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

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1 PAIN Ò 153 (2012) Consequences of the ablation of nonpeptidergic afferents in an animal model of 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 1Y6 b Alan Edwards Centre for Research on Pain, McGill University, Montreal, Quebec, Canada H3A 0G1 c 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. article info abstract Article history: Received 23 December 2011 Received in revised form 15 February 2012 Accepted 21 March 2012 Keywords: Nonpeptidergic afferents IB4-saporin P2X 3 Neuropathic pain Chronic constriction injury Trigeminal Damage to peripheral nerves causes significant remodeling of peripheral innervation and can lead to neuropathic pain. Most nociceptive primary afferents are unmyelinated (C fibers) and subdivided into peptidergic and nonpeptidergic fibers. Previous studies have found nerve injury in the trigeminal system to induce changes in small-diameter primary afferent innervation and cause significant autonomic sprouting into the upper dermis of the lower-lip skin of the rat. In this study, we used the ribosomal toxin, saporin, conjugated to the lectin IB4 to specifically ablate the nonpeptidergic nociceptive C fibers, to see if loss of these fibers was enough to induce autonomic fiber sprouting. IB4-saporin treatment led to specific and permanent ablation of the IB4-positive, P2X 3 -immunoreactive fibers and led to sprouting of parasympathetic fibers into the upper dermis, but not of sympathetic fibers. These changes were associated with significant increase in glial-derived nerve growth factor levels in the lower-lip skin. While IB4-saporin treatment had no effect on evoked mechanical thresholds when von Frey hairs were applied to the lower-lip skin, ablation of nonpeptidergic fibers in a chronic constriction injury model caused significant sympathetic and parasympathetic fiber sprouting, and led to an exacerbated pain response. This was an unexpected finding, as it has been suggested that nonpeptidergic fibers play a major role in mechanical pain, and suggests that these fibers play a complex role in the development of neuropathic pain. Ó 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 subsets of thinly myelinated and unmyelinated primary afferents in the skin. The unmyelinated (C) nociceptive fibers have been further divided into 2 subclasses [3,26]. The peptidergic C fibers express peptides such as calcitonin gene-related peptide (CGRP) and substance P, terminate mainly in lamina I and outer lamina II of the spinal dorsal horn, and depend on nerve growth factor (NGF) [6,34]. The nonpeptidergic C fibers express the purinergic receptor P2X 3, bind the plant lectin IB4, terminate in inner lamina II of the dorsal horn, and rely on glial-derived nerve growth factor (GDNF) [8,35]. The extent of the differences between these 2 nociceptive populations has suggested that they may play distinct roles in normal and/or pathological pain processing [10,11,42]. Corresponding author. Address: Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G 1Y6. Tel.: ; fax: address: alfredo.ribeirodasilva@mcgill.ca (A. Ribeiro-da-Silva). Damage to peripheral sensory nerves can lead to a chronic pain condition called neuropathic pain. While the causes of this aberrant pain sensation are largely unknown, previous studies have described significant remodeling of peripheral innervation of the skin following nerve injury. For example, partial nerve injuries of the sciatic and trigeminal nerve are characterized by partial reinnervation of peripheral tissues by primary sensory afferents [18,32,36].A concomitant sprouting of autonomic fibers into the upper dermis has also been described [19,39,41,52]. These ectopic autonomic fibers were found in close apposition to the regenerating sensory fibers, and it has been suggested that substances released by these ectopic autonomic fibers sensitize nociceptive endings, contributing to neuropathic pain. The peripheral remodeling that occurs following nerve injury has been postulated to be due to an increased availability of growth factors in the skin following a nerve lesion (for review see [49,53]). Accordingly, growth factors such as NGF and GDNF have been shown to be upregulated in Schwann cells and keratinocytes in areas distal to the nerve injury [4,21 23,33,37]. Exogenous application of NGF and GDNF has also increased peripheral regeneration of primary afferents [13]. A similar dichotomy in /$36.00 Ó 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

