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w w w . e l s e v i e r . c o m / l o c a t e / p a i n

Consequences of the ablation of nonpeptidergic afferents in an animal modelof trigeminal neuropathic pain

Anna M.W. Taylor a,b, Maria Osikowicz a,b, Alfredo Ribeiro-da-Silva a,b,c,⇑a Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1Y6b Alan Edwards Centre for Research on Pain, McGill University, Montreal, Quebec, Canada H3A 0G1c Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 0C7

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

a r t i c l e i n f o

Article history:Received 23 December 2011Received in revised form 15 February 2012Accepted 21 March 2012

Keywords:Nonpeptidergic afferentsIB4-saporinP2X3Neuropathic painChronic constriction injuryTrigeminal

0304-3959/$36.00 � 2012 International Associationhttp://dx.doi.org/10.1016/j.pain.2012.03.023

⇑ Corresponding author. Address: Department of Ptics, McGill University, 3655 Promenade Sir-WilliaCanada H3G 1Y6. Tel.: +1 514 398 3619; fax: +1 514

E-mail address: alfredo.ribeirodasilva@mcgill.ca (A

a b s t r a c t

Damage to peripheral nerves causes significant remodeling of peripheral innervation and can lead to neu-ropathic pain. Most nociceptive primary afferents are unmyelinated (C fibers) and subdivided into pep-tidergic and nonpeptidergic fibers. Previous studies have found nerve injury in the trigeminal system toinduce changes in small-diameter primary afferent innervation and cause significant autonomic sprout-ing into the upper dermis of the lower-lip skin of the rat. In this study, we used the ribosomal toxin, sapo-rin, conjugated to the lectin IB4 to specifically ablate the nonpeptidergic nociceptive C fibers, to see if lossof these fibers was enough to induce autonomic fiber sprouting. IB4-saporin treatment led to specific andpermanent ablation of the IB4-positive, P2X3-immunoreactive fibers and led to sprouting of parasympa-thetic fibers into the upper dermis, but not of sympathetic fibers. These changes were associated withsignificant increase in glial-derived nerve growth factor levels in the lower-lip skin. While IB4-saporintreatment had no effect on evoked mechanical thresholds when von Frey hairs were applied to thelower-lip skin, ablation of nonpeptidergic fibers in a chronic constriction injury model caused significantsympathetic and parasympathetic fiber sprouting, and led to an exacerbated pain response. This was anunexpected finding, as it has been suggested that nonpeptidergic fibers play a major role in mechanicalpain, and suggests that these fibers play a complex role in the development of neuropathic pain.

� 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

1. Introduction

Nociceptive stimuli in the environment are detected by subsetsof thinly myelinated and unmyelinated primary afferents in theskin. The unmyelinated (C) nociceptive fibers have been furtherdivided into 2 subclasses [3,26]. The peptidergic C fibers expresspeptides such as calcitonin gene-related peptide (CGRP) and sub-stance P, terminate mainly in lamina I and outer lamina II of thespinal dorsal horn, and depend on nerve growth factor (NGF)[6,34]. The nonpeptidergic C fibers express the purinergic receptorP2X3, bind the plant lectin IB4, terminate in inner lamina II of thedorsal horn, and rely on glial-derived nerve growth factor (GDNF)[8,35]. The extent of the differences between these 2 nociceptivepopulations has suggested that they may play distinct roles in nor-mal and/or pathological pain processing [10,11,42].

for the Study of Pain. Published by

harmacology and Therapeu-m-Osler, Montreal, Quebec,221 3207.. Ribeiro-da-Silva).

Damage to peripheral sensory nerves can lead to a chronic paincondition called neuropathic pain. While the causes of this aber-rant pain sensation are largely unknown, previous studies have de-scribed significant remodeling of peripheral innervation of the skinfollowing nerve injury. For example, partial nerve injuries of thesciatic and trigeminal nerve are characterized by partial reinnerva-tion of peripheral tissues by primary sensory afferents [18,32,36]. Aconcomitant sprouting of autonomic fibers into the upper dermishas also been described [19,39,41,52]. These ectopic autonomicfibers were found in close apposition to the regenerating sensoryfibers, and it has been suggested that substances released by theseectopic autonomic fibers sensitize nociceptive endings, contribut-ing to neuropathic pain.

The peripheral remodeling that occurs following nerve injuryhas been postulated to be due to an increased availability ofgrowth factors in the skin following a nerve lesion (for reviewsee [49,53]). Accordingly, growth factors such as NGF and GDNFhave been shown to be upregulated in Schwann cells and keratino-cytes in areas distal to the nerve injury [4,21–23,33,37]. Exogenousapplication of NGF and GDNF has also increased peripheralregeneration of primary afferents [13]. A similar dichotomy in

Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.pain.2012.03.023mailto:alfredo.ribeirodasilva@mcgill.cahttp://www.elsevier.com/locate/pain

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growth factor dependence exists in the autonomic system as withthe peptidergic and nonpeptidergic C fibers, in that sympatheticand peptidergic fibers rely on NGF for growth and survival,whereas the parasympathetic and nonpeptidergic fibers rely onGDNF [1,7,15,17]. Given this reliance on similar growth factor sys-tems and the previous evidence implicating peripheral reinnerva-tion with the presence of growth factors, we hypothesize thatthe loss of C fibers results in overproduction of NGF and GDNF,which provides a permissive environment for autonomic fibers togrow.

