These experiments were approved by our institute’s Subcommittee on Animal Studies and followed the guidelines of the International Association for the Study of Pain (IASP) . Adult (9-month-old) male Sprague Dawley rats (Simonsen Laboratories, Gilroy, CA, USA) were used in all experiments. The animals were housed individually in isolator cages with solid floors covered with 3 cm of soft bedding and were fed and watered ad libitum. During the experimental period the animals were fed Lab Diet 5012 (PMI Nutrition Institute, Richmond, IN, USA), which contained 1.0% calcium, 0.5% phosphorus, and 3.3 IU/g of vitamin D3, and were kept under standard conditions with a 12-h light–dark cycle.
Tibial fracture was performed under isoflurane anesthesia as we have previously described . The right hindlimb was wrapped in stockinet (2.5 cm wide) and the distal tibia was fractured using pliers with an adjustable stop that had been modified with a 3-point jaw. The hindlimb was wrapped in casting tape so the hip, knee and ankle were flexed. The cast extended from the metatarsals of the hindpaw up to a spica formed around the abdomen. To prevent the animals from chewing at their casts, the cast material was wrapped in galvanized wire mesh. The rats were given subcutaneous saline and buprenorphine (0.03 mg/kg) immediately after the procedure and on the next day after fracture for postoperative hydration and analgesia. At 4 weeks the rats were anesthetized with isoflurane and the cast removed with a vibrating cast saw. All rats used in this study had union at the fracture site after 4 weeks of casting.
The anti-NGF antibody muMab 911 (Rinat Laboratories, Pfizer Inc, SF, CA, USA) is a TrkA immunoglobulin G (TrkA-IGG) fusion molecule that binds to the NGF molecule, thus blocking the binding of NGF to the TrkA and p75 NGF receptors and inhibiting TrkA autophosphorylation . Pharmacokinetic and behavioral experiments in rodents indicate that muMab 911 has a terminal half-life of 5 to 6 days in plasma and that a 10 mg/kg dose administered every 5 or 6 days reduces nociceptive behavior in a variety of rodent chronic pain models [21–23].
The NK1 receptor antagonist LY303870 was a generous gift from Dr. L. Phebus (Eli Lily Company, Indianapolis, IN, USA). This compound has nanomolar affinity for the rat NK1 receptor, has no affinity for 65 other receptors and ion channels, has no sedative, cardiovascular or core body temperature effects in rats at systemic doses up to 30 mg/kg, and is physiologically active for 24 h after a single systemic dose of 10 mg/kg [24–26].
To measure mechanical allodynia in the rats an up-down von Frey testing paradigm was used as we have previously described [2, 15, 27]. Rats were placed in a clear plastic cylinder (20 cm in diameter) with a wire mesh bottom and allowed to acclimate for 15 minutes. The paw was tested with one of a series of eight von Frey hairs ranging in stiffness from 0.41 g to 15.14 g. The von Frey hair was applied against the hindpaw plantar skin at approximately midsole, taking care to avoid the tori pads. The fiber was pushed until it slightly bowed and then it was jiggled in that position for 6 seconds. Stimuli were presented at an interval of several seconds. Hindpaw withdrawal from the fiber was considered a positive response. The initial fiber presentation was 2.1 g and the fibers were presented according to the up-down method of Dixon to generate six responses in the immediate vicinity of the 50% threshold. Stimuli were presented at an interval of several seconds. An incapacitance device (IITC Inc. Life Science, Woodland, CA, USA) was used to measure hindpaw unweighting. The rats were manually held in a vertical position over the apparatus with the hindpaws resting on separate metal scale plates and the entire weight of the rat was supported on the hindpaws. The duration of each measurement was 6 s and 10 consecutive measurements were taken at 60-second intervals. Eight readings (excluding the highest and lowest ones) were averaged to calculate the bilateral hindpaw weight-bearing values.
The room temperature was maintained at 23°C and humidity ranged between 25 and 45%. The temperature of the hindpaw was measured using a fine wire thermocouple (Omega, Stanford, CT, USA) applied to the paw skin, as previously described [2, 15, 27]. The investigator held the thermistor wire using an insulating Styrofoam block. Three sites were tested over the dorsum of the hindpaw; the space between the first and second metatarsals (medial), the second and third metatarsals (central), and the fourth and fifth metatarsals (lateral). After a site was tested in one hindpaw the same site was immediately tested in the contralateral hindpaw. The testing protocol was medial dorsum right then left, central dorsum right then left, lateral dorsum right then left, medial dorsum left then right, central dorsum left then right, and lateral dorsum left then right. The six measurements for each hindpaw were averaged for the mean temperature.
