In total, 60 male Sprague-Dawley rats (Charles River Canada), weighing between 150-175 g, were used for this study. All animals were maintained on a 12 hour light/dark cycle and allowed access to food and water ad libitum. All animal protocols were approved by the McGill University Animal Care Committee and followed the guidelines of the Canadian Council on Animal Care and the International Association for the Study of Pain.
Peripheral Nerve Lesions
Animals were randomly assigned to receive either a traditional chronic constriction injury (CCI) or sham operation. All animals were deeply anaesthetized with isoflurane. In total, 30 rats received the sham operation and 30 received the CCI injury to the common sciatic nerve, as described in detail by Bennett & Xie . Briefly, the left common sciatic nerve was exposed at the mid-thigh through the biceps femoris. All surrounding fascia was removed to allow free passage to blunt glass probes. Proximal to the trifurcation of the sciatic nerve, four ligatures (4-0 chromic gut, Ethicon) were tied loosely around the nerve with ~1 mm spacing in between. The incision was closed in layers using absorbable sutures (Vicryl, Ethicon). The animals assigned to sham-operated groups underwent the above mentioned procedures with the exception of the application of the chromic gut ligatures.
Animal perfusion and histological or Western blot processing
Animals were sacrificed after periods of one, two, and four weeks post-surgery. Rats were administered 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. Three animals per time point were perfused for immunohistochemical analysis. Once animals were deeply anesthetized, they were quickly transcardially perfused with vascular rinse (0.1% w/v sodium nitrite in a phosphate buffer) followed by histological fixatives (3% paraformaldehyde, 15% saturated picric acid in 0.1 M phosphate buffer pH 7.4) for 30 minutes. The glabrous skin from the left hind paw, specifically the thin skin surrounded by tori, was removed, post-fixed for one hour in the same fixative used for the perfusion and placed in 30% sucrose in 0.1 M phosphate buffer for cryoprotection overnight at 4°C. Tissue was then embedded in an optimum cutting temperature medium (OCT, TissueTek), cut using a cryostat (Leica) at a thickness of 50 μm and processed as free floating sections. A second cohort of six animals per time point were perfused for 30 seconds with ice cold saline to clear contaminating blood. The same region of glabrous skin as that used for immunohistochemistry was excised from the rat hind paw, snap frozen in liquid nitrogen and stored at -20°C for no more than ten days until processed for Western blot analysis.
Sections were washed in PBS for 30 minutes. In all subsequent steps, phosphate buffered saline (PBS) with 0.2% Triton X-100 (PBS-T) was used to dilute the immunoreagents and for washing. Sections were treated with 50% ethanol for 30 minutes followed by one hour incubation in PBS-T containing 10% (v/v) normal serum of the species used to generate the secondary antibody to reduce non-specific staining. Subsequently, sections were incubated with primary antibodies (diluted in PBS-T) at 4°C overnight (or 48 hours in the case of S100). The following primary antibodies were used: polyclonal guinea-pig anti-human calcitonin gene related peptide (CGRP; 1:4000, Bachem; Torrance CA), monoclonal mouse anti-dopamine β-hydroxylase (DBH; 1:5 MediMabs, gift of Dr. A. Claudio Cuello), polyclonal rabbit anti-proNGF (1:500, Alomone Labs, Israel), monoclonal mouse anti-p75 (MC192; 1:5, Novus Biologicals), polyclonal rabbit anti-S100β (1:5000; Swant Switzerland) and polyclonal rabbit anti-PGP 9.5 antibody (1:800, Cedarlane; Burlington Ont.). To assess by immunohistochemistry the specificity of the anti-proNGF antibody, we pre-adsorbed it with the control antigen supplied by Alomone (3 μg of control peptide to 0.8 μg of antibody) for three hours over ice prior to incubation with control tissue from naïve rats. After washing, the sections were incubated with secondary antibody diluted in PBS-T for two hours at room temperature. The following secondary antibodies were used: a) for those antibodies raised in mouse, a highly cross-adsorbed goat anti-mouse IgG conjugated to Alexa 596 (1:800, Molecular Probes, Eugene, OR); b) for those raised in rabbit or guinea-pig, a goat anti-rabbit or anti-guinea-pig conjugated to Alexa 488 (1:800, Molecular Probes, Eugene, OR); c) in the case of the triple labeling a highly cross-adsorbed donkey anti-mouse conjugated to Cy-5 (1:800, Molecular Probes, Eugene, OR). Sections were washed and mounted on gelatin-coated slides and coverslipped using Aquapolymount (Polysciences; Warrington, PA). In the case of S100 immunostaining, an hour incubation with an goat anti-rabbit biotin conjugated secondary (1:400; BA-1000; Vector) was used. Following this, sections were incubated for one hour with the A and B reagents (1:400 Vectastain ABC kit; Vector) followed by a two hour incubation with a streptavidin antibody conjugated to Alexa 488 (1:200; Molecular Probes, Eugene, OR). For the S100/p75/proNGF triple labeling, the anti-proNGF antibody was added following the completion of the staining with S100 and p75. In the case of bright field images using anti-proNGF antibodies, sections were prepared as described above except for a ten minute incubation with 0.1% H2O2 prior to blocking and following incubation with primary antibody, incubated for two hours with a biotinylated goat anti-rabbit secondary antibody (1:200, Vector). The sections were washed with PBS and incubated for one hour with the A and B reagents (4 μl of A + 4 μl of B per mL of PBS, ABC Vectastain Elite kit). The sections were then washed in PBS and incubated for 5 minutes in a solution of 3-3'-diaminobenzidine (Sigma, 5 mg for 10 ml of PBS-T). Hydrogen peroxide was then added to the solution to reach a final concentration of 0.01%. The reaction was terminated after ten minutes by the addition of PBS. The sections were washed in PBS, mounted on gelatin-subbed slides and air dried. Slides were rehydrated and briefly exposed to a 1% solution of toluidine blue, rinsed and dehydrated in ascending alcohols and xylene, then coverslipped with Entellan (EMD, Gibbstown, NJ).
Primary Antibody Characterization
CGRP: The anti-CGRP antibody raised in guinea pig was purchased from Bachem (T-5027, lot A00098-2). It was generated against a synthetic peptide from human α-CGRP with the following sequence: H-Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Al a-Phe-NH2. The antibody has 100% reactivity with human and rat α-CGRP, human CGRP (8-37), chicken CGRP, and human β-CGRP. It has 0.04% crossreactivity with human amylin and 0% crossreactivity with rat amylin and with human and salmon calcitonin as determined by radioimmunoassay (manufacturer's technical information). Although Western blot information was not available, the immunostaining has been localized in the skin to IB4 negative C-fibers, and fibers innervating blood vessels in rats [75, 76] and in monkeys .
DβH: The anti-dopamine β-hydroxylase monoclonal antibody was received as a gift from Dr. Claudio Cuello (clone DBH 4; MediMabs, Montreal, Canada). It was generated against rat purified DBH. The specificity was tested by Western blot, ELISA and immunocytochemistry and it was found that it is highly specific for rat DβH, and does not cross-react with DβH in mouse, human, rabbit, bovine, guinea pig or cat tissues .
S100β: A rabbit polyclonal antibody purchased from Swant (Code Number 37A). It was generated against purified bovine brain S100β and shows < 0.5% cross-reactivity with S100α and reacts with S100β from all species (i.e. bovine, rat, chicken and human) (data obtained from supplier). This antibody has been used as a marker for Schwann cells .
P75: The monoclonal antibody against the low-affinity neurotrophin receptor (MC192, Novus Biologicals, Littleton, CO) was generated from solubilized rat PC12 cell membranes and was found to be selective towards rat p75 to the exclusion of gerbil, hamster, guinea pig or mouse p75 . Its distribution in rat skin was described at both light microscopic and electron microscopic levels 
TrkA: Monoclonal anti-TrkA (R&D Systems MAB1056 Clone: 315104 Lot #WPO0108021) raised against recombinant rat TrkA extracellular domain (a.a. 33-418). This antibody recognized rrTrkA by ELISA and Western Blot with no cross-reactivity towards recombinant human TrkA, TrkB, TrkC, recombinant mouse TrkB and TrkC (information provided by supplier).