To this end, we used the ribosomal toxin, saporin, conjugated toIB4, to specifically and permanently ablate the nonpeptidergic C fi-bers. Changes in evoked behavior and presence of autonomic fibersprouting were assessed. Levels of GDNF in the skin following spe-cific ablation of the nonpeptidergic C fibers were determined. Toidentify the role of nonpeptidergic fibers in a neuropathic pain state,animals were treated with IB4-saporin and a partial nerve lesion,and behavior and degree of autonomic sprouting were assessed.

2. Materials and methods

Male Sprague-Dawley rats (275–300 g; Charles River Laborato-ries, St-Constant, QC, Canada) were housed in groups of 2–4 andmaintained on a 12-hour cycle and allowed access to food andwater ad libitum. All protocols were approved by the McGill Uni-versity Animal Care Committee and complied with the policiesand guidelines outlined by the Canadian Council on Animal Careand the International Association for the Study of Pain.

2.1. Surgeries

2.1.1. Bilateral injections of IB4-saporin into the mental nervesRats were anesthetized with isoflurane and the mental nerve

was bilaterally exposed at its point of exit from the mental fora-men. A calibrated glass micropipette was inserted into the nerveand 4 lL of 800 lg/mL IB4-saporin (Advanced Targeting Systems,San Diego CA, USA), diluted in 0.2 M phosphate buffer (PB) and FastGreen Dye (Sigma, St. Louis, MO, USA), was injected. The dye FastGreen was used to visualize the injection to ensure accurateadministration within the nerve. The incision was closed with 4–0 vicryl sutures (Ethicon Inc, Somerville, NJ, USA), and animalswere allowed to recover for 3 weeks. Saporin control animalsunderwent a similar surgical procedure but were injected with4 lL of 800 lg/mL unconjugated saporin diluted in 0.2 M PB andFast Green Dye.

2.1.2. Bilateral modified chronic constriction injury lesionThree weeks after IB4-saporin or unconjugated saporin injec-

tion, animals were reanesthetized for the nerve ligation surgery.A modified version of the chronic constriction injury (CCI) lesion[9], as described previously [19], was used to induce a nerve injuryof the mental nerve. Briefly, rats were anesthetized with isoflurane,and 6 mm of the mental nerve was exposed and freed of adheringconnective tissue. Two ligatures were loosely tied around the men-tal nerve between the mental nerve foramen and its bifurcation.The incision was closed with vicryl sutures (Ethicon) and animalswere allowed to recover for at least 1 week. Sham animals under-went a similar surgical procedure to isolate the mental nerve, butno sutures were applied to the nerve. Wounds were closed with vi-cryl sutures as before, and allowed to recover for at least 1 week.

2.2. Behavior: mechanical allodynia

Each group consisted of at least 5 animals. All animals weretested at 3 weeks following IB4-saporin or unconjugated saporin

injection, and 2 and 4 weeks following CCI. Calibrated von Frey fil-aments of increasing stiffness were applied to the lower lip todetermine the mechanical withdrawal thresholds to innocuouspunctate stimulation. Rats were placed in a transparent Plexiglascage atop a wire mesh and allowed to acclimate to their surround-ings for at least 20 minutes. Von Frey filaments were applied per-pendicularly to the lower lip, and a positive reaction wasrecorded if the animal exhibited a vigorous head retraction. Theup-and-down method as described by Dixon [16] was used, where-by filaments with increasing stiffness were applied until a positivereaction was observed. Following the first positive reaction, thenext less stiff filament was applied. If no reaction, the next stifferfilament was applied; if a reaction was observed, the next less stifffilament was applied. This was repeated 6 times per animal and the50% withdrawal threshold was calculated according to the methodoutlined by Chaplan and collaborators [12]. Mechanical allodyniawas considered as a significant reduction in withdrawal thresholdwhen compared to Sham animals, as measured by a 1-way analysisof variance (ANOVA) with Dunnett post hoc test.

