2.1. Animals
Electrophysiology and molecular biology experiments were conducted using age-matched adult (4–6 weeks) male Swiss Webster mice (Hilltop Farms, Scottdale, PA). All animals were housed in group cages, maintained in a 12h light-dark cycle with a temperature controlled environment and given food and water ad libitum. All procedures used in these experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh. Animals were cared for and used in accordance with guidelines of the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, the NIH Guide for the Care and Use of Laboratory Animals and following institutional AAALAC approved practices.
2.2. Injection of Complete Freund’s Adjuvant (CFA)
Mice were anesthetized by isofluorane. A syringe containing a 1:1 mixture of CFA and saline and a 30g needle was used to inject CFA into the hairy hindpaw skin. Approximately 20 μL of CFA mixture was injected directly under the hairy skin starting slightly above the ankle and slowly retracting the needle as the CFA was introduced under the skin. The wound was cleaned and animals were allowed to survive for 3 and 5 days after nerve injection for immunocytochemical, electrophysiological, western blotting and/or realtime PCR analysis.
2.3. siRNA injections
siRNAs were designed and conjugated to Penetratin-1 as described previously [
4;
9;
12]. Mice were anesthetized as described. A small incision made in the mid-thigh region exposed the saphenous nerve to be injected. Pen-siRNAs were heated to 65°C for 5 min prior to injection. 0.1–0.2 μL of 90 μM Penetratin-1 linked control (PenCON) or P2Y1 targeting (PenY1) siRNAs were pressure injected into the saphenous nerve using a quartz microelectrode connected to a pico-spritzer immediately prior to CFA injection. This strategy for
in vivo siRNA-mediated inhibition does not cause significant injury to the sensory afferents being studied even though it requires direct injection of Penetratin-1 modified siRNAs into the saphenous nerve [
12].
2.4. RNA Isolation and Realtime PCR
Animals were deeply anesthetized with a mixture of ketamine and xylazine (Ketamine: 90 mg/kg and Xylazine: 10 mg/kg). The mice were then intracardially perfused with ice cold 0.9% NaCl prior to dissection of DRGs. RNA isolation from the L2 and L3 DRGs was performed using Qiagen RNeasy mini kits for animal tissues using the supplied protocol. Purified RNA was treated with DNase I (Invitrogen) and 1 μg of DNased RNA was reverse transcribed using Superscript II Reverse Transcriptase (Invitrogen). For realtime PCR, 20 ng samples of cDNA were added to a SYBR Green MasterMix (Applied Biosystems) and run in triplicate on an Applied Biosystems Imager. Forward and reverse primer sequences used in realtime PCR reactions for TRPV1 and GAPDH were obtained from Elitt et al. [
6], and primer sequences for P2X3, P2Y2 and P2Y1 were obtained from Jankowski et al., [
10]. mx2 primer sequences were obtained from Jankowski et al. [
11]. This gene was chosen for analysis under the experimental conditions in order to verify that there was no anti-viral related off-target effects associated with the siRNA injections [
11]. Values were normalized to GAPDH and changes in expression are calculated as a ΔΔCt value that is determined by subtracting the cycle time (Ct) values of the gene of interest from the GAPDH internal control for each sample and compared among samples. Fold change is described as 2
ΔΔCt (Applied Biosystems) and 2-fold change equals 100% change.
2.5. Western Blot
DRGs from naïve and inflammed mice were homogenized in lysis buffer containing 1% sodium dodecyl sulfate (SDS), 10 mM Tris–HCl (pH 7.4), and protease inhibitors (1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mM sodium orthovanadate and 100 μg/ml phenylmethylsulfonyl fluoride; Sigma Biochemicals). Samples (10 μg) were centrifuged, boiled 10 min in a denaturing buffer containing β-mercaptoethanol and SDS, separated on a 10% polyacrylamide SDS-PAGE gel and transferred to a PVDF (Hybond) membrane (Amersham) that was blocked in 5% milk (in 0.1M tris buffered saline with 0.1% tween-20) and then incubated with primary antibodies overnight at 4°C (P2Y1: 1:800; Alamone; actin: 1:500; Ab Cam). Antibody binding was visualized using horseradish peroxidase-conjugated goat anti-rabbit or donkey anti-goat secondary antibodies (1:10,000) and chemiluminescent detection (Pierce Biochemical). Immunoreactive bands were analyzed by densitometry and intensity quantified using NIH Image J software. Band intensity was normalized to actin and reported as a percent change.