A laser sensor technique was used to determine the dorsal-ventral thickness of the hindpaw, as we have previously described . Before baseline testing the bilateral hindpaws were tattooed with a 2 to 3 mm spot on the dorsal skin over the midpoint of the third metatarsal. For laser measurements each rat was briefly anesthetized with isoflurane and then held vertically so the hindpaw rested on a table top below the laser. The paw was gently held flat on the table with a small metal rod applied to the top of the ankle joint. Using optical triangulation, a laser with a distance measuring sensor was used to determine the distance to the table top and to the top of the hindpaw at the tattoo site and the difference was used to calculate the dorsal-ventral paw thickness. The measurement sensor device used in these experiments (4381 Precicura, Limab, Goteborg, Sweden) has a measurement range of 200 mm with a 0.01 mm resolution.
Homogenization procedure and enzyme immunoassay for TNF-α, IL-1β, IL-6 and NGF
Rat hindpaw dorsal skin was collected after behavioral testing or at time points as indicated and frozen immediately on dry ice. Skin tissue was cut into fine pieces in ice-cold phosphate buffered saline (PBS), pH 7.4, containing protease inhibitors (aprotinin (2 μg/ml), leupeptin (5 μg/ml), pepstatin (0.7 μg/ml), and PMSF (100 μg/ml); Sigma, St. Louis, MO, USA) followed by homogenization using a rotor/stator homogenizer. Homogenates were centrifuged for 5 minutes at 14,000 g, and at 4°C. Supernatants were transferred to fresh pre-cooled Eppendorf tubes. Triton X-100 (Boehringer Mannheim, Germany) was added at a final concentration 0.01%. The samples were centrifuged again for 5 minutes at 14,000 g at 4°C. The supernatants were aliquoted and stored at −80°C. TNF-α, IL-1β, and IL-6 protein levels were determined using EIA kits (R&D Systems, Minneapolis, MN, USA). The NGF concentrations were determined using the NGF Emax® ImmunoAssay System kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The optical density (OD) of the reaction product was read on a microplate reader at 450 nm. The concentrations of TNF-α, IL-1β, IL-6, and NGF proteins were calculated from the standard curve at each assay. Positive and negative controls were included in each assay. Each protein concentration was expressed as pg/mg total protein. Total protein contents in all tissue extracts were measured by the Coomassie Blue Protein Assay Kit (Pierce, Rockford, IL, USA).
Tissue processing and keratinocyte immunofluorescence confocal microscopy
Animals were euthanized and immediately perfused with 4% paraformaldehyde (PFA) in PBS, pH 7.4, via the ascending aorta; the hindpaw skin including sub-dermal layers was removed and post-fixed in 4% PFA for 2 h, and then the tissues were treated with 30% sucrose in PBS at 4°C before embedding in the optimal cutting temperature compound (OCT) (Sakura Finetek USA, Inc., Torrance, California, USA). Following embedding, 10-μm thick slices were made using a cryostat, mounted onto Superfrost microscope slides (Fisher Scientific, Pittsburgh, PA, USA), and stored at −80°C.
To examine the effects of intraplantar SP on inflammatory mediator production and NK1 receptor expression in epidermal keratinocytes, double immunolabeling was performed as previously described . Briefly, frozen skin sections were permeabilized and blocked with PBS containing 10% donkey serum and 0.3% Triton X-100, followed by exposure to the primary antibodies overnight at 4°C in PBS containing 2% serum. Upon detection of the first antigen, a primary antibody from a different species against the second antigen was applied to the sections and visualized using an alternative fluorophore-conjugated secondary antibody. Sections were then rinsed in PBS and incubated with fluorophore-conjugated secondary antibodies against the immunoglobulin of the species from which the primary antibody was generated. After three washes, the sections were mounted with anti-fade mounting medium (Invitrogen, Grand Island, NY, USA). With regard to primary antibodies, goat anti-rat IL-1β (R&D Systems, 1:200), goat anti-rat NGF-β (R&D Systems, 1:100), rabbit anti-rat NK1 receptor (Sigma-Aldrich, diluted 1:8000) and monoclonal mouse anti-rat keratin (clone AE1/AE3) (Thermo Fisher Scientific, Waltham, MA 02454, USA, diluted 1:50) were used. Double-labeling immunofluorescence was performed with a series of conjugated secondary antibodies (Jackson ImmunoResearch Laboratories), that is, donkey anti-mouse IgG (1:500) conjugated with fluorescein isothiocyanate (FITC) and donkey anti-goat IgG (1:500) conjugated with cyanine dye 3 (Cy3) for co-immunostaining of keratin and IL-1β or NGF, and donkey anti-mouse IgG (1:500) conjugated with Cy3 and donkey anti-rabbit IgG (1:500) conjugated with FITC for co-staining of keratin and NK1 receptor, respectively.