2.3. Immunocytochemistry

Following final behavioral testing, animals were deeply anes-thetized with Equithesin (6.5 mg chloral hydrate and 3 mg sodiumpentobarbital in a volume of 0.3 mL, i.p., per 100 g body weight)and transcardially perfused with 3% paraformaldehyde and 15%saturated picric acid (v.v) in 0.1 M PB, pH 7.4, for 1 hour. Hairy low-er-lip skin and medulla oblangata were isolated and postfixed for1 hour in the above fixative and cryoprotected in 30% sucrose in0.1 M PB for 24 hours at 4�C. Tissue was embedded in an optimumcutting temperature medium (Tissue Tek OCT; Sakura Finetek Eur-ope, Alphen aan den Rijn, The Netherlands), and 35-lm and 50-lmmedulla oblongata and lip sections, respectively, were cut at �20�Con a cryostat (Leica, Wetzlar, Germany).

2.3.1. Labeling in the skinSections to be labeled for P2X3 were processed for immunoflu-

orescence using the Tyramide Signal Amplification technique, aspreviously described [47]. Briefly, skin and caudal medulla oblon-gata sections were blocked for nonspecific binding of the second-ary antibody by an incubation with 10% Normal Donkey Serum(NDS) solution diluted in phosphate-buffered saline and 0.1% Tri-ton-X 100 (PBS-T) for 1 hour. Sections were then incubated for48 hours at 4�C with a guinea pig polyclonal anti-P2X3 antibody(1:25,000; Neuromics, Edina, MN, USA), diluted in PBS-T. Followingprimary antibody incubation, sections were treated with a biotin-conjugated donkey anti-guinea pig immunoglobulin (IgG) (1:200,Jackson ImmunoResearch Laboratories, West Grove, PA, USA) di-luted in PBS-T for 90 minutes, followed by further signal amplifica-tion via application of tyramide (1:75; Perkin Elmer, Waltham, MA,USA) diluted in PBS-T for 7 minutes. Finally, sections were incu-bated in streptavidin conjugated to Alexa Fluor 488 (1:200; Molec-ular Probes, Life Technologies, Grand Island, NY, USA) for 2 hours.Sections were washed in PBS, mounted on gelatin-coated slides,and coverslipped with Aqua-Polymount (Polysciences, Warrington,PA, USA).

Sections singly labeled for CGRP, vesicular monoamine trans-porter 2 (VMAT2), and vesicular acetylcholine transporter (VAChT)were pretreated with 10% Normal Goat Serum (NGS) or 10% NDSdiluted in PBS-T for 1 hour, followed by incubation with the follow-ing primary antibodies: 1) rabbit anti-CGRP (1:2000, Sigma); 2)rabbit anti-VMAT2 (1:7500; Synaptic Systems, Goettingen, Ger-many); 3) goat anti-VAChT (1:500, Chemicon, Millipore, Billerica,MA, USA). Following 24-hour incubation at 4�C, sections labeledwith CGRP and VMAT2 were labeled with highly cross-adsorbedgoat anti-rabbit IgG conjugated to Alexa Fluor 594 diluted in

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PBS-T (1:400, Molecular Probes). Sections labeled with VAChTwere incubated with a highly cross-adsorbed donkey anti-goatIgG conjugated to an Alexa Fluor 594 diluted in PBS-T (1:400,Molecular Probes). All sections were incubated for 2 hours at roomtemperature in the dark, washed in PBS, mounted on gelatin-subbed slides, and coverslipped, as described above.

2.3.2. Labeling in the trigeminal subnucleus caudalisIn order to confirm the IB4-saporin lesion was restricted to

nonpeptidergic afferents, trigeminal subnucleus caudalis sectionswere colabeled for either IB4 and CGRP or IB4 and P2X3. First, sec-tions were washed in PBS-T and pretreated with 10% Normal GoatSerum or 10% NDS for 1 hour. IB4 binding was detected using theisolectin GS-IB4 isolated from Griffonia simplicifolia conjugated toAlexa Fluor 488 (1:200, Molecular Probes). P2X3 and CGRP labelingwas detected as described above, with the same primary and sec-ondary antibodies.

2.3.3. QuantificationAt least 4 experimental animals and 3 sham animals were used

for each treatment group (IB4-saporin and unconjugated saporin)and time point (1, 2, and 4 weeks after CCI lesion) to quantify thechanges in autonomic and sensory innervation in the skin. Imageswere taken on a Zeiss Axioplan 2 imaging fluorescence microscope(Carl Zeiss Microscopy LLC, Thornwood, NY, USA), with a 40� oil-immersion objective. Images were acquired with a high-resolutioncolor digital camera with Zeiss Axiovision 4.5 software.

2.3.4. Sensory nerve quantificationChanges in P2X3-immunoreactive (IR) and CGRP-IR innervation

in the lower-lip skin were determined by analyzing the density offibers within the upper dermis. Several images were taken at serialfocal planes (z-stack) from 50-lm-thick sections and merged intoone horizontal projection by the Axiovision software, using the ex-tended focus feature. Six randomly chosen fields per lip section oneach of 3 sections were captured, totaling 18 images per animal.Images were exported in TIFF format and adjusted for brightnessand contrast using Adobe Photoshop 7.0.1 (Adobe Systems, SanJose, CA, USA). Quantification was performed using an MCID Eliteimage analysis system (Imaging Research Inc, St. Catharines, ON,Canada). The total fiber length per unit area was determined, and1-way ANOVA with Dunnett post hoc test was used to determinesignificance between the means of the groups. Statistical signifi-cance was accepted at P < 0.05.

