Male Sprague–Dawley rats (250–300 g, Harlan Bioproducts for Science, Indianapolis, IN) were housed in cages on a standard 12:12 h light/dark cycle. Water and food were available ad libitum until rats were transported to the laboratory approximately 1 h before experiments. All experimental procedures received prior approval from the Animal Care and Use Committee at the Johns Hopkins University. Animal procedures were consistent with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain and the ethical guidelines to investigate experimental pain in a conscious animal. Efforts were made to minimize animal suffering and to reduce the number of animals used. The experimenters were blind to the treatment condition during the behavioral testing.
DRG microinjection was carried out as described [17–19]. Briefly, the DRG was exposed by laminectomy. A midline incision was made in the lower lumbar back region, and the L5 vertebral body was exposed. The lamina was then removed with small ronguers. After the DRG was exposed, viral solution (2 μl/DRG, 1012 particles/ml) was injected into one site in the L5 DRG with a glass micropipette connected to a Hamilton syringe. The pipette was removed 10 min after injection. The surgical field was irrigated with sterile saline and the skin incision closed with wound clips.
L5 SNL-induced neuropathic pain model
L5 SNL was carried out according to our previous protocols [20–23]. Briefly, after the rats were anesthetized with isoflurane, the left L6 transverse process was removed to expose the L4 and L5 spinal nerves. The L5 spinal nerve was then carefully isolated, tightly ligated with 3–0 silk thread, and transected just distal to the ligature. The surgical procedure for the sham group was identical to that of the SNL group, except that the spinal nerve was not transected or ligated.
Sciatic nerve axotomy
After the rats were anesthetized with isoflurane, the left sciatic nerve was exposed, and the nerve was cut at a point approximately 1 cm distal to the exit point of spinal nerve roots according to the method described previously . Proximal and distal stumps were separated to ensure full transection. In the sham group, the surgical procedure was identical, except that the sciatic nerve was not transected.
Capsaicin-induced acute pain
The experimental procedure used for the capsaicin test was carried out as described previously . Briefly, fifty μl of vehicle (0.7% alcohol) or capsaicin (2 μg) was injected intradermally under the dorsal surface of the rat left hindpaw by use of a microsyringe with a 26-gauge needle. The amount of time that animals spent licking and/or lifting the injected paw was measured with a stopwatch and was considered as an indicator of the nocifensive response. The animal was observed individually for 5 min immediately after the injection of vehicle or capsaicin. Mechanical, cold, and thermal tests as described below were carried out 30 min after the injection of vehicle or capsaicin.
Mechanical paw withdrawal thresholds were measured with the up–down testing paradigm 1 day before surgery and on days 3, 7, and 14 after SNL or sham surgery according to our previous reports [20–22]. Briefly, the rat was placed in a Plexiglas chamber on an elevated mesh screen. Von Frey hairs in log increments of force (0.407, 0.692, 1.202, 2.041, 3.63, 5.495, 8.511, 15.14 g) were applied to the plantar surface of the left and right hind paws. The 2.041-g stimulus was applied first. If a positive response occurred, the next smaller von Frey hair was used; if a negative response was observed, the next higher von Frey hair was used. The test was ended when (i) a negative response was obtained with the 15.14-g hair, (ii) four stimuli were applied after the first positive response, or (iii) nine stimuli were applied to one hind paw.
Paw withdrawal latencies to cold were measured with a cold plate, the temperature of which was monitored continuously. A differential thermocouple thermometer (Harvard Apparatus, South Natick, MA) attached to the plate provided temperature precision of 0.1°C. Each animal was placed in a Plexiglas chamber on the cold plate, which was set at 0°C. The length of time between the placement of the hind paw on the plate and the animal jumping, with or without paw licking and flinching, was defined as the paw withdrawal latency. Each trial was repeated three times at 10-min intervals for the paw on the ipsilateral side. A cutoff time of 60 s was used to avoid paw tissue damage.
Paw withdrawal latencies to heat were measured with a Model 336 Analgesia Meter (IITC Life Science Instruments, Woodland Hills, CA, USA). Each animal was placed in a Plexiglas chamber on a glass plate (at 25°C) located above a light box. Radiant heat was applied by aiming a beam of light through a hole in the light box through the glass plate to the middle of the plantar surface of each hind paw. When the animal lifts its paw in response to the heat, the light beam is turned off. The length of time between the start of the light beam and the foot lift was defined as the paw withdrawal latency. Each trial was repeated five times at 5-minute intervals for each paw. A cutoff time of 20 seconds was used to avoid paw tissue damage.
The following locomotor function tests were performed: (1) Placing reflex: The rat was held with the hind limbs slightly lower than the forelimbs, and the dorsal surfaces of the hind paws were brought into contact with the edge of a table. The experimenter recorded whether the hind paws were placed on the table surface reflexively; (2) Grasping reflex: The rat was placed on a wire grid and the experimenter recorded whether the hind paws grasped the wire on contact; (3) Righting reflex: The rat was placed on its back on a flat surface and the experimenter noted whether it immediately assumed the normal upright position. Scores for placing, grasping, and righting reflexes were based on counts of each normal reflex exhibited in five trials.