2 1312 A.M.W. Taylor et al. / PAIN Ò 153 (2012) growth factor dependence exists in the autonomic system as with the peptidergic and nonpeptidergic C fibers, in that sympathetic and peptidergic fibers rely on NGF for growth and survival, whereas the parasympathetic and nonpeptidergic fibers rely on GDNF [1,7,15,17]. Given this reliance on similar growth factor systems and the previous evidence implicating peripheral reinnervation with the presence of growth factors, we hypothesize that the loss of C fibers results in overproduction of NGF and GDNF, which provides a permissive environment for autonomic fibers to grow. To this end, we used the ribosomal toxin, saporin, conjugated to IB4, to specifically and permanently ablate the nonpeptidergic C fibers. Changes in evoked behavior and presence of autonomic fiber sprouting were assessed. Levels of GDNF in the skin following specific ablation of the nonpeptidergic C fibers were determined. To identify 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 ( g; Charles River Laboratories, St-Constant, QC, Canada) were housed in groups of 2 4 and maintained on a 12-hour cycle and allowed access to food and water ad libitum. All protocols were approved by the McGill University Animal Care Committee and complied with the policies and guidelines outlined by the Canadian Council on Animal Care and the International Association for the Study of Pain Surgeries Bilateral injections of IB4-saporin into the mental nerves Rats were anesthetized with isoflurane and the mental nerve was bilaterally exposed at its point of exit from the mental foramen. A calibrated glass micropipette was inserted into the nerve and 4 ll of 800 lg/ml IB4-saporin (Advanced Targeting Systems, San Diego CA, USA), diluted in 0.2 M phosphate buffer (PB) and Fast Green Dye (Sigma, St. Louis, MO, USA), was injected. The dye Fast Green was used to visualize the injection to ensure accurate administration within the nerve. The incision was closed with 4 0 vicryl sutures (Ethicon Inc, Somerville, NJ, USA), and animals were allowed to recover for 3 weeks. Saporin control animals underwent a similar surgical procedure but were injected with 4 ll of 800 lg/ml unconjugated saporin diluted in 0.2 M PB and Fast Green Dye Bilateral modified chronic constriction injury lesion Three weeks after IB4-saporin or unconjugated saporin injection, 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 injury of the mental nerve. Briefly, rats were anesthetized with isoflurane, and 6 mm of the mental nerve was exposed and freed of adhering connective tissue. Two ligatures were loosely tied around the mental nerve between the mental nerve foramen and its bifurcation. The incision was closed with vicryl sutures (Ethicon) and animals were allowed to recover for at least 1 week. Sham animals underwent a similar surgical procedure to isolate the mental nerve, but no sutures were applied to the nerve. Wounds were closed with vicryl sutures as before, and allowed to recover for at least 1 week Behavior: mechanical allodynia Each group consisted of at least 5 animals. All animals were tested at 3 weeks following IB4-saporin or unconjugated saporin injection, and 2 and 4 weeks following CCI. Calibrated von Frey filaments of increasing stiffness were applied to the lower lip to determine the mechanical withdrawal thresholds to innocuous punctate stimulation. Rats were placed in a transparent Plexiglas cage atop a wire mesh and allowed to acclimate to their surroundings for at least 20 minutes. Von Frey filaments were applied perpendicularly to the lower lip, and a positive reaction was recorded if the animal exhibited a vigorous head retraction. The up-and-down method as described by Dixon [16] was used, whereby filaments with increasing stiffness were applied until a positive reaction was observed. Following the first positive reaction, the next less stiff