The CAP response amplitudes from contralateral, naïve control nerves, and ipsilateral sham nerves were similar and therefore grouped together. 2 weeks after SNE, the amplitudes of both A- and C-fiber CAPs were significantly increased in ipsilateral sciatic nerves when compared to control, sham or contralateral nerves (Fig. 1). The increases were observed in the plateau responses to maximal stimulation (Fig. 1A, B) Although leftward shifts in the stimulus-response relationsips were also detected for the A-fiber and to a lesser extent the C-fiber CAP after normalizing responses to maximal amplitudes, these shifts were not statistically significant (Fig. 1A and 1B insets). The conduction velocity (CV) of the peak A-fiber CAP was not different between nerves ipsilateral to SNE (32.4 ± 1.5 m/s) and control, sham or contralateral nerves (29.9 ± 1.7 m/s). The CV of the ipsilateral C-fiber CAP 1.2 ± 0.1 m/s) was also similar to that of control, sham and contralateral nerves (1.1 ± 0.1 m/s).

Concentration-dependent decreases in CAP amplitude were detected within 30 sec after TTX (0.1–1 μM) was applied in the recording chamber (p < 0.05, two-way ANOVA with Tukey post-hoc) and measurements were obtained after 20–25 min to ensure a full drug response. The C-and A-fiber CAPs in both injured and uninjured nerves were blocked completely in the presence of 1 μM TTX (not shown). At lower concentrations, the TTX-resistance of both the A-fiber and C-fiber CAPs was significantly increased in nerves ipsilateral to SNE (p < 0.05, two-way ANOVA with Tukey post-hoc) (Fig. 2A, B).

At two weeks after SNE injury, NaV1.8-like immunoreactivity in the ipsilateral L4 and L5 dorsal root ganglia (DRG) was reduced (Fig. 3A, B). Specifically, the staining intensities of NaV1.8+ small-, medium-, and large diameter neurons were all significantly decreased (p < 0.05, one-way ANOVA with Tukey post-hoc; Fig. 3B). In marked contrast to the DRG, NaV1.8-like immunoreactivity increased more than two-fold in ipsilateral nerves (p < 0.05, t-test; Fig. 3C, D).

Real-time PCR detected the presence of NaV1.8 mRNA in the sciatic nerve and revealed a significant up-regulation in the ipsilateral nerve as compared to the contralateral and uninjured nerve (p < 0.05, one-way ANOVA with Tukey post-hoc; Fig. 4A). In contrast, ipsilateral, contralateral, and uninjured DRG showed no significant differences in NaV1.8 mRNA levels (Fig. 4B). To confirm that the NaV1.8 PCR signals represent mRNA and not genomic DNA contamination, 3'RACE PCR was performed. Southern blot hybridization analysis showed a positive band at the predicted size of 830 bp in both contralateral and ipsilateral sciatic nerve samples (Fig. 4C). After agarose gel separation and cloning of amplicons, DNA sequencing positively identified the 3' end of the coding region and the 3' untranslated region of rat Nav1.8 as well as an additional poly A tail (data not shown).

We hypothesized that if sciatic nerve NaV1.8 mRNA was of non-neuronal origin, it would be detectable in cultures of dissociated sciatic nerves where there are no neuronal somata and neuronal axons have degenerated. No real-time PCR signal was detected for NaV1.8 in sciatic nerve cultures (Fig 5A, C), whereas robust NaV1.8 expression was observed in DRG cultures that served as a positive control (Fig. 5B, C). However, similar signals for 18s rRNA were detected in the sciatic nerve and DRG cultures (Fig. 5D), suggesting similar concentrations of template cDNA. Additionally, we were able to detect strong expression of s100B, a Schwann cell marker, in both sciatic nerve and DRG cultures, with Ct values ranging from 26–30. Interestingly, although s100B expression was strong in nerve cultures, it was still stronger in DRG cultures (Fig. 5E).