2.3.5. Autonomic nerve quantificationQuantification of the changes in autonomic innervation con-

sisted of counting the number of VMAT2-IR and VAChT-IR fibers inthe upper dermis (defined as the area above the opening of the seba-ceous glands to the dermal-epidermal junction), an area normallydevoid of these fibers. To quantify the level of autonomic fibers inthe upper dermis, we obtained images from 6 randomly chosenfields per lip section on each of 4 sections per animal, totaling 24images per animal. The mean number of fibers in the upper dermiswas compared between groups using 1-way ANOVA and a Dunnettpost hoc test. Statistical significance was accepted at P < 0.05.

2.4. Protein extraction and Western blot

Changes in GDNF levels in the skin following ablation of thenonpeptidergic afferents were measured using Western blots. Ani-mals were injected with unconjugated saporin or IB4-saporin intothe mental nerve as described earlier. Three weeks following injec-tion, animals were sacrificed with an overdose of Equithesin(0.4 mL/100 g body weight, i.p.) and the lower lip was extractedand flash frozen with liquid nitrogen. Each lip section was mechan-

ically homogenized using a razor blade and transferred to anEppendorf tube with cold radioimmunoprecipitation assay buffercontaining protease inhibitors. Samples were further homogenizedusing a shearing homogenizer and incubated overnight at 4�C. Sol-ubilized proteins were extracted by a 45-minute centrifugation at13,000 RPM at 4�C. The protein concentration was determined byDC Protein Assay (Bio-Rad, Berkeley, CA, USA) with bovine serumalbumin as the protein standard.

Ten micrograms of protein was prepared in sample buffer underreducing conditions, and electrophoresed on 4%–10% polyacryl-amide gels. Gels were blotted onto a nitrocellulose membraneand rinsed in tris-buffered saline with Triton-X100 (TBS-T) andblocked with 5% milk powder in TBS-T for 1 hour at room temper-ature. Membranes were incubated overnight with goat anti-GDNFantibody (1:500; R&D Systems, Minneapolis, MN, USA) in 2.5% milkpowder in TBS-T at 4�C. Membranes were rinsed thoroughly withTBS-T and incubated with peroxidase-conjugated donkey anti-goatIgG (1: 2500) in 2.5% milk powder in TBS-T for 2 hours at roomtemperature. After thorough washing with TBS-T, immunoreactiv-ities were visualized using the enhanced ECL detection system(Pierce, Thermo Fisher Scientific, Rockford, IL, USA). Membraneswere rinsed and incubated with a mouse anti-b-actin antibody(1:40,000; Sigma) diluted in 2.5% milk in TBS-T for 1 hour at roomtemperature, washed with TBS-T, and incubated with peroxidaseconjugated donkey anti-mouse IgG (1:5000; Santa Cruz Biotech-nology, Santa Cruz, CA, USA) in 2.5% milk powder in TBS-T for1 hour. Membranes were rinsed and immunoreactivities werevisualized as above. Results were analyzed by computer-assisteddensitometry and levels of GDNF immunoreactivity were normal-ized with respect to the b-actin levels in each sample.

2.5. Biochemistry statistical analyses

All values are given as mean ± SEM. Values were normally dis-tributed and so parametric statistical analysis was performed.Groups were compared using an unpaired t-test. Differences wereconsidered statistically significant when P < 0.05.

3. Results

3.1. Behavior

Animals receiving IB4-saporin injection into the mental nervesdisplayed no changes in spontaneous behavior, including feedingor grooming patterns, and had similar body weight to age-matchedcontrols. This group also showed no change in evoked thresholds tomechanical punctate stimuli at 3 weeks following IB4-saporininjection when compared to control-operated animals receivingunconjugated saporin (Fig. 1). These thresholds were not signifi-cantly different from naïve animals receiving no surgical interven-tion (data not shown). This is in contrast to bilateral chronicconstriction of the mental nerve (CCI), which resulted in significantreduction in mechanical thresholds at 2 and 4 weeks following sur-gery. Sham surgery resulted in no change in mechanical thresholdsat any time point, so were pooled together (CCI Sham). CCI Shamanimals were also not significantly different from the IB4-sapo-rin-treated animals. Animals receiving IB4-saporin treatment fol-lowed by CCI of the mental nerve exhibited heightenedsensitivity to light touch at 4 weeks following CCI lesion (Fig. 1).This sensitivity was not only significantly lower when comparedto sham controls, but also significantly lower than age-matchedanimals receiving only the CCI lesion. Most animals in the IB4-saporin + CCI group consistently responded to the lowest von Freyfilament, effectively reaching the minimum threshold of thisbehavioral testing technique. Injection of unconjugated saporin

