phase, while the icv administration has hyperalgesic effect on theinterphase of the formalin test. General activity and grooming behavior are not affectedeither by peripheral or by central administration of BoNTs. Our results show for the firsttime a central effect of BoNTs that differently modulate inflammatory pain depending bothon serotype and on route of administration. Such data suggest BoNTs as a useful tool in thestudies aimed at the comprehension of the mechanisms of inflammatory pain.

© 2006 Elsevier B.V. All rights reserved.

Keywords:BotulinumSNARE proteinNeurotransmissionInflammatory painBehaviorMice

1. Introduction

Different serotypes (A–G) of botulinum neurotoxins (BoNTs)target the presynaptic nerve terminals where they selectivelyproteolyze SNAP-25 (BoNT/A, /C1, /E), syntaxin1 (/C1), andVAMP/synaptobrevin (/B, /D, /F, /G), all proteins belonging tothe SNARE complex responsible for neurotransmitter release

4.(F. Pavone).

er B.V. All rights reserved

(Humeau et al., 2000; Schiavo et al., 2000). Cleavage of one ofthese proteins results in incomplete formation of the SNAREcomplex, preventing the fusion of vesicles with the pre-synaptic membrane and thereby inhibiting the release ofneurotransmitter (Hua and Scheller, 2001; Sudhof, 2000).

This specialized action on nerve terminals made BoNTsuseful both in basic and clinical science (Jahn et al., 1995;

.

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Pearce et al., 1997; Schiavo et al., 1994). Intramuscularinjections of BoNTs are fast becoming the treatment of choicefor a variety of disorders caused by hyperfunction of cho-linergic terminals (Jankovic and Brin, 1997; Jost and Kohl, 2001;Munchau and Bhatia, 2000; Silberstein and Aoki, 2003). Somereports evidenced the efficacy of BoNTs in relieving pain withlong-lasting effects with respect to the relaxation of theinjected muscle (Freund and Schwartz, 2003; Lange et al.,1987). These findings, together with pain alleviation ofmyofascial pain, low back pain, and tension-type headache(Difazio and Jabbari, 2002; Gobel et al., 2001; Loder and Biondi,2002), suggest an activation of more complex analgesicprocesses than the simple consequence of muscle relaxation(Aoki, 2003; Argoff, 2002; Guyer, 1999; Lang, 2003; Lew, 2002;Voller et al., 2003).

The formalin test, a preclinical model used to investigatethe analgesic effect of drugs, may be useful to confirm thishypothesis (Dubuisson and Dennis, 1977). Subcutaneousinjection of formalin into the animal paw elicits an immediateincrease in C-afferent fiber's activity and evokes a series ofbehavioral responses directly involving the injured limb (Porro

Fig. 1 – Effects of sc-injection of saline, BoNT/A, or BoNT/B on then = 12), BoNT/A, or BoNT/Bwas done 3 days before formalin. Time1 (0–5 min) and phase 2 (10–40 min) of formalin test in: (A) salinemouse) sc-injected mice and (B) saline or BoNT/B (●, 3.75, n = 11significance: (*) P < 0.05, (**) P < 0.01, vs. saline sc-injected mice (F

and Cavazzuti, 1993). These responses are biphasic with anearly short-lasting phase (phase 1), due to direct stimulation ofnociceptors and initiation of inflammation, followed by asecond prolonged phase (phase 2) reflecting pain sensitizationprocesses (Coderre et al., 1990; Dickenson and Sullivan, 1987;Tjolsen et al., 1992). Between these two phases, a quiescentinterphase is observed which seems to be an inhibitory phasedue to a central inhibitory modulation of nociceptive behavior(Green and Dickenson, 1997; Henry et al., 1999; Matthies andFranklin, 1992).

Recently, Cui et al. (2004) showed that subcutaneousinjection of BoNT/A (BOTOX®) into the rat paw significantlyreduced formalin pain during phase 2, and suggested that theantinociceptive effect of BoNT/A would be a consequence ofan inhibition of neuronalmechanisms involving the release ofexcitatory substances during pain sensitization processes.