After confirmation of the presence of NaV1.8 mRNA in the sciatic nerve, we compared steady-state levels of mRNA for NaV1.3, NaV1.5, NaV1.6, NaV1.7, NaV1.8, and NaV1.9 between ipsilateral and contralateral segments of the sciatic nerve immediately proximal to the injury site (Fig. 6A). Ipsilateral NaV1.8 mRNA was significantly up-regulated by 10.8-fold (p < 0.05, Mann-Whitney). Ipsilateral NaV1.6 mRNA expression was increased by 3.48-fold, but this difference did not reach statistical significance (p > 0.05, Mann-Whitney). None of the other sodium channels showed significant up-regulation in the sciatic nerve (p > 0.05, Mann-Whitney). We further examined mRNA expression of the same sodium channels in the DRG at the same time point. Ipsilateral NaV1.5, NaV1.6, NaV1.7, and NaV1.9 mRNA levels decreased by 28.6%, 39.8%, 54.1%, and 54.9%, respectively, however only the decrease was only statistically significant for NaV1.7 (p < 0.05, Mann-Whitney; Fig. 6B).

To investigate potential inhibition of peripheral NaV1.8 mRNA degradation by polyadenylation, we conducted the polyA tail length assay as originally described by Salles and Strickland 23. NaV1.8 mRNA isolated from both injured nerve and uninjured DRG primarily exhibited a discrete polyA tail of about 50 bases, with additional polyadenylation occurring out to 200 bases (Fig. 7). However, there were no apparent differences in the maximum polyA tail length or smearing pattern.

Surgery was performed as described previously 22 using adult male Sprague-Dawley rats weighing 200–250 g. Briefly, three Tygon^® cuffs (length = 1 mm, outer diameter = 2.28 mm, inner diameter = 0.76 mm) were placed around the exposed sciatic nerve proximal to the trifurcation.

At 2 weeks after injury, rats were anesthetized with pentobarbital (100 mg/kg, i.p.) and fixed by intracardiac perfusion of 500 ml ice-cold 4% paraformaldehyde and 0.14% picric acid in 0.1 M phosphate buffer (PB), pH 7.4. Ipsilateral and contralateral L4 and L5 DRG were harvested along with sciatic nerve segments. Tissue was post-fixed for 24 hrs and cryoprotected overnight in 30% sucrose in 0.1 M PB. Samples were mounted in Tissue-Tek (Sakura Finetek USA, Inc., Torrance, CA), and cryosectioned at 25 μm (DRG) or 20 μm (sciatic nerve). Free-floating sections were incubated overnight with an anti-NaV1.8 antibody (1:2000 dilution, in PBS containing 5% NGS and 0.3% Triton X-100). The NaV1.8 antibody, which was previously extensively characterized, ¹⁵ was generously provided by Dr. John Wood, London, UK. Sections were then incubated in a FITC-labeled goat anti-rabbit secondary antibody (Molecular Probes, Carlsbad, CA) at 1:500 dilution. The sections were mounted in an anti-fade medium (ProLong, Molecular Probes, Carlsbad, CA). To minimize variability between specimens, ipsilateral and contralateral tissues were processed simultaneously and representative sections grouped on the same slides.

Confocal fluorescence images were taken at 20× magnification on a Leica TCS-SP confocal fixed-stage upright microscope (Heidelberg, Germany) equipped with argon (488 nm excitation: JDS Uniphase) and DPSS diode (561 nm excitation: Melles-Griot) lasers. All laser excitation and detection parameters were kept constant across all samples. Image stacks were averaged and given identical high and low intensity thresholds using LCS lite software (Leica, Heidelberg, Germany). For DRG images, neuronal area was measured by outlining NaV1.8 immunoreactive somata and then calculating the area with ImageJ software http://rsb.info.nih.gov/ij/. Fluorescence intensity was measured by outlining the cytosolic region of NaV1.8 immunoreactive somata; outlines were in the shape of an annulus, to exclude the darker nuclei. Average cytosolic fluorescence intensity was then measured using ImageJ software. When overlapping NaV1.8-immunoreactive somata were detected, the overlapping regions were excluded from intensity analysis. For sciatic nerve analyses, 1–2 111 × 320 pixel rectangles were drawn on each image such they did not cover any gap, break, or artifact. The average intensity within each rectangle was measured using ImageJ software. For both DRG and nerve sections, output intensity values were on a 0–255 scale based on the 255 shades of grey present in the image, with 0 representing black and 255 representing white. The output numerical intensity value was defined as "relative fluorescence."