Plasmid construction, cell culture and transfection, and virus production
To construct the full-length Kv1.2 expression plasmid, total RNA was extracted from rat DRG tissue using Trizol. The reverse transcription was performed with specific primer (5′-GGGTGACTCTCATCTTTGGA-3′). Then the full-length sequence of Kv1.2 cDNA was amplified using primers (Forward: 5′-ATCCACCGGTGCCACCATGACAGTGGCTACC GGAGA-3′; Reverse: 3′-ATAGTTTAGCGGCCGCTCAGACATCAGTTAACATTTTGG-5′). The PCR products were subsequently digested using AgeI and NotI and cloned into the restriction sites of the proviral plasmids (pHpa-trs-SK, provided by Dr. R.J. Samulski, University of North Carolina, Chapel Hill) to replace enhanced GFP (EGFP) sequence. The cDNA sequence and recombinant clones were verified by using DNA sequencing. The resulting two vectors expressed EGFP and Kv1.2 RNA under the control of the cytomegalovirus promoter. AAV5 viral particles carrying the two cDNAs were produced at the University of North Carolina Vector Core.
Human embryonic kidney 293 (HEK293) cells (1 × 106) were seeded in 6-well culture plates and cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (10,000 U/ml and 10 mg/ml, respectively). After 24 hours of culture, 2.5 μg of the plasmids (pHpa-trs- Kv1.2 or pHpa-trs-EGFP) were transfected into the HEK-293 T cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. After the 4-day incubation, the cells were collected and subjected to Western blot analysis as described below.
Total RNA preparation and quantitative real-time RT-PCR
RNA extraction and real-time RT-PCR were performed according to our previously published protocols [20, 21]. Briefly, rats were decapitated, and bilateral L4/5 DRGs and L5 spinal cord were rapidly collected. To obtain enough RNA, L4 or L5 DRGs from one side of three rats per time point were pooled. Total RNA was extracted with the Trizol method (Invitrogen, Carlsbad, CA), precipitated with isopropanol, treated with RNase-free DNase I (1 μL/20 μL; Promega Corp., Madison, WI), and reverse-transcribed by using the Omniscript kit (Qiagen, Valencia, CA) with specific primers [Kv1.2: 5′-GGG TGA CTC TCA TCT TTG GA-3′, Kv1.2 AS: 5′-CGTCACACCTCCTGAGGACAG-3′, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, an internal control for normalization): 5′-GAG CAC AGG GTA CTT TAT TGA T -3′]. cDNA was amplified by real-time PCR by using the probes for Kv1.2/Kv1.2 AS (5′-/56-FAM/TGC TGT TGG AAT AGG TGT GGA AGG T/BHQ_1/-3′) (Integrated DNA Technologies, Coralville, IA), and GAPDH [probe/primers were obtained from Applied Biosystems (catalog number: 4331182), Foster City, CA]. The Kv1.2 PCR primer sequences were 5′-AGA AAG GGT CGG TGA AGG AGG T-3′ (forward) and 5′-GTG TGG CTT CTC TTT GAA TAC C-3′ (reverse). Kv1.2 AS PCR primer sequence were 5′-GTG TGG CTT CTC TTT GAA TAC C-3′ (forward) and 5′-AGA AAG GGT CGG TGA AGG AGG T-3′ (reverse). Real-time PCR for each sample was run in quadruplicate in a 20-μL reaction with TaqMan Universal PCR Master Mix (Applied Biosystems). Reactions were performed in an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). The amplification protocol was: 3 min at 95°C, followed by 45 cycles of 10 sec at 95° for denaturation and 45 sec at 58° for annealing and extension. Ratios of ipsilateral-side mRNA levels to contralateral-side mRNA levels were calculated by using the ΔCt method (2−ΔΔCt) at a threshold of 0.02. All data were normalized to GAPDH, which has been demonstrated to be stable even during peripheral nerve injury [20, 21, 25].
Western blot analysis
The protocol for Western blot analysis has been described previously [26, 27]. In brief, cultured cells and bilateral L5 DRGs were collected and rapidly frozen in liquid nitrogen. The cultured cells were sonicated and tissues homogenized in chilled lysis buffer (50 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 μM leupeptin). The crude homogenate was centrifuged at 4°C for 15 min at 1,000 × g. The supernatant was collected and the pellet (nuclei and debris fraction) discarded. After protein concentration was measured, the samples were heated at 99°C for 5 min and loaded onto a 4% stacking/7.5% separating SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA). The proteins were then electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, MA). The membranes were blocked with 3% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h and incubated with primary mouse anti-Kv1.2 (NeuroMab, Davis, CA), primary mouse anti-Kv1.4 (NeuroMab), or primary mouse anti-β-actin (Santa-Cruz Biotechnology, Santa Cruz, CA) overnight under gentle agitation. β-actin was used as a loading control. The proteins were detected by horseradish peroxidase-conjugated anti-mouse secondary antibody and visualized by chemiluminescence regents (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) and exposure to film. The intensity of blots was quantified with densitometry. The blot density from the contralateral side of the EGFP-treated group was set as 100%. The relative density values from the ipsilateral side of the EGFP group and the ipsilateral and contralateral sides of Kv1.2-treated groups after sham or SNL were determined by dividing the optical density values from these groups by the value of the contralateral side in the EGFP-treated group after they were normalized to the corresponding β-actin.