Fig. 1. Behavioral characterization of mechanical thresholds using von Freyfilaments. Unconjugated Saporin (SAP Control) did not affect mechanical thresholds(measured as a significant change in the 50% threshold compared to naive animals),and was not significantly different from chronic constriction injury (CCI) Sham ornaive animals. CCI Sham animals were tested at 2 and 4 weeks after sham surgery.The mechanical thresholds were found to not differ significantly, so were pooledinto one group (CCI sham). Three weeks after IB4-saporin (IB4SAP) injection into themental nerve, mechanical thresholds were not significantly different from any ofthe control groups. CCI of the mental nerve resulted in significant reduction ofmechanical thresholds at 2 and 4 weeks after lesion. Injection of unconjugatedSaporin did not cause significant changes in the already reduced mechanicalthresholds 2 or 4 weeks after nerve lesion. In animals treated with IB4SAP followedby CCI of the mental nerve, mechanical thresholds were significantly lower than theCCI group at the 4-week time point (n = 4–6, ⁄P < 0.05, ⁄⁄P < 0.01). Error barsrepresent SEM.

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followed by CCI lesion resulted in significantly lowered mechanicalthresholds compared to CCI Sham and Saporin controls, but werenot significantly different from CCI alone.

3.2. Changes in C-fiber innervation of trigeminal subnucleus caudalisand lower-lip skin

Specificity of the IB4-saporin-induced ablation of nonpeptider-gic afferents was confirmed via immunocytochemical labeling ofIB4, P2X3, and CGRP in the trigeminal subnucleus caudalis. By3 weeks after injection of IB4-saporin into the mental nerves, acomplete loss of IB4 binding and P2X3 labeling in the most medialaspect of the trigeminal subnucleus caudalis was observed(Fig. 2C, D). This region correlates with the somatotopic locationof the central afferents of the mental nerve in the trigeminal sub-nucleus caudalis. The lesion was shown to persist to all timepoints measured, up to 8 weeks after the initial IB4-saporin injec-tion. Staining for peptidergic fibers, using the antibody directedagainst CGRP, showed minimal loss of these fibers following IB4-saporin treatment (Fig. 2D). Similarly, treatment with unconju-gated saporin produced no reduction in IB4, P2X3, or CGRP labeling(Fig. 2A, B).

In the skin, IB4-saporin treatment caused near-complete loss ofP2X3-IR fibers in the entire dermal and epidermal layers, persistingto all time points measured (Fig. 3A, B). As observed in the trigem-inal subnucleus caudalis, IB4-saporin treatment caused a small butnot significant loss of CGRP-IR fibers in the skin (Fig. 3C, D).

In animals receiving IB4-saporin treatment followed by CCI ofthe mental nerve, a consistent and complete loss of nonpeptidergic

C fibers (as labeled with IB4 and P2X3) in both the trigeminal sub-nucleus caudalis and skin was observed (Fig. 3A, B). CCI in IB4-saporin-treated animals led to a significant loss of CGRP-IR fibersin the upper dermis of the lower-lip skin (Fig. 3C, D). This losswas most substantial at 1 week post lesion, after which fibersslowly re-innervated the deafferented area.

3.3. Autonomic sprouting

Previous studies have demonstrated an aberrant sprouting ofautonomic fibers into the upper dermis following neuropathic in-jury, an area where they are normally absent [19]. Since the facialarea is innervated by both parasympathetic and sympathetic fi-bers, we explored whether specific ablation of nonpeptidergic C fi-bers via IB4-saporin treatment was able to induce this sprouting.While IB4-saporin treatment led to complete ablation of the nonp-eptidergic afferents, it also led to a concomitant sprouting of para-sympathetic (VAChT-IR) fibers into the upper dermis, an areawhere they are normally absent (Fig. 4A, B). The level of sproutedfibers was comparable to what was observed in a straight CCI le-sion [48]. IB4-saporin treatment, however, did not induce signifi-cant sprouting of sympathetic (VMAT2-IR) fibers into the upperdermis (Fig. 4C, D). Treatment with IB4-saporin followed by CCIof the mental nerve led to significant sprouting of both sympa-thetic and parasympathetic fibers into the upper dermis. These fi-bers persisted to all time points tested following CCI lesion, up to4 weeks (Fig. 4A–D).

3.4. GDNF protein levels

GDNF levels in the skin of the lower lip following specific abla-tion of the nonpeptidergic C fibers were measured using Westernblot. Three weeks after bilateral injection of IB4-saporin into themental nerves, GDNF levels were found to be significantly higherthan those from animals treated with unconjugated saporin(Fig. 5). This increase corresponds with the peak level of autonomicsprouting and sensory afferent regeneration as previouslydescribed.