At 2 weeks after injury, rats were anesthetized with sodium pentobarbital (50 mg/kg) and L4 and L5 DRG were excised with their dorsal roots, the ventral roots, the spinal nerves and a length of attached sciatic nerves cut just proximal to the site of entrapment. The preparations were further trimmed at 0–4°C in the low-Na⁺ artificial cerebrospinal fluid (ACSF) composed of (in mM): NaCl, 62; KCl, 3.5; NaH₂PO₄, 1.25; CaCl₂, 2; MgCl₂, 2; NaHCO₃, 26; glucose, 10 and sucrose, 124. Isolated DRG and sciatic nerve sections were then placed in cryogenic vials, frozen in liquid nitrogen and stered at -80°C. Alternatively, sections of sciatic nerve were then transferred to normal ACSF (20–23°C) composed of (in mM): NaCl, 124; KCl, 3.5; NaH₂PO₄, 1.25; CaCl₂, 2; MgCl₂, 2; NaHCO₃, 26 and glucose, 10. The ACSF was continuously bubbled with a 95/5% mixture of O₂/CO₂. For recording, preparations were transferred to a custom recording chamber (0.3 ml volume) that was perfused (2.7 ml/min) with oxygenated ACSF at 34.5°C. TTX (0.1–1 μM, Sigma) was dissolved and applied in ACSF.

Compound action potentials (CAPs) were recorded from the sciatic nerves using a suction recording electrode 5051. Signals from the recording electrode and the artifact suppression electrode were fed into a differential amplifier (Dam 50, WPI, Sarasota, FL) in AC mode. A calibration pulse (± 2 V across a 100 MΩ series resistor) was always included in the CAP recordings to estimate the resistance at the nerve/recording electrode junction 5051. Signals from CAP recordings were further amplified (Brownlee Precision Instruments, model 440, Santa Clara, CA). Amplified signals were digitized at 10–20 kHz via the Digidata 1200B interface (Molecular Devices, Union City, CA) and displayed on a computer screen using the pCLAMP 8 software package (Molecular Devices). Data were analyzed offline using the pCLAMP 8 software package (Molecular Devices). Since CAP amplitude/area varies linearly with changes in this resistance 5051, the CAP signal was adjusted based on the calibration pulse amplitude changes during analysis of any given recording. All data are presented as mean ± standard error of the mean (SEM).

Sciatic nerve sections and lumbar DRG were extracted as above and cut into small pieces in ice-cold Hank's balanced salt solution (HBSS) with 20% fetal bovine serum (FBS). The tissue was incubated for 75 min at 37°C in HBSS with 0.125% collagenase P (Roche, Indianapolis, IN), and 0.05% DNAse. 0.25% trypsin was added for 5 min, after which the tissue was centrifuged for 5 min at 2000 rpm. After washing with 20% FBS in HBSS, the cell pellet was resuspended in HBSS containing 0.295% MgSO₄ and 0.05% DNAse. The tissue was then triturated 10 times with a fire-polished siliconized Pasteur pipette and centrifuged for 5 min at 2000 rpm. The resulting cell pellet was resuspended in 1 ml of F12 nutrient mixture with 10% FBS, and 300 μl were plated into each of 3 wells of a 12-well cell culture plate. The plate was incubated at 37°C for 1 hr, and then another 700 μl of medium was added. The medium was then changed every 2 days. After 4 days, culture plates were washed in PBS, snap-frozen in liquid nitrogen, and then stored at -80°C. For RNA extraction, culture plates were thawed, and 350 μl of buffer RLT from the RNEasy Plus Mini Kit (Qiagen, Valencia, CA) were immediately added to each well. Wells were incubated for 15 minutes at room temperatue and scraped with pipette tips. The buffer RLT containing scraped cells from each well was then placed into separate RNAse-free 1.5 mL microcentrifuge tubes. The RNEasy Plus protocol with gDNA eliminator columns was then followed for RNA extraction.