PGP 9.5: The rabbit anti-PGP 9.5 antibody (Ultraclone code RA95101) was generated by repeated injection of purified whole human PGP 9.5 in Freund's adjuvant into rabbits. The antibody cross-reacts with PGP 9.5 protein in all mammalian species (manufacturer's technical information). By Western blot, it recognizes a band at 38 kDa from human and rat skin, and preadsorption with purified PGP 9.5 completely abolished this recognition [81, 82]. Immunohistochemical staining with this antibody in either rat or human skin was absent following preadsorption with purified human PGP 9.5 protein [81, 82].
proNGF: The rabbit anti-proNGF antibody (Alomone #ANT-005 Lot#AN-03) was generated by injection of a synthetic peptide corresponding to a.a. 84-104 of the precursor form of rat NGF. It cross-reacts with rat, mouse and human proNGF (information from supplier).
Samples were kept at -20°C until manually homogenized with liquid nitrogen and added to RIPA buffer (1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 25 mL Tris-HCl, pH 7.6) containing protease inhibitors (Complete, Roche Molecular Biochemicals, Indianapolis, IN). Samples were agitated overnight at 4°C, followed by 45 minute centrifugation at 13,200 rpm to separate supernatant. The supernatant was pipetted and protein quantified using Bio-Rad DC Protein Assay (Bio-Rad, Mississauga Ont). Samples were then prepared as either reduced (in the case of TrkA and proNGF) or not (in the case of p75) using 10% β-mercaptoethanol (Bio-Rad, Mississauga Ont) SDS-based protein loading buffer. Samples were then boiled for 5 min and 50 mg total protein for p75 westerns and 75 mg total protein for proNGF and TrkA westerns were loaded onto a 4% stacking and 12% separating acrylamide gel for SDS-PAGE at 90 V. Samples were transferred to nitrocellulose membranes for 90 minutes at 110 mA and blocked for one hour. Primary antibodies such as mouse anti-TrkA (1:500, MAB1056, R&D Systems, Minneapolis, MN), rabbit anti-proNGF (1:500, ANT-005, Alomone Labs, Isreal), or mouse anti-p75 (1:200, MC192, Novus Biologicals) were incubated overnight in blocking buffer at 4°C. The following day, membranes were washed with tris-buffered saline with 0.1% Tween-20 (TBS-T) and incubated for two hours at room temperature with secondary antibodies raised against either mouse (Product no.: 715-035-151) or rabbit (Product no.: 711-035-152) IgG, conjugated to horseradish peroxidase (1:5000, Jackson ImmunoResearch, West Grove PA), and incubated for one minute with ECL substrate (Western Lighting, Perkin Elmer, Montreal, Qc). Following x-ray film exposure, membranes were incubated for one hour with stripping buffer, blocked and incubated with mouse anti β-actin primary antibody (1:400, A5441, Sigma) in blocking buffer for one hour washed with TBS-T and incubated with anti-mouse IgG-HRP (1:5000). Immunoreactive bands were quantified by simultaneously obtaining equally sized bands and quantification was based on optical density and normalized against the loading control (β-actin) using MCID Image Analysis software (MCID4 Image Analysis System; Imaging Research Inc.; St Catherine's ON, Canada).
Images for the Figures
Pictures for the figures of this publication were taken as Z-stacks of confocal optical sections using a Zeiss LSM 510 confocal microscope equipped with argon and helium neon lasers applying a 40X water-immersion objective. Images were originally saved in the Zeiss format, then exported directly into the TIFF format and adjusted for brightness and contrast only, using Adobe Photoshop CS4. Bright field images were taken using a Zeiss Axioplan 2 imaging microscope equipped with a 40 × Plan-Fluotar oil-immersion objective. Images were acquired with a high-resolution color digital camera using the Zeiss AxioVision software. Representative images of Western Blots were taken using an Epson scanner, saved as TIFF format and realigned using Adobe Photoshop CS4.