Impaired neuropathic pain and preserved acute pain in rats overexpressing voltage-gated potassium channel subunit Kv1.2 in primary afferent neurons
© Fan et al.; licensee BioMed Central Ltd. 2014
Received: 17 November 2013
Accepted: 30 December 2013
Published: 29 January 2014
Voltage-gated potassium (Kv) channels are critical in controlling neuronal excitability and are involved in the induction of neuropathic pain. Therefore, Kv channels might be potential targets for prevention and/or treatment of this disorder. We reported here that a majority of dorsal root ganglion (DRG) neurons were positive for Kv channel alpha subunit Kv1.2. Most of them were large and medium, although there was a variety of sizes. Peripheral nerve injury caused by lumbar (L)5 spinal nerve ligation (SNL) produced a time-dependent reduction in the number of Kv1.2-positive neurons in the ipsilateral L5 DRG, but not in the contralateral L5 DRG. Such reduction was also observed in the ipsilateral L5 DRG on day 7 after sciatic nerve axotomy. Rescuing nerve injury-induced reduction of Kv1.2 in the injured L5 DRG attenuated the development and maintenance of SNL-induced pain hypersensitivity without affecting acute pain and locomotor function. This effect might be attributed to the prevention of SNL-induced upregulation of endogenous Kv1.2 antisense RNA, in addition to the increase in Kv1.2 protein expression, in the injured DRG. Our findings suggest that Kv1.2 may be a novel potential target for preventing and/or treating neuropathic pain.
Peripheral nerve injury often causes persistent neuropathic pain that is characterized by spontaneous ongoing or intermittent burning pain, allodynia, and hyperalgesia . Treatment options are limited in part because our understanding of the mechanisms that underlie the induction and maintenance of neuropathic pain is incomplete [2, 3]. Peripheral nerve injury causes several changes in the nervous system, including abnormal ectopic firing from neuromas and dorsal root ganglion (DRG) neurons [1, 4, 5]. These abnormal neuronal activities play a critical role in the development and maintenance of neuropathic pain.
DRG neurons express a variety of ion channels, including voltage-gated potassium (Kv) channels [6–8]. Kv channels are tetramers of superfamily-specific channel subunits that comprise ion-conducting integral protein α subunits and auxiliary cytoplasmic β subunits. More than a dozen α subunits of the Kv channel have been isolated from mammalian cells and divided into 12 subfamilies, Kv1–12; many subfamilies consist of more than one subunit [9, 10]. Because Kv channels are critical for establishing resting membrane potential and controlling neuronal excitability , changes in the expression levels and operating characteristics of Kv channels in the DRG after peripheral nerve injury may contribute to abnormal activities of DRG neurons in neuropathic pain.
The Kv1.2 subunit may participate in the formation of Kv channel tetramers in most DRG neurons. The mRNA for Kv1.1 and Kv1.2 is highly abundant, whereas that of Kv1.3, Kv1.4, Kv1.5, and Kv1.6 is present at lower levels in the DRG . Some controversy exists regarding the subpopulation distribution of Kv1.2 in rat DRG. For example, Ishikawa et al.  showed that Kv1.2 was expressed in small DRG neurons, whereas Rasband et al.  revealed that Kv1.2 was predominantly distributed in large DRG neurons. Interestingly, peripheral nerve injury down-regulated the expression of Kv1.2 in the injured DRG [12–15]. This down-regulation may be responsible for the nerve injury-induced increase in the ectopic discharge activity observed in DRG neurons . Therefore, DRG Kv.1.2 might be a potential target for neuropathic pain treatment.
In the present study, we first characterized the subpopulation distribution patterns and neurochemical properties of Kv1.2-positive cells. Second, we used immunohistochemistry to further examine the temporal change in the number of K v1.2-positive neurons in the injured DRG in two peripheral nerve injury models: lumbar (L)5 spinal nerve ligation (SNL) and sciatic nerve transection (axotomy). Finally, we tested whether rescuing Kv1.2 downregulation by over-expressing Kv1.2 RNA in the injured DRG affected SNL-induced neuropathic pain, acute pain, and locomotor functions.
Materials and methods
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.
