All animal procedures were conducted in accordance with the National Institutes of Health policy on the use of animals in research and the University of Saskatchewan animal care committee guidelines (protocol 19920164). A total of 32 young adult male Wistar rats (Charles River Laboratories, Wilmington, MA) weighing 250–300 g were used. Animals were anesthetized for surgery with sodium pentobarbital (Somnitol, 65 mg/kg; MTC Pharm, Cambridge, Ontario, Canada). Pre- and post-operative (for 24 h) subcutaneous injections of buprenorphine (Temgesic, 0.1–0.2 mg/kg) were given to alleviate any post-operative discomfort. To examine the effect of NT-3 on the expression of Nav
1.8 and Nav
1.9, 40 rats were used: 17 underwent 7 d unilateral CCI of the sciatic nerve (Bennett and Xie, 1988
), 3 received sham CCI surgeries whereby the sciatic nerve was exposed but not ligated, 17 received 7 d unilateral CCI with intrathecal infusion of NT-3 for the duration of the injury, and 3 received 7 d unilateral CCI with sham pump implantation whereby the dorsal roots were exposed, the dura opened as with the CCI + NT-3 procedure, but no pump was implanted.
NT-3 (generously supplied by Regeneron Pharmaceuticals, Tarrytown, NY) was delivered intrathecally for 7 d via mini-osmotic pumps (model 2001; Alza, Cupertino, CA) inserted at the lumbar sacral junction as per Verge et al. (1989a)
at a concentration and rate of 600 ng/μl/hr (Karchewski et al., 2002
) in a solution of PBS containing rat serum albumin (1 mg/ml), streptomycin (100 U/ml), and penicillin (100 U/ml). This dose of NT-3 was the minimum dose found to selectively reverse injury-associated gene expression in injured trkC-expressing neurons (Verge et al., 1996
; Jongsma Wallin et al., 2001
; Karchewski et al., 2002
). At the conclusion of the experiments, rats were killed, and tissue was dissected and processed for in situ
hybridization and/or immunohistochemistry as described below. Previous studies have demonstrated a lack of influence ipsilateral and contralateral to injury when vehicle is infused intrathecally (Verge et al., 1989a
; Verge et al., 1995
; Jongsma Wallin et al., 2001
; Wilson-Gerwing et al., 2005
In situ hybridization
Deeply anesthetized animals were perfused via the aorta with 0.1 M PBS, pH 7.4, followed by 4% paraformaldehyde in 0.1M PBS. The right and left L4 and L5 DRG were rapidly dissected, postfixed for 1 hour in the same fixative, and cryoprotected in 20% sucrose in 0.1M PBS overnight. Paired experimental and control tissues were mounted in the same cryomold (to ensure processing under identical conditions), covered with OCT compound (Tissue Tek; Miles Laboratories, Elkhart, IN, USA) and frozen in cooled isopentane. Transverse sections were cut at 6 μm on a Micron cryostat (Zeiss, Canada), thaw mounted onto Probe-On+ slides (Fisher Scientific, Edmonton, AB, Canada) and stored with desiccant at − 20 °C until hybridization.
Prior to hybridization, slides were air dried, fixed in 4% paraformaldehyde, and washed in 1X PBS. Sections were then treated with proteinase K (20 μg/ml) containing 10 ml 1M Tris-HCl (pH 7.6), 2 ml 0.5 M EDTA, 200 μl proteinase K stock (20 mg/ml) and 188 μl ddH20, rinsed in 1 X PBS, and post-fixed in 4% paraformaldehyde. Slides were then rinsed and dehydrated in ascending alcohols.
Oligonucleotide probes complementary to and selective for Nav
1.8 mRNA [complementary to bases 640–687 (Akopian et al., 1996
1.9 mRNA [complementary to bases 2811–2858 (Dib-Hajj et al., 1998
)] and p75 mRNA [complementary to bases 873–920 (Radeke et al., 1987
)] were synthesized (University of Calgary DNA services, Alberta, Canada). The probes were checked against the GenBank database (NIH) to ensure no greater than 60% homology was found to sequences other than the cognate transcript. The probes were labeled at the 3′-end with α-[35
S]dATP (New England Nuclear, Boston, MA, USA) using terminal deoxynucleotidyl-transferase (Amersham Pharmacia Biotech, Piscataway, NJ, USA) in a buffer containing 10 mM CoCl2
, 1 mM dithiothreitol, 300 mM Tris base and 1.4 M-potassium cacodylate (pH 7.2), and purified through Bio-Spin®
Disposable Chromatograph Columns (Bio-Rad laboratories, Hercules, CA, USA) containing 200 mg of NENSORB™
PREP Nucleic Acid Purification Resin (NEN®
, Boston, MA, USA). Dithiothreitol was added to a final concentration of 10 nM.