In this research, by investigating the central effects ofBoNT/A and BoNT/B, we extended the study of BoNTs effectson formalin-induced inflammatory pain. The results demon-strate, for the first time, a central modulation of BoNTs onformalin-induced inflammatory pain and the comparison

formalin-induced licking behavior. The injection of saline (○,course of licking duration and total licking time during phaseor BoNT/A (●, 3.75, n = 11; ■, 7.5, n = 12; ▲, 15, n = 12; pgtox/; ■, 7.5, n = 10; pgtox/mouse) sc-injected mice. Statisticalisher's PLSD).

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with peripheral effects show that antinociceptive effects ofBoNTs are dependent on the route of administration andBoNTs serotype considered. Some of these results have beenpresented in abstract form (Pavone et al., 2003).

2. Results

Fig. 1 shows the time course of the licking response (left part)and the cumulative licking time (right part) during phase 1(0–5 min) and phase 2 (10–40 min) of formalin-induced pain indifferent groups of saline, BoNT/A (panel A), or BoNT/B (panelB) sc-injected mice. Compared to licking responses observedin saline sc-injected mice, sc-injection of BoNT/A (3.75, 7.5, 15pgtox/mouse) did not significantly alter the licking timeduring phase 1 (F3,43: 2.061; P = 0.1195), although a tendencyto reduce licking response was observed at the highest doseadministered. All doses of BoNT/A induce a significantreduction of licking time during phase 2 (F3,43: 2.978;P < 0.05), mainly between 10 and 20 min from the beginningof the test. On the other hand, the analysis of variance showed

Fig. 2 – Effects of icv-injection of saline, BoNT/A, or BoNT/B on th(○, n = 11), BoNT/A, or BoNT/Bwas done 3 days before formalin. Tiphase 1 (0–5 min), the interphase (5–10 min) and the phase 2 (10(■, 1.875, n = 11; ●, 3.75, n = 12; pgtox/mouse) icv-injected micepgtox/mouse) icv-injected mice. Statistical significance: (*) P < 0.0

a significant decrease of the licking time during phase 1 (F2,30:4.862; P < 0.05) in BoNT/B sc-injected mice (3.75, 7.5 pgtox/mouse) and no significant changes during phase 2 (F2,30: 1.057;P = 0.3601), even if a tendency to reduce licking response wasobserved.

Additional experiments to evaluate the local effects ofBoNTs were done by injecting either saline or 3.75 pgtox/mouse of BoNT/A or BoNT/B into the plantar surface of thecontralateral hindpaw (left hindpaw). Injection of BoNTs intothe contralateral hindpaw did not alter pain behavior elicitedby injection of 5% formalin in the right hindpaw. When micewere injected with either saline, BoNT/A, or BoNT/B incontralateral hindpaw, the formalin-induced licking times ofipsilateral hindpawwere 136 ± 18, 117 ± 18, 125 ± 15 for phase 1(P > 0.05) and 425 ± 52, 460 ± 45, 430 ± 33 for phase 2,respectively (P > 0.05).

Fig. 2 shows the time course of the licking response (leftpart) and the cumulative licking time (right part) during phase1 (0–5 min) and phase 2 (10–40 min) of the formalin-inducedpain in different groups of saline, BoNT/A (panel A), or BoNT/B(panel B) icv-injected mice. Fig. 2 also shows the cumulative

e formalin-induced licking behavior. The injection of salineme course of licking duration and total licking time during the–40 min) of formalin test in: (A) saline or BoNT/Aand (B) saline or BoNT/B (■, 1.875, n = 12; ●, 3.75, n = 12;5, (**) P < 0.01, vs. saline icv-injected mice (Fisher's PLSD).