4. Discussion

Injection of IB4-saporin into the mental nerve caused completeloss of IB4+ terminals in the trigeminal subnucleus caudalis. Be-cause of the known problems with IB4 labeling in the skin [43],an antibody directed against the P2X3 receptor was used that la-bels nonpeptidergic fibers in the skin [47]. As expected, IB4-sapo-rin injection led to complete and permanent loss of P2X3-IR fibersin the skin. A previous study examining IB4-saporin injection intothe sciatic nerve described the loss of IB4+ neurons in the spinalcord and ganglia to begin around 3 days after injection and wascompleted by 10–21 days [51]. In the present study, we examinedthe animals 21 days after IB4-saporin injection, as we could be as-sured that no ongoing nerve degeneration was occurring.

4.1. IB4-Saporin and autonomic sprouting

Ablation of IB4+ neurons led to significant sprouting of para-sympathetic fibers into the upper dermis, an area where they arenormally absent. This response is similar to what is observed fol-lowing a nerve lesion, where autonomic fibers sprout into theupper dermis, and were found in close apposition to injured affer-ents [19,48]. In contrast to the nerve injury model, ectopic sympa-thetic fibers were rarely observed in the upper dermis followingIB4-saporin treatment, which suggests that parasympatheticsprouting is related to the loss of nonpeptidergic C fibers. This is

Fig. 2. Depletion of central terminals of nonpeptidergic C fibers in the trigeminal subnucleus caudalis as identified by IB4 binding and P2X3 labeling. (A) P2X3 (green) and IB4(red) labeling in unconjugated Saporin group. Note the near-complete overlap between P2X3 and IB4 labeling. (B) CGRP (green) and IB4 (red) labeling in unconjugated Saporingroup. Note that this does not affect peptidergic or nonpeptidergic terminals in the trigeminal subnucleus caudalis. (C) P2X3 (green) and IB4 (red) labeling in IB4-saporingroup. Note the loss of nonpeptidergic terminals in the most medial aspect of the trigeminal subnucleus caudalis, corresponding to mental nerve afferents (indicated byarrows). (D) CGRP (green) and IB4 (red) labeling in IB4-saporin group. The CGRP-IR fibers are not significantly affected. Scale bar = 50 lm.

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supported by the fact that GDNF levels were found to be signifi-cantly elevated in the skin following IB4-saporin treatment. Thatsympathetic fibers, which respond to NGF, did not sprout following

IB4-saporin injection, suggests that the relationship between C-fi-ber loss and autonomic sprouting is a specific one connected via re-sponse to growth factors.

Fig. 3. Innervation of peptidergic and nonpeptidergic peripheral afferents in IB4-saporin (IB4-SAP) and IB4-saporin + CCI (chronic constriction injury) groups. (A)Photomicrographs depicting the innervation of P2X3-IR fibers in the lower-lips skin of rat, clockwise from top left, from unconjugated Saporin (SAP), IB4-SAP, IB4-SAP + 2 week CCI, and IB4-SAP + 4 week CCI. Scale bar = 50 lm. (B) Bar graph showing average density of P2X3-IR fibers in the upper dermis (n = 6, ⁄P < 0.05, ⁄⁄P < 0.01). Errorbars represent SEM. (C) Photomicrographs depicting the innervation of CGRP-IR fibers in the upper dermis, from top left, SAP, IB4SAP, IB4SAP + 2 week CCI, IB4SAP + 4 weekCCI. Scale bar = 50 lm. (D) Bar graph showing average density of CGRP-IR fibers in the upper dermis (n = 6, ⁄P < 0.05). Error bars represent SEM. Epi, epidermis; Ud, upperdermis.

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Presence of ectopic autonomic fibers in the upper dermis fol-lowing nerve injury has been proposed to contribute to the hyper-sensitivity of nociceptors by releasing factors that directly sensitizethe surrounding neurons [5,19,41]. While the role of the sympa-thetic nervous system in neuropathic pain has been thoroughlyinvestigated [38], the parasympathetic system has been less ex-plored, although it is plausible that it may play a role in chronicpain. Primary afferents express both nicotinic and muscarinicreceptors [14], and application of acetylcholine and nicotinecaused primary afferent discharge associated with pain sensations[5,20,24,28,44]. Application of nicotinic antagonists blocked theneurogenic flare following nociceptive stimulation [20,31].

It is also possible that the excess GDNF in the skin directly sen-sitizes the remaining primary afferents. GDNF overexpressingtransgenic mice have lowered mechanical thresholds when com-pared to wild-type littermates [29]. However, while IB4-saporinresulted in significant increase in GDNF protein levels and concom-itant sprouting of parasympathetic fibers into the upper dermis,this treatment did not result in any changes in evoked mechanicalthresholds. In fact, it was not until a nerve lesion was applied (IB4-saporin + CCI) that the mechanical thresholds were significantlyreduced, despite the continued presence of ectopic parasympa-thetic afferents. This would argue against the role of GDNF and/or parasympathetic fibers in nerve injury-related pain. It is alsopossible that the presence of nonpeptidergic C fibers is necessaryfor the sensitization to occur.