To assess LY303870 effects on epidermal thickness at 4 weeks post-fracture, the sections of hindpaw skin were permeabilized and blocked as described above, followed by exposure to monoclonal anti-rat keratin (clone AE1/AE3) (Thermo Fisher Scientific, diluted 1:50) overnight at 4°C in PBS containing 2% serum. The primary antibody was detected using FITC-conjugated donkey anti-mouse IgG (H + C) antibody (Jackson Immuno Reasearch Laboratories, West Grove, PA, USA, diluted 1:500). Images were obtained using confocal microscopy (Zeiss LSM/510 Upright 2 photon; Carl Zeiss, Thornwood, NY, USA) and stored on digital media. Control experiments included incubation of slices in primary and secondary antibody-free solutions both of which led to low intensity non-specific staining patterns in preliminary experiments (data not shown).
In vivo bromodeoxyuridine (BrdU) labeling and BrdU immunohistochemistry
Labeling with BrdU was done to evaluate keratinocyte proliferation. At 3 weeks after tibial fracture, animals were injected intraperitoneally (i.p.) once daily with 50 mg/kg BrdU (Sigma-Aldrich) for 8 days . Hindpaw skin was harvested and fixed one day after the last injection and processed for immunostaining. Skin sections were pretreated in 2 N HCl for 30 minutes at 37°C, followed by neutralization in 0.1 M borate buffer (pH 8.5) for 10 minutes and blocking with 10% normal donkey serum for 1 h at room temperature, after which immunohistochemistry was performed using a rat anti-BrdU monoclonal antibody (1:300, Accurate Chemical, WESTBURY, NY, USA) and donkey anti-rat fluorescein isothiocyanate secondary antibody (1:400, Jackson Immuno Research Laboratories). After three rinses with PBS, the sections were immunostained with the monoclonal anti-rat keratin as mentioned above. BrdU immunostaining was observed using a Leica DM 2000 fluorescent microscope and imaged using a Spot Camera (version 4.0.8, Diagnostic Instruments, Sterling Heights, MI, USA). The number of BrdU-positive cells was counted, specifically those in keratin-positive cells in the area of the epidermis with a minimum of six sections per animal from seven intact and five fractured animals. Cell densities were calculated by dividing cell numbers by the area. Representative images were obtained using confocal microscopy (Zeiss LSM/510 Upright 2 photon; Carl Zeiss).
Microcomputed tomography (μCT)
Ex vivo scanning was performed for assessment of trabecular and cortical bone architecture using micro computer tomography (μCT) (VivaCT 40, Scanco Medical AG, Basserdorf, Switzerland). Specifically, trabecular bone architecture was evaluated at the distal femur and cortical bone morphology was evaluated at the midshaft femur. CT images were reconstructed in 1024 × 1024 pixel matrices for distal femur and mid-femur samples and stored in 3-dimensional arrays. The resulting grayscale images were segmented using a constrained Gaussian filter to remove noise, and a fixed threshold (25.5% of the maximal grayscale value for the vertebrae and the distal femur, and 35% for the mid-femoral cortical bone) was used to extract the structure of the mineralized tissue. The μCT parameters were set at threshold = 255, σ = 0.8, support = 1 for vertebral samples, threshold = 255, σ = 0.8, support = 1 for distal femur, and threshold = 350, σ = 1.2, and support = 2 for mid-femur evaluation analysis. A single operator outlined the trabecular bone region within the distal femur, the vertebral body and the cortical bone region in the mid-femoral shaft. The trabecular bone region was manually identified and all slices containing trabecular bone between the growth plates were included for analysis. In the distal femur 150 transverse slices of 21 μm thickness (21-μm isotropic voxel size) encompassing a length of 3.15 mm were acquired, but only 100 slices encompassing 2.1 mm of the distal femur were evaluated, starting where the growth plate bridge across the middle of the metaphysis ends. The region of interest (ROI) was manually outlined on each CT slice, extending proximally from the growth plate. The bone parameters analyzed included the bone volume fraction (BV/TV) (%), trabecular number (TbN) (mm-1), trabecular thickness (TbTh) (μm), trabecular separation (TbSp) (μm), and connectivity density (ConnD) (1/mm3). At the femoral mid-shaft, 10 transverse CT slices were obtained, each 21 μm thick totaling 0.21 mm in length (21 μm isotropic voxel size) and these were used to compute the cortical bone area (Bar) (mm2), total cross sectional area (TtAr) (mm2), medullary area (MeAr) (mm2), cortical thickness (CtTh) (μm), bone perimeter (BPm) (mm), and relative cortical bone area (BAr/TtAr) (%).