After being deeply anesthetized with isoflurane, the rats were perfused through the ascending aorta with 100 mL of 0.01 M phosphate-buffered saline (PBS, pH 7.4) followed by 400 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). L5 DRGs were removed, post-fixed in the same fixative for 2–4 h, and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer overnight at 4°C. Transverse sections (20 μm thickness) were cut on a cryostat. For single labeling, every fourth section was collected (at least 3–4 sections/DRG). For double labeling, five sets of sections (2–3 sections/set) were collected from each DRG by grouping every fifth serial section.
Single-label immunofluorescence histochemistry was carried out as described previously [28, 29]. After being blocked for 1 h at 37°C in PBS containing 10% goat serum and 0.3% TritonX-100, the sections were incubated alone with primary mouse monoclonal anti-Kv1.2 overnight at 4°C. The sections were then incubated with goat anti-mouse IgG conjugated with Cy2 (1:400, Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. Control experiments included substitution of normal mouse serum for the primary antiserum and omission of the primary antiserum. Finally, the sections were rinsed in 0.01 M PBS and mounted onto gelatin-coated glass slides. Cover slips were applied with a mixture of 50% glycerin and 2.5% triethylene diamine in 0.01 M PBS.
Double-label immunofluorescence histochemistry was carried out as described previously [28, 29]. Five sets of sections were incubated overnight at 4°C with primary mouse monoclonal anti-Kv1.2 and one each of the following primary antibodies: rabbit anti-NF200 (Sigma, St. Louis, MO), rabbit anti-P2X3 (Neuromics, Edina, MN), biotinylated IB4 (1:100, Sigma), rabbit anti- substance P (SP; Millipore), and rabbit anti- calcitonin gene-related peptide (CGRP; EMD, Gibbstown, NJ). The sections were then incubated for 1 h at 37°C with a mixture of goat anti-mouse IgG conjugated with Cy3 (1:400, Jackson ImmunoResearch) and donkey anti-rabbit IgG conjugated with Cy2 (1:400, Jackson ImmunoResearch) or with a mixture of goat anti-mouse IgG conjugated with Cy3 (1:400) and FITC-labeled avidin D (1:200, Sigma) for 1 h at 37°C. Control experiments as described above were performed in parallel. After the sections were rinsed in 0.01 M PBS, cover slips were applied as described above.
All immunofluorescence-labeled images were examined under a Nikon TE2000E fluorescence microscope (Nikon Co., Japan) and captured with a CCD spot camera. For single labeling, all labeled and unlabeled neurons with nuclei were counted. Cell profiles were outlined and cell area was calculated by using the imaging software Image-Pro Plus (Media Cybernetics, Silver Spring, MD). For double labeling, single-labeled and double-labeled neurons with nuclei were counted.
Whole-cell patch clamp recording
Kv1.2 current was recorded in HEK293 cells transfected with Kv1.2 or EGFP plasmid by using whole-cell patch clamp recording 3–4 days after transfection. Cover slips were placed into the perfusion chamber (Warner Instruments, Hamden, CT) positioned on the stage of a microscope (Nikon); Micropipettes were pulled from borosilicate glass with a micropipette puller (PP-830, Narishige, Japan), and the electrode resistances ranged from 2 to 4 MΩ. Cells were voltage-clamped via the whole-cell configuration of the patch clamp with an Axopatch-700B amplifier (Molecular Devices, Sunnyvale, CA). The intracellular pipette solution (ICS) contained (in mM): potassium gluconate 120, KCl 20, MgCl2 2, EGTA 10, HEPES 10, Mg-ATP 4 (pH = 7.3 with KOH, 310 mOsm). We minimized the Na+ and Ca2+ component in Kv current recording by using an extracellular solution composed of (in mM): choline-Cl 150, KCl 5, CdCl2 1, CaCl2 2, MgCl2 1, HEPES 10, glucose 10 (pH = 7.4 with Tris-base, 320 mOsm). Signals were filtered at 1 kHz and digitized by using a DigiData 1322A with pClamp 9.2 software (Molecular Devices). Series resistance was compensated by 60–80%. Cell membrane capacitances were acquired by reading the value for whole-cell capacitance compensation directly from the amplifier. An online P/ 4 leak subtraction was performed to eliminate leak current contribution. The data were stored on computer by a DigiData 1322A interface and were analyzed by the pCLAMP 9.2 software package (Molecular Devices).
The results from the behavioral tests, Western blot, and immunohistochemistry were statistically analyzed with a one-way or two-way analysis of variance (ANOVA). Data are presented as means ± SEM. When ANOVA showed a significant difference, pairwise comparisons between means were tested by the post hoc Tukey method. Significance was set at p < 0.05. The statistical software package SigmaStat (Systat, San Jose, CA) or GraphPad Prism 4.0 (GraphPad Software Inc., La Jolla, CA) was used to perform all statistical analyses.