4.2. IB4-saporin and behavioral response

Three weeks after IB4-saporin injection into the mental nerve,mechanical thresholds were unchanged. This is supported by previ-ous studies that reported a slight increase in mechanical and ther-mal thresholds shortly after IB4-saporin injection, but whichreturned to normal levels by 21 days [51]. This was surprising asIB4-saporin treatment caused a significant loss of primary afferentsin the skin, which would be expected to significantly alter nocicep-tive processing. One explanation is that only light mechanicalthresholds were measured in this study, due to the technical chal-lenges of behavioral testing in the lower-lip region. It is possible thatlight mechanical stimuli are mediated by fast-conducting myelin-ated nociceptors, and loss of C fibers may not result in changes tothis specific test. However, the fact that a previous study describedno permanent change in thermal nociceptive thresholds followingIB4-saporin injection into the sciatic nerve [51] suggests the lackof change in nociceptive thresholds pervasive across many stimulusmodalities. Given the coexpression of many transducers on bothpeptidergic and nonpeptidergic C fibers, such as acid-sensing ionchannels and the heat receptor TRPV1, it is possible there is consid-erable overlap in nociceptive function of these 2 populations of noci-ceptors [30]. Following loss of nonpeptidergic C fibers in this model,peptidergic C fibers are presumably able to mediate the normalnociceptive stimuli on their own. However, even though other stud-ies using IB4SAP have shown mechanical and thermal thresholds to

Fig. 4. Changes in parasympathetic (VAChT-IR) and sympathetic (VMAT2-IR) innervation in IB4-saporin (IB4SAP) and IB4SAP + CCI (chronic constriction injury) groups. (A)Photomicrographs depicting VAChT-IR fibers in the skin in unconjugated Saporin (SAP), IB4-SAP, and IB4-SAP + 4 week CCI groups. Arrows indicate ectopic VAChT-IR fibers inthe upper dermis in both IB4SAP and IB4SAP + CCI groups. Scale bar = 50 lm. (B) Bar graph showing the average number of VAChT-IR fibers counted in the upper dermis(n = 6, ⁄P < 0.05, ⁄⁄P < 0.01). (C) Photomicrographs depicting VMAT2-IR fibers in the upper dermis in SAP, IB4SAP, and IB4SAP + 4 week CCI. Scale bar = 50 lm. Arrows indicateectopic VMAT2-IR fibers in the upper dermis of IB4SAP + 4 week CCI. (D) Bar graph showing the average number of VMAT2-IR fibers counted in the upper dermis (n = 6,⁄⁄P < 0.01). Error bars represent ± SEM.

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remain relatively constant over time, we cannot be certain that IB4-saporin does not cause changes in mechanical thresholds beyondthe 1-month time point. These changes might still be undetectableat 4 weeks, but what underlies them might contribute to the low-ered mechanical thresholds we detected in IB4-saporin + CCI rats.

The lack of changes in mechanical thresholds following IB4-saporin treatment also contradicts the previous observation thatnonpeptidergic fibers specifically mediate mechanical pain,

whereas peptidergic C fibers mediate thermal pain [42]. As thisprevious study was performed in mice, it suggests that the strictdichotomy between C fiber populations and nociceptive modalitiesdoes not apply to higher-order species such as rats and humans.This is supported by the distribution of the heat receptor TRPV1,which in mice is found specifically on peptidergic C fibers, but islocated on both peptidergic and nonpeptidergic C fibers in ratsand higher-order primates [50].

Fig. 5. Changes in glial-derived nerve-growth factor (GDNF) protein levels in thelower-lip skin following specific ablation of the nonpeptidergic C fibers. (A)Representative Western blot of GDNF levels (18 kDa) taken from the lower lip ofanimals injected with unconjugated Saporin (SAP) or IB4-saporin (IB4SAP) in themental nerves. All GDNF levels normalized to b-actin (42 kDa). (B) GDNF blotdensity, expressed as arbitrary units, was normalized to the reference protein, bactin (42 kDa). GDNF levels were significantly higher in IB4SAP-injected animalscompared to SAP. Error bars represent ± SEM. ⁄P < 0.05. n = 4 per group.