filament was applied. If no reaction, the next stiffer filament was applied; if a reaction was observed, the next less stiff filament was applied. This was repeated 6 times per animal and the 50% withdrawal threshold was calculated according to the method outlined by Chaplan and collaborators [12]. Mechanical allodynia was considered as a significant reduction in withdrawal threshold when compared to Sham animals, as measured by a 1-way analysis of variance (ANOVA) with Dunnett post hoc test Immunocytochemistry Following final behavioral testing, animals were deeply anesthetized with Equithesin (6.5 mg chloral hydrate and 3 mg sodium pentobarbital 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 lower-lip skin and medulla oblangata were isolated and postfixed for 1 hour in the above fixative and cryoprotected in 30% sucrose in 0.1 M PB for 24 hours at 4 C. Tissue was embedded in an optimum cutting temperature medium (Tissue Tek OCT; Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands), and 35-lm and 50-lm medulla oblongata and lip sections, respectively, were cut at 20 C on a cryostat (Leica, Wetzlar, Germany) Labeling in the skin Sections to be labeled for P2X 3 were processed for immunofluorescence using the Tyramide Signal Amplification technique, as previously described [47]. Briefly, skin and caudal medulla oblongata sections were blocked for nonspecific binding of the secondary antibody by an incubation with 10% Normal Donkey Serum (NDS) solution diluted in phosphate-buffered saline and 0.1% Triton-X 100 (PBS-T) for 1 hour. Sections were then incubated for 48 hours at 4 C with a guinea pig polyclonal anti-p2x 3 antibody (1:25,000; Neuromics, Edina, MN, USA), diluted in PBS-T. Following primary antibody incubation, sections were treated with a biotinconjugated donkey anti-guinea pig immunoglobulin (IgG) (1:200, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted in PBS-T for 90 minutes, followed by further signal amplification via application of tyramide (1:75; Perkin Elmer, Waltham, MA, USA) diluted in PBS-T for 7 minutes. Finally, sections were incubated in streptavidin conjugated to Alexa Fluor 488 (1:200; Molecular 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 transporter 2 (VMAT2), and vesicular acetylcholine transporter (VAChT) were pretreated with 10% Normal Goat Serum (NGS) or 10% NDS diluted in PBS-T for 1 hour, followed by incubation with the following primary antibodies: 1) rabbit anti-cgrp (1:2000, Sigma); 2) rabbit anti-vmat2 (1:7500; Synaptic Systems, Goettingen, Germany); 3) goat anti-vacht (1:500, Chemicon, Millipore, Billerica, MA, USA). Following 24-hour incubation at 4 C, sections labeled with CGRP and VMAT2 were labeled with highly cross-adsorbed goat anti-rabbit IgG conjugated to Alexa Fluor 594 diluted in

3 A.M.W. Taylor et al. / PAIN Ò 153 (2012) PBS-T (1:400, Molecular Probes). Sections labeled with VAChT were incubated with a highly cross-adsorbed donkey anti-goat IgG conjugated to an Alexa Fluor 594 diluted in PBS-T (1:400, Molecular Probes). All sections were incubated for 2 hours at room temperature in the dark, washed in PBS, mounted on gelatinsubbed slides, and coverslipped, as described above Labeling in the trigeminal subnucleus caudalis In order to confirm the IB4-saporin lesion was restricted to nonpeptidergic afferents, trigeminal subnucleus caudalis sections were colabeled for either IB4 and CGRP or IB4 and P2X 3. First, sections were washed in PBS-T and pretreated with 10% Normal Goat Serum or 10% NDS for 1 hour. IB4 binding was detected using the isolectin GS-IB4 isolated from Griffonia simplicifolia conjugated to Alexa Fluor 488 (1:200, Molecular Probes). P2X 3 and CGRP labeling was detected as described above, with the same primary and secondary antibodies Quantification At 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 the changes in autonomic and sensory innervation in the skin. Images were taken on a Zeiss Axioplan 2 imaging fluorescence microscope (Carl Zeiss Microscopy LLC, Thornwood, NY, USA), with a 40 oilimmersion objective. Images were acquired with a high-resolution color digital camera with Zeiss Axiovision 4.5 software Sensory nerve quantification Changes in P2X 3 -immunoreactive (IR) and CGRP-IR innervation in the lower-lip skin were determined by analyzing the density of fibers within the upper dermis. Several images were taken at serial focal planes (z-stack) from 50-lm-thick sections and merged into one horizontal projection by the Axiovision software, using the extended focus feature. Six randomly chosen fields per lip section on each of 3 sections were captured, totaling 18 images per animal. Images were exported in TIFF format and adjusted for brightness and contrast using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Quantification was performed using an MCID Elite image analysis system (Imaging Research Inc, St. Catharines, ON, Canada). The total fiber length per unit area was determined, and 1-way ANOVA with Dunnett post hoc test was used to determine significance between the means of the groups. Statistical significance was accepted at P < Autonomic nerve quantification Quantification of the changes in autonomic innervation consisted of counting the number of VMAT2-IR and VAChT-IR fibers in the upper dermis (defined as the area above the opening of the sebaceous glands to the dermal-epidermal junction), an area normally devoid of these fibers. To quantify the level of autonomic fibers in the upper dermis, we obtained images from 6 randomly chosen fields per lip section on each of 4 sections per animal, totaling 24 images per animal. The mean number of fibers in the upper dermis was compared between groups using 1-way ANOVA and a Dunnett post hoc test. Statistical significance was accepted at P < Protein extraction and Western blot Changes in GDNF levels in the skin following ablation of the nonpeptidergic afferents were measured using Western blots. Animals were injected with unconjugated saporin or IB4-saporin into the mental nerve as described earlier. Three weeks following injection, animals were sacrificed with an overdose of Equithesin (0.4 ml/100 g body weight, i.p.) and the lower lip was extracted and flash frozen with liquid nitrogen. Each lip section was mechanically homogenized using a razor blade and transferred to an Eppendorf tube with cold radioimmunoprecipitation assay buffer containing protease inhibitors. Samples were further homogenized using a shearing homogenizer and incubated overnight at 4 C. Solubilized proteins were extracted by a 45-minute centrifugation at 13,000 RPM at 4 C. The protein concentration was determined by DC Protein Assay (Bio-Rad, Berkeley, CA, USA) with bovine serum albumin as the protein standard. Ten micrograms of protein was prepared in sample buffer under reducing conditions, and electrophoresed on 4% 10% polyacrylamide gels. Gels were blotted onto a nitrocellulose membrane and rinsed in tris-buffered saline with Triton-X100 (TBS-T) and blocked with 5% milk powder in TBS-T for 1 hour at room temperature. Membranes were incubated overnight with goat anti-gdnf antibody (1:500; R&D Systems, Minneapolis, MN, USA) in 2.5% milk powder in TBS-T at 4 C. Membranes were rinsed thoroughly with TBS-T and incubated with peroxidase-conjugated donkey anti-goat IgG (1: 2500) in 2.5% milk powder in TBS-T for 2 hours at room temperature. After thorough washing with TBS-T, immunoreactivities were visualized using the enhanced ECL detection system (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). Membranes were 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 room temperature, washed with TBS-T, and incubated with peroxidase conjugated donkey anti-mouse IgG (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 2.5% milk powder in TBS-T for 1 hour. Membranes were rinsed and immunoreactivities were visualized as above. Results were analyzed by computer-assisted densitometry and levels of GDNF immunoreactivity were normalized with respect to the b-actin levels in each sample Biochemistry statistical analyses All values are given as mean ± SEM. Values were normally distributed and so parametric statistical analysis was performed. Groups were compared using an unpaired t-test. Differences were considered statistically significant when P < Results 3.1. Behavior Animals receiving IB4-saporin injection into the mental nerves displayed no changes in spontaneous behavior, including feeding or grooming