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licking time during the interphase (5–10 min). Compared tolicking response observed in saline icv-injected mice, analysisof variance showed for BoNT/A icv-injected mice a significantreduction of the licking time during phase 2 (F2,31: 5.785;P < 0.01), while the phase 1 and the interphase were notsignificantly modified (F2,31: 0.252; P = 0.7785 and F2,31: 1.156;P = 0.3280, respectively). On the other hand, icv-injection ofBoNT/B did not significantly alter the licking time during bothphases (F2,31: 0.436; P = 0.6505 and F2,31: 2.053; P = 0.1454, forphase 1 and phase 2, respectively) but, more surprisingly, atthe higher dose considered, it significantly enhanced thelicking time during the interphase (F2,31: 3.934; P < 0.05).

In Fig. 3, a comparison between the antinociceptiveeffects of BoNT/A icv or sc administered is shown. Dataare reported as percentage variation with respect to saline-injected mice and expressed either as picograms of toxinper mouse (A) or molar concentration (B). The obtaineddose–response curve (Fig. 3A) showed a similar magnitudeof attenuation whether BoNT/A was administered icv or sc.Moreover, BoNT/A exerted a very peculiar antinociceptiveeffect with a very narrow increase of % antinociception forthe range 0–1.875 pgtox/mouse, reaching a plateau at doseshigher than 1.875 pgtox/mouse. The maximal antinocicep-tive level was equal to 35%. However, taking into accountthat the same amount of toxin is diluted in differentinjection volume (sc: 20 μl; icv: 1 μl), the sc effects weremore potent than icv effects (Fig. 3B).

Fig. 4 shows the time course of activity and groomingbehavior during formalin injection recorded in saline, BoNT/A, or BoNT/B sc- or icv-injected mice. Comparisons betweenthe two different routes of administration were done for thedose of 3.75 pgtox/mouse. In saline sc- or icv-injected mice, asimilar trend was observed: (1) a progressive decline of timespent in activity during the formalin test indicating habitu-ation in mice and, (2) an almost constant grooming behaviorduring the formalin test. The BoNT/A and BoNT/B-injectedmice did not differently behave compared to the saline-injected mice: only during the first 10 min a higher, but notsignificant, activity level in BoNT/B sc-injected and in BoNT/A

Fig. 3 – Dose–response curve of the antinociceptive effect of BoNantinociception was calculated by using the equation: %Antinocthe cumulative licking time recorded in BoNT/A- or saline-injecteof BoNT/A are expressed as: (A) picograms of toxin per mouse (p

icv-injected mice was recorded. The absence of any signifi-cant alterations of activity and grooming indicates that thedepression of licking time observed during phase 2 offormalin test, in icv- and sc-injected BoNT/A mice, or theincrease of licking response during formalin-induced inter-phase, in icv-injected BoNT/B mice, was not a consequence ofa generally depressed healthy status of mice. Taken together,these results suggest that the alterations of the lickingbehavior in BoNTs mice were due to a direct interaction ofthe BoNTs with the nociceptive processes and pain transmis-sion mechanisms.

3. Discussion

Themain result of the present experiments is that BoNTsmaymodulate the inflammatory pain at the central level. Inparticular, it has been observed that BoNT/A and BoNT/Bexert different effects on the behavioral responses induced bythe long-lasting nociceptive stimulation of formalin and thatthese differences depend on route of administration andserotype considered.

In sc-injected mice, an antinociceptive effect of BoNT/Ahas been observed mainly on the phase 2 of the formalintest; in contrast, BoNT/B does not significantly affect phase 2while it induces antinociception on the phase 1. Theanalgesic effects induced by BoNT/A in mice are comparableto that already observed in rats (Cui et al., 2004). By usingconcentrations of BoNT/A (BOTOX®) in a range of 3.5–15 U/kg,the authors reported a reduction of both formalin-inducedlicking response and local glutamate release during thephase 2 of the formalin test. Assuming that 100-units equalsto 4.8 ng of neurotoxin (Cui et al., 2004), our range of 3.75–15pgtox/mouse approximately corresponds to the range of 3.5–15 U/kg in rats.

In icv-injected mice, formalin-induced pain is againreduced by BoNT/A during the phase 2 of the test. Moresurprisingly, BoNT/B abolishes the interphase inhibitionoccurring between the two phases of formalin-induced pain.