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4.3. IB4-saporin + CCI and behavioral response

IB4-saporin followed by CCI of the mental nerves led to com-plete loss of IB4+, P2X3-IR fibers, and a significant but transient lossof CGRP-IR fibers. A significant reduction of mechanical thresholdswas also observed. The IB4-saporin + CCI group had significantlylowered mechanical thresholds at 4 weeks following nerve lesionwhen compared to the equivalent time point of animals receivingonly the CCI lesion. The heightened pain response in IB4-sapo-rin + CCI animals is a curious observation, as loss of nonpeptidergicC fibers would be expected to reverse or delay the onset of neuro-pathic pain. Indeed, a previous study performing a nerve injury onthe sciatic nerve followed by intrasciatic IB4-saporin injectioncaused a transient delay of mechanical allodynia and hyperalgesia[46]. There are several reasons for this discrepancy. First, the pre-vious study tested only the presence of mechanical allodynia orhyperalgesia, whereas this study used the up-down method todetermine the mechanical nociceptive threshold following injury.Also, the previous study performed the nerve lesion before IB4-saporin injection, whereas in this study we performed the IB4-saporin injection prior to the nerve lesion, in order to ensure allnonpeptidergic fibers were completely destroyed before inducinga nerve lesion.

The decreased mechanical thresholds in neuropathic animalslacking nonpeptidergic afferents is intriguing. It has been proposedthat initial inflammatory stimulus triggers long-lasting hypersensi-tivity to inflammatory cytokines in primary afferents, leading to astate of hyperalgesic priming [40]. The hyperalgesic priming wasproposed to cause an increased response of primary afferent neu-

rons to subsequent nociceptive stimuli, and is thought to be med-iated via the protein kinase C epsilon signaling [2]. In our model,IB4-saporin injection leads to complete destruction of nonpeptid-ergic fibers, which is known to recruit a strong immune response.It is possible that the inflammatory response is enough to inducethe exacerbated pain response observed in our model, and wouldexplain the heightened pain response in animals receiving IB4-saporin followed by a nerve lesion.

While ablation of nonpeptidergic afferents using the IB4-saporinapproach has produced relatively consistent behavioral results pub-lished here and elsewhere [46,51], inhibition of nonpeptidergicafferents via P2X3 receptor manipulation produced varying behav-ioral results. P2X3 knockout mice had significantly lowered re-sponse to acute thermal pain, but thermal hyperalgesia waspotentiated in an inflammation model [45]. However, P2X3 knock-down using antisense oligonucleotides had no effect on acute painbehaviors and reduced neuropathic and inflammatory pain behav-iors in rats [25]. Application of the P2X3 receptor antagonist, A-317491, also had no effect on acute pain behaviors, but reduced neu-ropathic and inflammatory pain behaviors [27]. The cause of thesediscrepant results is unclear; however, the specificity of both P2X3antagonist and antisense oligonucleotides has been questioned,and these approaches may produce significant off-target effects. Inany case, it is clear that specifically manipulating the P2X3 receptoris not equivalent to ablating the nonpeptidergic afferents, and so it isimpossible to compare the behavioral results between these studies.

4.4. Conclusions

Overall, the results of this study highlight some intriguingperipheral adaptations following a nerve injury. Specifically, ablat-ing the nonpeptidergic fibers led to significant increase of GDNFprotein levels in the skin followed by specific sprouting of para-sympathetic fibers into the upper dermis. This demonstrates animportant link between loss of nonpeptidergic C fibers and para-sympathetic sprouting, mediated through GDNF levels in the skin.Furthermore, while IB4-saporin treatment alone did not cause anylong-term changes in mechanical thresholds, IB4-saporin followedby a nerve lesion led to an exacerbated pain response characterizedby lowered mechanical thresholds. This suggests loss of nonpeptid-ergic fibers before a nerve lesion produces important changes inthe peripheral nervous system that renders this system vulnerableto future injury-induced changes.

Conflict of interest statement

The authors disclose no conflict of interest in respect of thiswork.

Acknowledgements

This work was supported by Canadian Institute of Health Re-search (CIHR) Grant MOP-53278 (to A.R.-da-S.). A.M.W.T is the re-cipient of a CIHR Frederick Banting and Charles Best CanadaGraduate Scholarship Doctoral Award.

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Consequences of the ablation of nonpeptidergic afferents in an animal model of trigeminal neuropathic pain1 Introduction2 Materials and methods2.1 Surgeries2.1.1 Bilateral injections of IB4-saporin into the mental nerves2.1.2 Bilateral modified chronic constriction injury lesion

2.2 Behavior: mechanical allodynia2.3 Immunocytochemistry2.3.1 Labeling in the skin2.3.2 Labeling in the trigeminal subnucleus caudalis2.3.3 Quantification2.3.4 Sensory nerve quantification2.3.5 Autonomic nerve quantification

2.4 Protein extraction and Western blot2.5 Biochemistry statistical analyses

3 Results3.1 Behavior3.2 Changes in C-fiber innervation of trigeminal subnucleus caudalis and lower-lip skin3.3 Autonomic sprouting3.4 GDNF protein levels

4 Discussion4.1 IB4-Saporin and autonomic sprouting4.2 IB4-saporin and behavioral response4.3 IB4-saporin+CCI and behavioral response4.4 Conclusions

Conflict of interest statementAcknowledgementsReferences