patterns, and had similar body weight to age-matched controls. This group also showed no change in evoked thresholds to mechanical punctate stimuli at 3 weeks following IB4-saporin injection when compared to control-operated animals receiving unconjugated saporin (Fig. 1). These thresholds were not significantly different from naïve animals receiving no surgical intervention (data not shown). This is in contrast to bilateral chronic constriction of the mental nerve (CCI), which resulted in significant reduction in mechanical thresholds at 2 and 4 weeks following surgery. Sham surgery resulted in no change in mechanical thresholds at any time point, so were pooled together (CCI Sham). CCI Sham animals were also not significantly different from the IB4-saporin-treated animals. Animals receiving IB4-saporin treatment followed by CCI of the mental nerve exhibited heightened sensitivity to light touch at 4 weeks following CCI lesion (Fig. 1). This sensitivity was not only significantly lower when compared to sham controls, but also significantly lower than age-matched animals receiving only the CCI lesion. Most animals in the IB4- saporin + CCI group consistently responded to the lowest von Frey filament, effectively reaching the minimum threshold of this behavioral testing technique. Injection of unconjugated saporin

4 1314 A.M.W. Taylor et al. / PAIN Ò 153 (2012) C fibers (as labeled with IB4 and P2X 3 ) in both the trigeminal subnucleus caudalis and skin was observed (Fig. 3A, B). CCI in IB4- saporin-treated animals led to a significant loss of CGRP-IR fibers in the upper dermis of the lower-lip skin (Fig. 3C, D). This loss was most substantial at 1 week post lesion, after which fibers slowly re-innervated the deafferented area Autonomic sprouting Fig. 1. Behavioral characterization of mechanical thresholds using von Frey filaments. 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 or naive 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 pooled into one group (CCI sham). Three weeks after IB4-saporin (IB4SAP) injection into the mental nerve, mechanical thresholds were not significantly different from any of the control groups. CCI of the mental nerve resulted in significant reduction of mechanical thresholds at 2 and 4 weeks after lesion. Injection of unconjugated Saporin did not cause significant changes in the already reduced mechanical thresholds 2 or 4 weeks after nerve lesion. In animals treated with IB4SAP followed by CCI of the mental nerve, mechanical thresholds were significantly lower than the CCI group at the 4-week time point (n = 4 6, P < 0.05, P < 0.01). Error bars represent SEM. followed by CCI lesion resulted in significantly lowered mechanical thresholds compared to CCI Sham and Saporin controls, but were not significantly different from CCI alone Changes in C-fiber innervation of trigeminal subnucleus caudalis and lower-lip skin Specificity of the IB4-saporin-induced ablation of nonpeptidergic afferents was confirmed via immunocytochemical labeling of IB4, P2X 3, and CGRP in the trigeminal subnucleus caudalis. By 3 weeks after injection of IB4-saporin into the mental nerves, a complete loss of IB4 binding and P2X 3 labeling in the most medial aspect of the trigeminal subnucleus caudalis was observed (Fig. 2C, D). This region correlates with the somatotopic location of the central afferents of the mental nerve in the trigeminal subnucleus caudalis. The lesion was shown to persist to all time points measured, up to 8 weeks after the initial IB4-saporin injection. Staining for peptidergic fibers, using the antibody directed against CGRP, showed minimal loss of these fibers following IB4- saporin treatment (Fig. 2D). Similarly, treatment with unconjugated saporin produced no reduction in IB4, P2X 3, or CGRP labeling (Fig. 2A, B). In the skin, IB4-saporin treatment caused near-complete loss of P2X 3 -IR fibers in the entire dermal and epidermal layers, persisting to all time points measured (Fig. 3A, B). As observed in the trigeminal subnucleus caudalis, IB4-saporin treatment caused a small but not significant loss of CGRP-IR fibers in the skin (Fig. 3C, D). In animals receiving IB4-saporin treatment followed by CCI of the mental nerve, a consistent and