At 2 weeks after injury, rats were anesthetized (50 mg/kg pentobarbital, i.p.), and ipsilateral L4 and L5 DRG were harvested together with sciatic nerve segments. RNA was extracted using a combination of phenol/chloroform extraction and glass fiber purification (Trizol, Invitrogen, Carlsbad, CA, and RNAqueous, Ambion, Austin, TX), DNAse treated (Ambion, Austin, TX), and reverse-transcribed (Superscript III, Invitrogen, Carlsbad, CA) using random hexamer primers. Samples were subjected in triplicate to real-time PCR under default cDNA conditions (Applied Biosystems, Foster City, CA) using a ready-made 18s rRNA primer and probe set (Applied Biosystems, Foster City, CA) as an internal control. Ready-made Taqman expression assay sets (Applied Biosystems, Foster City, CA) were used for PCR amplification of various sodium channel cDNAs; the expression assay IDs were: NaV1.3, Rn00565438_m1; NaV1.5, Rn00565502_m1; NaV.16, Rn00570506_m1; NaV1.7, Rn00581647_m1; NaV1.8, Rn_00568393; NaV1.9, Rn00570487_m1.

At 2 weeks after injury, total RNA (800 ng) extracted separately from ipsilateral and contralateral sciatic nerve was converted to first strand cDNA and then subjected to 3'RACE PCR using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA). The anchoring primer for the 3'RACE PCR reaction was 5'-GTTTATGGCGACAATCTCTCCAAAG-3' (Tm = 65°C), located approximately 830 bp upstream of the NaV1.8 mRNA polyA tail. Approximately 18 ng of first strand cDNA template was consistently used for all samples. Samples were denatured at 94°C for 30 sec, annealed at 68°C for 30 sec, and elongated at 72°C for 3 min. First strand cDNA was amplified for 35 cycles with the anchoring primer and oligo d(T) primer. Southern blot hybridization analysis was used to identify positive PCR amplicons containing NaV1.8 3'end sequence. The DIG-labeled DNA probe against the NaV1.8 3' end coding sequence was synthesized using the PCR DIG Probe Synthesis Kit (Roche, Indianapolis, IN) with forward primer 5'-GGAAGACCTCTCAGCCACAG-3' and reverse primer 5'-CAGGGTGTTGGAGAGTGTCA-3'. After hybridization, the membrane was washed, blocked, incubated with anti-DIG antibody, and detected according to the manufacturer's instructions. Chemiluminescence was detected by a LAS-3000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). 18S ribosomal RNA was amplified from the contralateral and ipsilateral sciatic nerve RNA samples for use as an internal control during PCR.

At 2 weeks after injury, total RNA was extracted as described above. Reverse transcription was performed using 5'-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3' as the primer. Reaction mixtures were prepared on a block heated to 94°C and then subjected to 35 cycles of PCR amplification using high fidelity Ex Taq polymerase (Takara, Otsu, Japan). Each cycle consisted of 30 sec denaturation, 1 min annealing, and 2 min extension. The primers were 5'-ACTCCTCCATTCTGACGTCCCTTCC-3' and 5'-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3,' and the annealing temperature was 68°C.

Digoxigenin-labeled probes were generated by PCR using a standard kit and protocol (Roche). The primers were 5'-TTACCGGCTGAGTGTCCATAACAGG-3' and 5'-CATTCTGACGTCCCTTCCGAGTTCC-3', and the annealing temperature was 66°C. PCR amplicons from the PolyA tail length assay were run on a 1.5% agarose gel, transferred and cross-linked to a positively charged nylon membrane by UV light. After probe hybridization, the membrane was washed and blocked with a standard kit (Roche, Indianapolis, IN), and 1:10000 Anti-Digoxigenin-AP conjugate was applied. The membrane was washed again and developed with CDP-Star (Roche, Indianapolis, IN). The membrane was exposed for 0.5–15 min and developed.

Ipsilateral and contralateral data sets were checked for normality using Shapiro-Wilkes and Liliefors tests, with p < 0.05 accepted as a non-normal distribution. Comparisons of two groups with normal distributions were made using Student's t-test. Comparisons of a non-normally distributed group with any other group were made using the Mann-Whitney U test. Comparisons of more than two groups, all of which were normally distributed, were made using 1-way ANOVA with Tukey post-hoc tests for pairwise comparisons. Two-way ANOVA with Tukey post-hoc tests was used for behavioral and electrophysiological data where a second variable (i.e. stimulus strength or time point) was analyzed simultaneously with laterality (i.e. ipsilateral vs. contralateral).