2.6. Ex-vivo preparation
The
ex-
vivo somatosensory system preparation has been described in detail previously [
19;
10;
12]. Briefly, mice were anesthetized via injection of ketamine and xylazine (90 and 10 mg/kg, respectively) and perfused transcardially with oxygenated (95% O2–5% CO2) artificial CSF (aCSF; in mM: 1.9 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 26.0 NaHCO3, and 10.0 D-glucose) containing 253.9 mM sucrose at 12–15°C. The spinal cord and the right hindlimb was excised and placed in a bath of aCSF. Hairy skin of the right hindpaw, saphenous nerve, DRGs and spinal cord were isolated. Following dissection, the preparation was transferred to a separate recording chamber containing chilled oxygenated aCSF in which the sucrose was replaced with 127.0 mM NaCl. The skin was pinned out on a stainless steel grid located at the bath/air interface, such that the dermal surface remained perfused with the aCSF while the epidermis stayed dry. The platform served to provide stability during applied thermal and mechanical stimuli. The bath was then slowly warmed to 31°C before recording.
2.7. Recording and Stimulation
Sensory neuron somata were impaled with quartz microelectrodes (impedance >150 MΩ) containing 5% Neurobiotin (Vector Laboratories, Burlingame, CA) in 1 M potassium acetate. Orthograde electrical search stimuli were delivered through a suction electrode on the nerve to locate sensory neuron somata innervating the skin. Peripheral receptive fields (RF) were localized with a blunt glass stylus and von Frey hairs. When cells were driven by the nerve but had no mechanical RF, a thermal search was conducted. This was accomplished by applying hot (~ 52°C) and/or cold (~ 0°C) physiological saline to the skin. There was some concern that the brief but multiple applications of hot saline might cause sensitization of nociceptors during the course of an experiment. We examined this possibility in two recent studies [
19;
12] and found no change in average heat thresholds obtained at the onset of the experiment when compared to the average heat thresholds of the last fibers recorded. We have made a similar comparison of the data from these experiments in mice following CFA and also found no change in the average heat thresholds during the course of these experiments (data not shown).
The response characteristics of DRG cells were determined by applying digitally controlled mechanical and thermal stimuli. The mechanical stimulator consisted of a tension/length controller (Aurora Scientific) attached to a 1 mm diameter plastic disc. Computer controlled 5s square waves of 1, 5, 10, 25, 50 and 100 mN were applied to the cell’s RF. After mechanical stimulation, a controlled thermal stimulus was applied using a 3 mm2 contact area peltier element (Yale Univ. Machine Shop). The temperature stimulus consisted of a 12s heat ramp from 31–52°C followed by a 5s plateau at 52°. The stimulus then ramped back down to 31°C in 12s. Adequate recovery times (approx. 30s) were employed between stimulations. While recording from myelinated nociceptors in many cases, multiple heat applications were made and in some cases the heat ramp was continued to 54°C and held for 5s. In other instances, fibers that were unable to be characterized by computer controlled mechanical or thermal stimulation but were characterized by von Frey and/or saline stimuli were not included in the determination of thresholds. All elicited responses were recorded digitally for offline analysis (Spike2 software, Cambridge Electronic Design). After physiological characterization, select cells were labeled by iontophoretic injection of Neurobiotin (2–3 cells per DRG). Peripheral conduction velocity was then calculated from spike latency and the distance between stimulating and recording electrodes (measured directly along the nerve). Thermal thresholds were determined to be the temperature at which the first spike was elicited during the temperature change for fibers that did not exhibit ongoing activity prior to thermal stimulation. For those fibers that did have some degree of ongoing activity, threshold was determined as the temperature at the second spike of two where the instantaneous frequency exceeded that present in a 30 second window prior to the thermal stimulation.
2.8 Classification of cutaneous A- and C-fiber sensory neurons
Sensory neurons with a conduction velocity of < 1.2 m/s were classified as C-fibers [
17], and all others were classified as A-fibers. Conduction velocities between 1.2 and 10 m/s were considered to be in the Aδ range and those ≥10 m/s were in classified as conducting in the Aβ range [
16;
24]. For the purposes of these experiments we have focused our recordings and analyses specifically on the population of inflamed C-fibers. In addition, in all inflamed preparations we encountered a number of cells that were driven by the electrical nerve stimulus, but were found to be both mechanically and thermally unresponsive; however, only cells that had a response to cutaneous stimulation (mechanical or thermal) were included in the analysis.
The C-fiber classes were as follows: 1) C-polymodal (CPM), meaning those that responded to mechanical and heat stimuli (CMH) and sometimes cool/cold stimuli (CMHC); 2) C-mechano (CM), those that responded only to mechanical stimulation of the skin; 3) C-mechano cool/cold (CMC), those that responded to mechanical and cooling stimuli (but not heating); 4) C-heat (CH), those that were mechanically insensitive but heat sensitive, and 5) C-cooling/cold (CC), those that were mechanically insensitive but responded to cooling of the skin.