complete loss of nonpeptidergic Previous studies have demonstrated an aberrant sprouting of autonomic fibers into the upper dermis following neuropathic injury, an area where they are normally absent [19]. Since the facial area is innervated by both parasympathetic and sympathetic fibers, we explored whether specific ablation of nonpeptidergic C fibers via IB4-saporin treatment was able to induce this sprouting. While IB4-saporin treatment led to complete ablation of the nonpeptidergic afferents, it also led to a concomitant sprouting of parasympathetic (VAChT-IR) fibers into the upper dermis, an area where they are normally absent (Fig. 4A, B). The level of sprouted fibers was comparable to what was observed in a straight CCI lesion [48]. IB4-saporin treatment, however, did not induce significant sprouting of sympathetic (VMAT2-IR) fibers into the upper dermis (Fig. 4C, D). Treatment with IB4-saporin followed by CCI of the mental nerve led to significant sprouting of both sympathetic and parasympathetic fibers into the upper dermis. These fibers persisted to all time points tested following CCI lesion, up to 4 weeks (Fig. 4A D) GDNF protein levels GDNF levels in the skin of the lower lip following specific ablation of the nonpeptidergic C fibers were measured using Western blot. Three weeks after bilateral injection of IB4-saporin into the mental nerves, GDNF levels were found to be significantly higher than those from animals treated with unconjugated saporin (Fig. 5). This increase corresponds with the peak level of autonomic sprouting and sensory afferent regeneration as previously described. 4. Discussion Injection of IB4-saporin into the mental nerve caused complete loss of IB4+ terminals in the trigeminal subnucleus caudalis. Because of the known problems with IB4 labeling in the skin [43], an antibody directed against the P2X 3 receptor was used that labels nonpeptidergic fibers in the skin [47]. As expected, IB4-saporin injection led to complete and permanent loss of P2X 3 -IR fibers in the skin. A previous study examining IB4-saporin injection into the sciatic nerve described the loss of IB4+ neurons in the spinal cord and ganglia to begin around 3 days after injection and was completed by days [51]. In the present study, we examined the animals 21 days after IB4-saporin injection, as we could be assured that no ongoing nerve degeneration was occurring IB4-Saporin and autonomic sprouting Ablation of IB4+ neurons led to significant sprouting of parasympathetic fibers into the upper dermis, an area where they are normally absent. This response is similar to what is observed following a nerve lesion, where autonomic fibers sprout into the upper dermis, and were found in close apposition to injured afferents [19,48]. In contrast to the nerve injury model, ectopic sympathetic fibers were rarely observed in the upper dermis following IB4-saporin treatment, which suggests that parasympathetic sprouting is related to the loss of nonpeptidergic C fibers. This is

5 A.M.W. Taylor et al. / PAIN Ò 153 (2012) Fig. 2. Depletion of central terminals of nonpeptidergic C fibers in the trigeminal subnucleus caudalis as identified by IB4 binding and P2X 3 labeling. (A) P2X 3 (green) and IB4 (red) labeling in unconjugated Saporin group. Note the near-complete overlap between P2X 3 and IB4 labeling. (B) CGRP (green) and IB4 (red) labeling in unconjugated Saporin group. Note that this does not affect peptidergic or nonpeptidergic terminals in the trigeminal subnucleus caudalis. (C) P2X 3 (green) and IB4 (red) labeling in IB4-saporin group. Note the loss of nonpeptidergic terminals in the most medial aspect of the trigeminal subnucleus caudalis, corresponding to mental nerve afferents (indicated by arrows). (D) CGRP (green) and IB4 (red) labeling in IB4-saporin group. The CGRP-IR fibers are not significantly affected. Scale bar = 50 lm. supported by the fact that GDNF levels were found to be significantly elevated in the skin following IB4-saporin treatment. That sympathetic fibers, which respond to NGF, did not sprout following IB4-saporin injection, suggests that the relationship between C-fiber loss and autonomic sprouting is a specific one connected via response to growth factors.