T/A on the phase 2 of formalin-induced licking behavior. Theiception = (1 − LT2BONTA/LT2SAL) × 100, where LT2 indicatesd mice during the phase 2 (10–40 min) of formalin test. Dosesgtox/mouse) or (B) molar concentration.

Fig. 4 – Effects of sc- or icv-injection of 3.75 pgtox/mouse of BoNT/A or /B on the time course of activity and groomingbehavior during the 40 min of the formalin test. Control groups were sc- or icv-injected with saline. Time spent in activity andgrooming was recorded during the same experiments as in Figs. 1 and 2.

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At difference, neither BoNT/A nor BoNT/B affects formalin-induced nociceptive behavior during the phase 1.

These results indicate a different mechanism of action ofBoNTs depending on both serotype and route of administra-tion. On one hand, both peripheral or central administration ofBoNT/A attenuates the formalin-induced pain during thephase 2 of formalin test; on the other hand, BoNT/B induceshyperalgesia on the interphase of formalin test when centrallyadministered while it induces analgesia during the phase 1 offormalin test when peripherally administered. These peculiarresponses raise two important questions that should beconsidered for the discussion. First, do the peripheral/centraleffects depend on BoNTs interaction with different neuro-transmitter systems? Second, do these different effectsdepend on different SNARE proteins cleaved?

It was already reported that BoNT/Amay inhibit the releaseof substance P, both in trigeminal nerve terminals of rabbit irissphincter muscle (Ishikawa et al., 1999) and in cultures ofdorsal root ganglia neurons (Purkiss et al., 2000; Welch et al.,2000), and the release of calcitonin gene-related peptide fromcultures of rat trigeminal ganglia (Durham et al., 2004). Inaddition, this botulinum serotype has been reported to inhibitalso the formalin-evoked release of glutamate in the rat's paw(Cui et al., 2004). These reports demonstrate that BoNT/A mayblock not only the ACh release from cholinergic neurons, asexpected by its canonical effect on the neuromuscularjunction, but also the release of excitatory molecules thatare involved in pain perception, vasodilation, and neurogenicinflammation.

We suggest that the analgesic effects induced by BoNT/Aon the phase 2 of formalin test, in sc and icv-injected mice,mainly depend on the blockade of excitatory neurotransmit-ter's release during peripheral and central pain sensitization,

while the analgesic effects of BoNT/B on the phase 1 in sc-injected mice may depend on its action on nociceptiveneurotransmitters released by primary sensory terminals.

The effects on the interphase of the formalin test inBoNT/B icv-injected mice suggest that this serotype maymodulate inhibitory pathways, in particular GABAergicsystem. This hypothesis is reinforced by considering thedifference between peripheral and central administration:BoNT/B is able to block the interphase only after centralinjection, in accordance with the absence of inhibitory GABAmodulation at peripheral nociceptive terminals. Thus, thelack of interphase following BoNT/B central administrationcan be discussed in terms of central modulation ofGABAergic system.

It has been demonstrated that, few minutes after theformalin tissue-injury, a release of GABA occurs together withan activation of GABAA receptors in the spinal cord thatcontributes to the period of quiescence between the twophases of formalin test (Green and Dickenson, 1997; Kanekoand Hammond, 1997). The spinal release of GABA, in responseto peripheral injection of formalin, may originate from manysources including GABAergic dorsal horn interneurons,GABAergic neurons in the ventromedialmedulla, andGABAer-gic neurons in the periaqueductal gray projecting to the spinalcord. GABA receptors are ubiquitously distributed and form acomplex system with multiple descending inhibitory controlswhich encompass a range of transmitters including mono-amines and opioids. Franklin and Abbott (1993) reported thatpentobarbital, diazepam, and alcohol eliminate the interphasedecline in pain response during formalin test, indicating aGABAA receptor's pain modulation. Hyperalgesia during theinterphase has also been reported in rats fed with drinkingwater containing α-difluoro-methylornithine (Kergozien et al.,

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1999) which directly interferes with GABAergic transmissioninvolved in the inhibition of central antinociceptive mecha-nisms (Ferchmin et al., 1993).