2.9. Tissue processing and analysis of recorded cells
Once a sensory neuron was characterized and intracellularly filled with Neurobiotin, the DRG containing the injected cell was removed and immersion fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 30 min at 4°C. Ganglia were then embedded in 10% gelatin, postfixed in 4% paraformaldehyde, and cryoprotected in 20% sucrose. Frozen sections (60 μm) were collected in PB and reacted with fluorescently-tagged (FITC) avidin to label Neurobiotin-filled cells (Vector Laboratories). Next, each section was processed for IB4 binding (AlexaFluor 647; Molecular Probes, Eugene, OR) and/or TRPV1 (1:500; CalBiochem), or CGRP (1:2000; Chemicon) immunohistochemistry. After incubation in primary antiserum, tissue was washed and incubated in Cy3 or Cy5 conjugated donkey anti-rabbit secondary antisera (1:200; Jackson Immunoresearch). Distribution of fluorescent staining was determined using Olympus FluoView™ 500 laser scanning confocal microscope (Olympus America Inc.). Sequential scanning was performed to prevent bleed-through of the different fluorophores.
2.10. Cell counts
The expression of TRPV1 and IB4 binding was analyzed in 3 L3 DRGs from naïve mice and 3 L3 DRGs from each CFA injected group at each time point. The DRGs taken after electrophysiological experiments and were processed for imunohstochemical analysis as described above. The numbers of positive cells were determined as previously reported [
3;
10]. In brief, 3 non-consecutive sections were randomly chosen and 15 μm stacks with 3 μm thick optical sections were captured using a 40x oil immersion objective. Multiple optical stacks were taken of each selected tissue section and visual confirmation was used to avoid analyzing cells twice. The number of TRPV1 positive cells or those that bound IB4 were counted and averaged in the top and bottom optical section of each stack and reported as mean ± SEM.
2.11. Primary Neuron Culture
DRGs from adult male mice were dissected into cold HBSS and then treated with 60U papain (Worthington), 1 mg of cysteine and 3 μL of NaHCO3 at 37°C for 10 min. Ganglia were then treated with 12 mg of collagenase II (Worthington) at 37°C for 10 min., washed and triturated with fire polished glass Pasteur pipettes in 1 mL of F12 complete media. Cells were then plated into poly-d-lysine/laminin coated wells, allowed to sit at 37°C/5% CO2 for 2h and then flooded with 1 mL of F12 media containing 10% fetal calf serum. Cultures were allowed to grow for 18–24 hours prior to calcium imaging analysis.
2.12. Calcium Imaging
Ca
++ imaging was performed as previously described [
22]. Cells were loaded with 2 mM fura-2-AM in HBSS with 5 mg/ml bovine serum albumin for 30 min at 37°C. The cells were then placed onto a microscope stage with constantly flowing HBSS at 5 ml/min. Temperature was maintained at 30°C using heated stage and in-line heating system (Warner Instruments). Treatments (capsaicin, ADP and K+) were delivered with a computer-controlled rapid-switching local perfusion system. Cells in regions of interest were identified with the Simple PCI, C-Imaging software. Absorbance data at 340 and 380 nm were collected once per second and the relative fluorescence (ratio 340/380) was plotted over time. A 50 mM K
+ stimulus in HBSS was applied to identify healthy neurons. To evaluate the activation of cells by P2Y1 and/or TRPV1, the percentage of neurons responding to capsaicin and/or ADP was calculated. Capsaicin was dissolved in 1-methyl-2-pyrrolidinone as a 10 mM stock solution; a working solution of 1 μM capsaicin was made fresh daily in HBSS. A 10 mM stock solution of ADP in HBSS was made fresh daily and applied as a 100 μM working solution.
2.13. Data analysis
One-way ANOVA tests and posthoc analysis (Tukey) were used to analyze differences in firing rate and instantaneous frequency along with mechanical and thermal thresholds of both A- and C-fibers. Average firing rates, maximal firing rates and firing rates during individual temperatures during the heat ramp were specifically analyzed. In order to test individual temperatures during the heat ramp, Student’s t test with Bonferroni correction was used. This information was sorted by neuronal functional type to examine whether or not certain classes of neurons have coherence with regard to the expression of any of the markers tested. Data recorded in response to the heat ramp was normalized as in our previous publications by multiplying the average spikes per degree by the percentage of cells responding at that temperature [e.g.
14]. Differences in fiber prevalence and immunostaining were determined by Fisher’s Exact analysis. Immunolabeling data from functionally identified cells was not found to be statistically different between the two time points analyzed and were combined for ease of presentation into a single table. Percent changes in mRNA were also determined to be statistically significant by ANOVA with post hoc analysis (Tukey). Statistical analyses were performed on the raw Ct data and represented for ease of presentation as a percent change. P-values were set at p < 0.05.
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