Hybridization was carried out according to published procedures (Dagerlind et al., 1992
) on a minimum of 5 slides/probe from each of the experimental and control groupings. Briefly, the sections were hybridized at 43 °C for 14–18 hours in a buffer containing 50% formamide (Sigma Aldrich, Oakville, ON, Canada), 4X SSC (1X SSC – 0.15 M NaCl, 0.015 sodium citrate), 1X Denhart’s solution (0.02% bovine serum albumin and 0.02% Ficoll), 1% sarcosyl (N-laurylsarcosine), 0.02 M phosphate buffer (pH 7.0), 10% dextran sulphate, 500 μg/ml heat-denatured sheared salmon sperm DNA, 200 mM dithiothreitol and 107
cpm/ml of probe. After hybridization, the slides were washed for 4 × 15 mins in 1X SSC at 55°C, dehydrated in ascending alcohols, processed for radioautography as per Karchewski et al., 2002
and exposed for 7 to 10 days before developing in D-19 (Kodak, Rochester, NY, USA).
Quantification and analysis
All slides from the 16 groupings of 7 d experimental and control animals were analyzed qualitatively and relative changes in hybridization signal from one group to another noted for sections mounted on the same slide to avoid bias due to the variance in hybridization signal observed from slide to slide. Representative slides were selected for further quantitative analysis. These slides had a similar number of neurons in all DRG sections. Photomontages were prepared and individual neurons with a visible nucleus were identified. Using a 40X light objective and a 2X optivar with an interactive computer-assisted image-analysis system (Richardson et al., 1989
), the cross-sectional area of individual neurons and the percentage of cytoplasmic area covered by silver grains was measured for each neuron with a visible nucleus in the DRG section. The area per grain was kept constant for all neurons and a correction for grain overlap was made to obtain a parameter linearly related to density of silver grains (Richardson et al., 1989
). Software for the image analysis system was Northern Eclipse, Version 7.0 (Empix Imaging, Mississauga, ON, Canada) and supplemented with Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA) and Prism 4.0 (Graph Pad Software, San Diego, CA). Cells were considered labeled if they had more than five times background levels of silver grains, as determined by averaging grain densities over defined areas of the neuropil devoid of positively labeled cell bodies. This criterion for determining labeled neuronal profiles correlates well with the identification of labeled versus unlabeled neurons as determined manually using a 63X oil immersion objective.
Analysis was performed in each instance on all neurons with a nucleus present in the section being quantified: for Nav
1.8 this represents 12 DRG sections or 2236 neuronal profiles (Intact: n = 3 animals, CCI: n = 3 animals, Intact + NT-3: n = 3 animals, CCI+NT-3: n = 3 animals); while for Nav
1.9 this represents 12 DRG sections or 2025 neuronal profiles (Intact: n = 3 animals, CCI: n = 3 animals, Intact + NT-3: n = 3 animals, CCI+NT-3: n = 3 animals). The contralateral intact DRG was used as an intact control as previous research from our lab has demonstrated that CCI does not induce bilateral hyperalgesia (Wilson-Gerwing et al., 2005
) and we have not discerned any qualitative differences between sham-operated or naive DRG and contralateral intact DRG with respect to Nav
1.8 or Nav
To determine whether alterations in the mean labeling index were significant, a nonparametric ANOVA test was employed (Kruskal-Wallis Test) since it could not be assumed that our data followed a Gaussian distribution. Following the Kruskal-Wallis test, Dunn’ Multiple Comparison test was used to determine significant differences between specific groups of data (p < 0.001). All statistical calculations were performed using Prism 4.0 (GraphPad Software, San Diego, CA).
Transverse 10 μm sections were cut on the cryostat, thaw-mounted onto Probe-ON+ slides (Fisher Scientific), and processed for immunohistochemistry. For Nav1.8: sections were washed in 0.1 M PBS, then permeabilized with 0.3% Triton X-100 in 0.1 M PBS for 45 minutes at room temperature. Sections were blocked overnight at 4 °C in 10% goat serum and 0.3% Triton X-100 in 0.1 M PBS, then incubated overnight at 4 °C with rabbit anti-Nav 1.8 Affinity Purified Polyclonal Antibody (1:200; Chemicon International, Temecula, CA, USA) diluted with 10% goat serum and 0.3% Triton X-100 in 0.1 M PBS. Sections were visualized with Alexa Fluor® 488 F(ab′)2 fragment of goat anti-rabbit IgG (H+L) (1:250; Molecular Probes, Eugene, OR, USA) in 2% goat serum in 0.1 M PBS for 2 hours at room temperature. Slides were washed and coverslipped with 50% glycerol/50% PBS. For Nav1.9: sections were washed in 0.1 M PBS, then permeabilized with 0.3% Triton X-100 in 0.1 M PBS for 45 minutes at room temperature. Sections were blocked overnight at 4 °C in 10% goat serum, 3% BSA, and 0.3% Triton X-100 in 0.1 M PBS, then incubated overnight at 4 °C with rabbit anti-Nav1.9 Affinity Purified Polyclonal Antibody (1:100; Chemicon International, Temecula, CA, USA) diluted with 10% goat serum, 3% BSA, and 0.3% Triton X-100 in 0.1 M PBS. Sections were visualized with Alexa Fluor® 546 goat anti-rabbit IgG (H+L), F(ab′)2 fragment conjugate (1:250; Molecular Probes, Eugene, OR, USA) in 2% goat serum for 2 hours at room temperature. Slides were washed and coverslipped with 50% glycerol/50% PBS. Control sections were processed in the same manner, but without the primary antibody. Results were visualized using a Zeiss Axioscope 50 microscope equipped with incident-light fluorescence optics and a digital camera.