Distribution of Kv1.2-positive neurons in DRG
Reduction in number of Kv1.2-positive neurons in the injured DRGs after peripheral nerve injury
To further verify the effect of SNL on the number of Kv1.2-positive neurons in the injured DRG, we examined Kv1.2 immunostaining in the ipsilateral L5 DRG on day 7 after sciatic nerve axotomy or sham surgery. As expected, sham surgery did not significantly change the number of Kv1.2-psotive neurons in the ipsilateral or contralateral L5 DRG (Figure 3D). However, as in the SNL model, the number of Kv1.2-positive neurons in the ipsilateral L5 DRG was reduced by 75.6% (p < 0.01; n = 4 rats) after axotomy compared to that after sham surgery (n = 4 rats) (Figure 3D).
Over-expressing Kv1.2 in the injured DRG blunted neuropathic pain without affecting basal nociceptive responses
Over-expressing Kv1.2 in the DRG did not alter capsaicin-induced acute pain
Over-expressing Kv1.2 RNA in the DRG did not affect locomotor functions
Mean (± SDM) changes in locomotor test
EGFP in naive
Kv1.2 in naive
EGFP in sham
Kv1.2 in sham
EGFP in SNL
Kv1.2 in SNL
Over-expressing Kv1.2 RNA in the DRG attenuated SNL-induced DRG Kv1.2 antisense (AS) RNA upregulation
Three major findings arise from the present study. First, Kv1.2 expressed in a majority of the large and medium DRG neurons is time-dependently downregulated in the injured DRG following peripheral nerve injury induced by L5 SNL and sciatic nerve axotomy. Second, rescuing this downregulation by over-expressing DRG Kv1.2 RNA blocked the development and maintenance of SNL-induced neuropathic pain. Finally, over-expressing DRG Kv1.2 did not affect basal acute pain, capsaicin-induced acute pain, and locomotor functions. These findings suggest that the DRG Kv1.2 channel may be a novel potential target for treating neuropathic pain.
Normal DRG expresses several Kv1 alpha subunits at various levels of basal expression. RT-PCR analysis showed that Kv1.1 and Kv1.2 mRNA was highly abundant, whereas Kv1.3, Kv1.4, Kv1.5, and Kv1.6 mRNA was present at lower levels . Immunohistochemistry further revealed that Kv1.1, Kv1.2, and Kv1.4 protein was detected highly in DRG neurons . In contrast, Kv1.3, Kv1.5, and Kv1.6 protein expression was very low or undetectable in the DRG . Consistent with these findings, we found that approximately 70% of DRG neurons expressed Kv1.2 protein in neuron profiles. These findings suggest that Kv1.2, Kv1.1, and Kv1.4 are key subunits in formation of heteromeric Kv channels in DRG neurons.
Kv1 alpha subunits are present in distinct classes of DRG neurons. An early study reported that Kv1.1 and Kv1.2 are expressed in small-sized DRG neurons . This finding was not confirmed in a subsequent study that showed Kv1.2 and Kv1.1 to be expressed predominantly in medium- and large-sized DRG neurons . We used specific cytochemical markers and the measurement of neuronal cell body area to further characterize Kv1.2 expression in functional classes of DRG neurons. In neuron profiles, approximately 72% of Kv1.2-positive neurons were large, 19% were medium, and 9% were small. Consistently, we found that most (80.3%) Kv1.2 co-localized with NF200, a marker for large, myelinated afferents, and some co-localized with P2X3 (11.11%) and CGRP (10.7%), markers of small nociceptive cells. Unexpectedly, few Kv1.2-positive neurons co-expressed two other markers of small nociceptive cells, IB4 (2.45%) and SP (3.97%). It is unclear why Kv1.2 showed different levels of co-localization with distinct nociceptive markers, but the observation may be related to different subpopulation distribution of these markers in rat DRG. CGRP and P2X3 are expressed not only in small DRG neurons but also in medium and/or large DRG neurons, whereas SP and IB4 are expressed exclusively in small DRG neurons [34–36]. In addition, CGRP is co-expressed with P2X3, but not with IB4, in some DRG neurons . Unique subpopulation distribution of Kv1.2 in DRG suggests a functional consequence of its down-regulation on pain-associated behaviors following peripheral nerve injury.