6 1316 A.M.W. Taylor et al. / PAIN Ò 153 (2012) 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 P2X 3 -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 P2X 3 -IR fibers in the upper dermis (n = 6, P < 0.05, P < 0.01). Error bars 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 week CCI. 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, upper dermis. Presence of ectopic autonomic fibers in the upper dermis following nerve injury has been proposed to contribute to the hypersensitivity of nociceptors by releasing factors that directly sensitize the surrounding neurons [5,19,41]. While the role of the sympathetic nervous system in neuropathic pain has been thoroughly investigated [38], the parasympathetic system has been less explored, although it is plausible that it may play a role in chronic pain. Primary afferents express both nicotinic and muscarinic receptors [14], and application of acetylcholine and nicotine caused primary afferent discharge associated with pain sensations [5,20,24,28,44]. Application of nicotinic antagonists blocked the neurogenic flare following nociceptive stimulation [20,31]. It is also possible that the excess GDNF in the skin directly sensitizes the remaining primary afferents. GDNF overexpressing transgenic mice have lowered mechanical thresholds when compared to wild-type littermates [29]. However, while IB4-saporin resulted in significant increase in GDNF protein levels and concomitant sprouting of parasympathetic fibers into the upper dermis, this treatment did not result in any changes in evoked mechanical thresholds. In fact, it was not until a nerve lesion was applied (IB4- saporin + CCI) that the mechanical thresholds were significantly reduced, despite the continued presence of ectopic parasympathetic afferents. This would argue against the role of GDNF and/ or parasympathetic fibers in nerve injury-related pain. It is also possible that the presence of nonpeptidergic C fibers is necessary for the sensitization to occur IB4-saporin and behavioral response Three weeks after IB4-saporin injection into the mental nerve, mechanical thresholds were unchanged. This is supported by previous studies that reported a slight increase in mechanical and thermal thresholds shortly after IB4-saporin injection, but which returned to normal levels by 21 days [51]. This was surprising as IB4-saporin treatment caused a significant loss of primary afferents in the skin, which would be expected to significantly alter nociceptive processing. One explanation is that only light mechanical thresholds were measured in this study, due to the technical challenges of behavioral testing in the lower-lip region. It is possible that light mechanical stimuli are mediated by fast-conducting myelinated nociceptors, and loss of C fibers may not result in changes to this specific test. However, the fact that a previous study described no permanent change in thermal nociceptive thresholds following IB4-saporin injection into the sciatic nerve [51] suggests the lack of change in nociceptive thresholds pervasive across many stimulus modalities. Given the coexpression of many transducers on both peptidergic and nonpeptidergic C fibers, such as acid-sensing ion channels and the heat receptor TRPV1, it is possible there is considerable overlap in nociceptive function of these 2 populations of nociceptors [30]. Following loss of nonpeptidergic C fibers in this model, peptidergic C fibers are presumably able to mediate the normal nociceptive stimuli on their own. However, even though other studies using IB4SAP have shown mechanical and thermal thresholds to

7 A.M.W. Taylor et al. / PAIN Ò 153 (2012) 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 in the 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 indicate ectopic 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. remain relatively constant over time, we cannot be certain that IB4- saporin does not cause changes in mechanical thresholds beyond the 1-month time point. These changes might still be undetectable at 4 weeks, but what underlies them might contribute to the lowered 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 that nonpeptidergic fibers specifically mediate mechanical pain, whereas peptidergic C fibers mediate thermal pain [42]. As this previous study was performed in mice, it suggests that the strict dichotomy between C fiber populations and nociceptive modalities does 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 is located on both peptidergic and nonpeptidergic C fibers in rats and higher-order primates [50].