To determine the time course of SP-induced pain behavior and signs of inflammation, normal rats received hindpaw intraplantar injection of SP (0, 10, 25, and 60 μg) in 50 μl of 0.9% saline. Baseline determinations were made of bilateral hindpaw mechanical nociceptive withdrawal thresholds to von Frey fibers, hindpaw temperature, and hindpaw thickness, and then the rats were injected and retested for behavior at 0.5, 1, 3, 6, 24, 48, and 72 h after SP injection (n = 6 per cohort). Based on the results of this behavioral study, a 25 μg dose of SP was selected for evaluating the time course of SP-induced inflammatory mediator expression. After normal rats underwent intraplantar injection with 25 μg SP in 50 μl of 0.9% saline, the hindpaw skin was collected at 0, 1, 3, 6, 24, and 48 h after injection for TNF-α, IL-1β, IL-6, and NGF protein level determination by EIA (n = 8 per cohort).
The effects of SP (25 μg) intraplantar injection on keratinocyte inflammatory mediator and NK1 receptor expression were evaluated by immunofluorescence confocal microscopy. Normal rats received hindpaw intraplantar injections with 25 μg SP, and then the injected hindpaw skin was collected at 0, 1, 3, and 6 h post-injection for immunostaining with anti-IL-1β, NGF, or NK1 primary antibody.
To evaluate the contribution of NGF signaling in SP-evoked allodynia, the NGF inhibitor muMab 911 (10 mg/kg) or vehicle was administered via i.p. injection in normal rats. Three days later, baseline hindpaw von Frey thresholds, temperature, and paw thickness were determined, and then the rats received an intraplantar injection with 25 μg SP. At 0.5, 1, 3, 6, 24, 48, and 72 h post-injection the animals underwent repeat tests.
To test the hypothesis that local SP signaling induces hindpaw pain and inflammation in the CRPS tibial fracture model, fracture rats were treated with either a systemic or locally administered NK1 receptor antagonist (LY303870). The rats were divided into four cohorts (n = 8 to 16 rats per cohort). Three cohorts underwent right distal tibial fracture with hindlimb cast immobilization for 4 weeks. The day after cast removal, all rats underwent bilateral hindpaw tests for von Frey thresholds, unweighting, warmth, and edema. One fracture cohort had no treatment, one cohort received daily i.p. injections of LY303870 (20 μg/kg) for 8 days prior to cast removal, and one cohort received a single intraplantar injection of LY303870 (50 μg/50 μl 0.9% saline) the day after cast removal. At one hour after intraplantar injection of LY303870 the rats were tested.
To evaluate the role SP signaling plays in post-fracture keratinocyte proliferation in the injured limb, tibial fracture rats were treated with vehicle or LY303870 (20 mg/kg i.p.) daily for 8 days prior to cast removal. All rats were injected with BrdU (50 mg/kg i.p.) daily for 8 days prior to cast removal. After cast removal the hindpaw skin was harvested bilaterally for co-immunostaining with anti-BrdU (DNA synthesis marker) and anti-keratin (keratinocyte marker) antibodies. Confocal microscopy was used to measure the number of BrdU-positive cells in the epidermis (n = 9 rats for the control, ipsilateral fracture (FX-IPSI), and contralateral fracture (FX-CONTRA) cohorts, and n = 4 for the LY303870 treatment group), and to measure epidermal thickness (n = 7 per cohort).
To test the hypothesis that SP signaling mediates fracture-induced inflammatory mediator expression in the hindpaw skin, three cohorts of rats were evaluated; controls, vehicle-treated fractured rats, and LY303870-treated fractured rats (n = 13 to 16 per cohort). LY303870 (20 mg/kg) was given by i.p. injection daily for 8 days prior to cast removal. After the casts were removed at 4 weeks post-fracture the animals were euthanized and the hindpaw skin collected for TNF-α, IL-1β, IL-6, and NGF protein level determination by EIA. The bilateral femurs were collected for ex vivo μCT of the proximal metaphyseal trabecular and the mid-femoral cortical bone to test the hypothesis that facilitated-SP signaling contributes to periarticular bone loss after fracture.
Statistical analysis was accomplished using a two-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests for the time course of the SP injection at various concentrations. One-way ANOVA was employed followed by Neuman-Keuls multiple comparison test to compare among the control, fractured and LY303870-treated fractured rats. All data are presented as the mean ± standard error (SE) of the mean, and differences are considered significant at a P-value less than 0.05 (Prism 5, GraphPad Software, San Diego, CA, USA).
Hindpaw temperature, thickness, and mechanical nociceptive thresholds data were analyzed as the difference between the treatment side and the contralateral untreated side. Right hindpaw weight bearing data were analyzed as a ratio between the right hindpaw weight and the sum of the right (R) and left (L) hindpaws values ((2R/(R + L)) × 100%).