A recent paper of Verderio et al. (2004) shows that thevesicle recycling in hippocampal glutamatergic neurons isblocked by BoNT/A and BoNT/B while the same processes inhippocampal GABAergic neurons are blocked by BoNT/B butnot by BoNT/A. Staining with various antibodies directedagainst SNAP-25 revealed the specific presence of this proteinin glutamatergic neurons. In contrast, GABAergic neuronslacked immunoreactivity for SNAP-25. Staining with antibo-dies directed against SNAP-23, homolog of SNAP-25, showedits presence in both glutamatergic and GABAergic neurons.SNAP-23 is not cleaved by BoNT/A and this explains whyBoNT/A is not able to block the GABAergic neurons. Because ofthe action of BoNT/B on another protein of the SNAREcomplex, VAMP/synaptobrevin, this serotype is able to blockGABAergic neurons. Considering all these evidences, wehypothesize that the effect of BoNT/B on the interphase ofthe formalin-induced pain could be partially ascribed to afunctional block of GABA presynaptic inhibition on theprimary afferent fibers in spinal dorsal horn.

In conclusion, the present research demonstrates that informalin-induced pain: (i) BoNT/A exerts, in vivo, an analgesiceffect on the phase 2 when locally or centrally administered;(ii) BoNT/B exerts an analgesic effect on the phase 1, whenlocally administered, while it exerts, a strong hyperalgesiceffect on the interphase of formalin-induced pain, whencentrally administered.

Three main points emerge from the present results thathave to be considered for future investigations and utiliza-tion of BoNTs. The first point is that BoNTs may act not onlyat peripheral but also at the central level; the second one isthat BoNTs serotypes differ in their action on pain modu-lation indicating that BoNTs serotypes are not interchange-able based on a simple dose ratio; the third point refers tothe involvement of neurotransmitter systems other thancholinergic in the neuronal effect of BoNTs. All these pointsmay be extremely important also in view of the growinginterest of BoNTs in clinical practice. Moreover, the lastobservation is fundamental for the comprehension of themechanism involved in the action of BoNTs: BoNT/A andBoNT/B may interact with inhibitory and/or excitatorysystems in modulating persistent inflammatory pain. Fur-ther experiments, in which the effects of pharmacologicalcombination of BoNTs with GABAergic and glutamatergicagonists/antagonists as well as neurochemical markers willbe assessed, will permit a better understanding of theinteraction of BoNTs with these neurotransmitter systemsin pain modulation.

4. Experimental procedures

4.1. Subjects

CD1 male mice (Charles River Labs, Como, Italy) weighingabout 35–40 g at the beginning of the experiments were used.Upon their arrival in the laboratory (at least 2 weeks before theexperiments), mice were housed in standard transparent

plastic cage, in groups of 4 per cage, under standard animalroom conditions (free access to food andwater, 12 L:12 D cycle,room temperature of 23 °C). The experiments were carried outbetween 12:00 and 16:00 h. Care and handling of the animalswere in accordance with the guidelines of the Committee forResearch and Ethical Issues of IASP (PAIN® 1983, 16, 109–110)and with the Italian National law (DL116/92, application of theEuropean Communities Council Directive 86/609/EEC) on theuse of animal for research.

4.2. Drugs

BoNTs, isolated and purified as described elsewhere (Rossettoet al., 1992; Schantz and Johnson, 1992), were a kind gift fromProf. C. Montecucco (University of Padova). The toxins werefreezed in liquid nitrogen and stored at −80 °C in 10 mM NaHEPES, 150 mM NaCl, pH 7.2. Stock solutions of purified 150-kDa di-chain molecule of BoNT/A and BoNT/B were tested foractivity in the ex vivo mouse hemidiagphram model and inthe in vitro cleavage of SNAP-25 and VAMP/synaptobrevin.Injectable solutions of BoNTs were freshly made by dilution insaline (0.9% NaCl). Ketamine and xylazine were purchasedfrom Sigma (St. Louis, MO).