8 1318 A.M.W. Taylor et al. / PAIN Ò 153 (2012) IB4-saporin followed by CCI of the mental nerves led to complete loss of IB4+, P2X 3 -IR fibers, and a significant but transient loss of CGRP-IR fibers. A significant reduction of mechanical thresholds was also observed. The IB4-saporin + CCI group had significantly lowered mechanical thresholds at 4 weeks following nerve lesion when compared to the equivalent time point of animals receiving only the CCI lesion. The heightened pain response in IB4-saporin + CCI animals is a curious observation, as loss of nonpeptidergic C fibers would be expected to reverse or delay the onset of neuropathic pain. Indeed, a previous study performing a nerve injury on the sciatic nerve followed by intrasciatic IB4-saporin injection caused a transient delay of mechanical allodynia and hyperalgesia [46]. There are several reasons for this discrepancy. First, the previous study tested only the presence of mechanical allodynia or hyperalgesia, whereas this study used the up-down method to determine 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 all nonpeptidergic fibers were completely destroyed before inducing a nerve lesion. The decreased mechanical thresholds in neuropathic animals lacking nonpeptidergic afferents is intriguing. It has been proposed that initial inflammatory stimulus triggers long-lasting hypersensitivity to inflammatory cytokines in primary afferents, leading to a state of hyperalgesic priming [40]. The hyperalgesic priming was proposed to cause an increased response of primary afferent neurons to subsequent nociceptive stimuli, and is thought to be mediated via the protein kinase C epsilon signaling [2]. In our model, IB4-saporin injection leads to complete destruction of nonpeptidergic fibers, which is known to recruit a strong immune response. It is possible that the inflammatory response is enough to induce the exacerbated pain response observed in our model, and would explain the heightened pain response in animals receiving IB4- saporin followed by a nerve lesion. While ablation of nonpeptidergic afferents using the IB4-saporin approach has produced relatively consistent behavioral results published here and elsewhere [46,51], inhibition of nonpeptidergic afferents via P2X 3 receptor manipulation produced varying behavioral results. P2X 3 knockout mice had significantly lowered response to acute thermal pain, but thermal hyperalgesia was potentiated in an inflammation model [45]. However, P2X 3 knockdown using antisense oligonucleotides had no effect on acute pain behaviors and reduced neuropathic and inflammatory pain behaviors in rats [25]. Application of the P2X 3 receptor antagonist, A , also had no effect on acute pain behaviors, but reduced neuropathic and inflammatory pain behaviors [27]. The cause of these discrepant results is unclear; however, the specificity of both P2X 3 antagonist and antisense oligonucleotides has been questioned, and these approaches may produce significant off-target effects. In any case, it is clear that specifically manipulating the P2X 3 receptor is not equivalent to ablating the nonpeptidergic afferents, and so it is impossible to compare the behavioral results between these studies Conclusions Fig. 5. Changes in glial-derived nerve-growth factor (GDNF) protein levels in the lower-lip skin following specific ablation of the nonpeptidergic C fibers. (A) Representative Western blot of GDNF levels (18 kda) taken from the lower lip of animals injected with unconjugated Saporin (SAP) or IB4-saporin (IB4SAP) in the mental nerves. All GDNF levels normalized to b-actin (42 kda). (B) GDNF blot density, expressed as arbitrary units, was normalized to the reference protein, b actin (42 kda). GDNF levels were significantly higher in IB4SAP-injected animals compared to SAP. Error bars represent ± SEM. P < n = 4 per group IB4-saporin + CCI and behavioral response Overall, the results of this study highlight some intriguing peripheral adaptations following a nerve injury. Specifically, ablating the nonpeptidergic fibers led to significant increase of GDNF protein levels in the skin followed by specific sprouting of parasympathetic fibers into the upper dermis. This demonstrates an important link between loss of nonpeptidergic C fibers and parasympathetic sprouting, mediated through GDNF levels in the skin. Furthermore, while IB4-saporin treatment alone did not cause any long-term changes in mechanical thresholds, IB4-saporin followed by a nerve lesion led to an exacerbated pain response characterized by lowered mechanical thresholds. 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