4.3. Surgical procedure

To perform intracerebroventricular (icv) injection, mice wereimplanted with chronic intracranial cannulae under anes-thesia induced by a mixture of ketamine (100 mg/kg, ip) andxylazine (5 mg/kg, ip), as previously described (Luvisetto etal., 2003, 2004). The mouse head was fixed in a stereotaxicapparatus and an incision was made in the atlanto-occipitalmembrane. Stainless steel guide cannulae were implantedaccording with the stereotaxic co-ordinates (anteroposterior,AP 0.0, mediolateral, ML ± 1.0 mm, from bregma) of the Atlasof mouse brain (Franklin et al., 1997). Cannulae were fixed tothe mouse skull with polycarbonate cement. Stainless steelinjection cannulae, connected via polyethylene tubing to aninfusion pump, were lowered into the lateral cerebralventricles (dorsoventral, DV 2.5 mm from bregma).

4.4. Drug injections

For peripheral injection, a volume of 20 μl of either saline (0.9%NaCl) or selected BoNTs solutions (BoNT/A: 0.937–15 pgtox/mouse; BoNT/B: 3.75, 7.5 pgtox/mouse) was subcutaneously(sc) injected into plantar surface of the right hindpaw of miceusing a microsyringe equipped with a 26-gauge needle. Thedoses of toxins were chosen on the basis of toxicity andbehavioral effects of icv-injected BoNTs in mice (Luvisetto etal., 2003, 2004). Preliminary results have moreover shown thatthe doses chosen did not induce any significant alteration ofspontaneous locomotor activity.We found that, in accordwithpreclinical studies on the safety margins of intramuscularinjection of BoNTs in mice (Aoki, 2001), BoNT/B administeredvia sc into paw of mice wasmore toxic than BoNT/A. For thesereasons, concentrations of BoNT/B higher than 7.5 pgtox/mouse were not tested.

For icv injection, a volume of 1 μl (single injection at 1 μl/min rate) of either saline (0.9% NaCl) or selected BoNTs (0.937–

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3.75 pgtox/mouse) solutions were icv-injected 6 days after thesurgical implantation of cannulae. As previously reported(Luvisetto et al., 2003), 3.75 pgtox/mouse was the maximalamount of BoNTs which can be injected via icv without toxiceffect. For this reason, concentrations of BoNTs higher than3.75 pg/tox were not tested.

4.5. Formalin test

The formalin test was used to measure inflammatory pain.Micewere tested3days after icv- or sc-injection.Wepreviouslyreported that this was the minimal time to minimize thepossible toxic effects of BoNTs injection (Luvisetto et al., 2003,2004). On the day of testing, one animal at a timewas placed for1 h in a standard plexiglass cage (30 × 12 × 13 cm) which servedas an observation chamber. After this adaptation period, 20 μlof formalin solution (5% in saline) was sc-injected into thedorsal surface of the right hindpaw of mice using a micro-syringe equipped with a 26-gauge needle. Mice were then putback into the chamber and the observation period started. Thelicking activity, i.e. the total amount of time the animal spentlicking and/or biting the injected paw, was taken as index ofpain. The licking activitywas recorded continuously for 40minand calculated in blocks of consecutive 5-minperiods. Amirrorplaced behind the cage allowed an unobstructed view of themice. In addition, general activity (time spent exploring theenvironment during walking, rearing and leaning) and self-grooming (time spent cleaning face and body) were continu-ously recorded for 40 min and calculated in blocks ofconsecutive 5-min periods.

4.6. Data analysis

Data from the formalin test were divided in three phases:phase 1 (0–5 min), interphase (5–10 min) and phase 2 (10–40 min), and separately analyzed by one-way ANOVAs foreach botulinum serotype. When appropriate, post hoc com-parisons were carried out using Fisher's PLSD test. Differenceswith P < 0.05 were considered significant.

Acknowledgment

This research was supported by the research grant FISR-CNRNeurobiotecnologia 2003 (D.M. 16/10/2000 MIUR—Fondo Inte-grativo Speciale per la Ricerca).

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