Nerve injury-induced Kv1.2 downregulation in the injured DRG may participate in neuropathic pain development and maintenance. Data from the present study and those of others [12–16, 38, 39] showed a time-dependent decrease in expression of Kv1.2 mRNA and protein in the injured DRG neurons following peripheral nerve injury. This decrease occurred predominantly in large and medium DRG neurons. We recently demonstrated that DRG Kv1.2 reduction in the large and medium DRG neurons decreased total voltage-gated potassium current, depolarized the resting membrane potential, decreased current threshold for activation of action potentials, increased the number of action potentials in these DRG neurons, and produced neuropathic pain symptoms . Blocking SNL-induced reduction of Kv1.2 expression in the injured DRG attenuated neuropathic pain during development and maintenance periods . The evidence indicates that DRG Kv1.2 is a key player in neuropathic pain genesis and may be a potential target for preventing and/or treating neuropathic pain. Consistent with this speculation, the present study demonstrated that rescuing Kv1.2 expression in the injured DRG diminished the induction and maintenance of SNL-induced mechanical, cold, and thermal pain hypersensitivities. These behavioral effects may be attributed to direct compensation of SNL-induced reduction of Kv1.2 protein and inhibition of SNL-induced upregulation of Kv1.2 AS RNA in the injured DRG. The latter may be related to the extensive overlap of complimentary regions between Kv1.2 mRNA and Kv1.2 AS RNA . Given that the nerve injury-induced increase in spontaneous ectopic activity in the injured myelinated afferents [40–42] is believed to play a leading role in the genesis of neuropathic pain [1, 43], rescuing Kv1.2 downregulation may maintain normal resting membrane potential and reduce abnormal ectopic activity in the injured DRG neurons. This effect may decrease primary afferent transmitter release and result in attenuation of spinal central sensitization formation and nerve injury-induced pain hypersensitivity. Interestingly, rescuing DRG Kv1.2 expression does not alter acute pain, which is supported by the observation that the altered DRG Kv1.2 expression did not change the threshold of action potential . Therefore, the strategy of rescuing DRG Kv1.2 expression may be a novel treatment for neuropathic pain.
This work was supported by the National Institutes of Health grants (MH084691 and GM078579) to M.L. and by the National Institutes of Health grants (NS072206, HL117684, DA033390) and the Rita Allen Foundation to Y.X.T.
- Campbell JN, Meyer RA: Mechanisms of neuropathic pain. Neuron 2006, 52: 77–92. 10.1016/j.neuron.2006.09.021PubMed CentralPubMedView ArticleGoogle Scholar
- Baron R, Binder A, Wasner G: Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Lancet Neurol 2010, 9: 807–819. 10.1016/S1474-4422(10)70143-5PubMedView ArticleGoogle Scholar
- Finnerup NB, Sindrup SH, Jensen TS: The evidence for pharmacological treatment of neuropathic pain. Pain 2010, 150: 573–581. 10.1016/j.pain.2010.06.019PubMedView ArticleGoogle Scholar
- Ji RR, Woolf CJ: Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain. Neurobiol Dis 2001, 8: 1–10. 10.1006/nbdi.2000.0360PubMedView ArticleGoogle Scholar
- Latremoliere A, Woolf CJ: Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain 2009, 10: 895–926. 10.1016/j.jpain.2009.06.012PubMed CentralPubMedView ArticleGoogle Scholar
- Altier C, Zamponi GW: Targeting Ca2+ channels to treat pain: T-type versus N-type. Trends Pharmacol Sci 2004, 25: 465–470. 10.1016/j.tips.2004.07.004PubMedView ArticleGoogle Scholar
- Shieh CC: Ion channels as therapeutic targets for neuropathic pain. Curr Pharm Des 2009, 15: 1709–1710. 10.2174/138161209788186317PubMedView ArticleGoogle Scholar
- Wang W, Gu J, Li YQ, Tao YX: Are voltage-gated sodium channels on the dorsal root ganglion involved in the development of neuropathic pain? Mol Pain 2011, 7: 16. 10.1186/1744-8069-7-16PubMed CentralPubMedView ArticleGoogle Scholar
- Coetzee WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, de Vega-Saenz ME, Rudy B: Molecular diversity of K + channels. Ann N Y Acad Sci 1999, 868: 233–285. 10.1111/j.1749-6632.1999.tb11293.xPubMedView ArticleGoogle Scholar
- Ocana M, Cendan CM, Cobos EJ, Entrena JM, Baeyens JM: Potassium channels and pain: present realities and future opportunities. Eur J Pharmacol 2004, 500: 203–219. 10.1016/j.ejphar.2004.07.026PubMedView ArticleGoogle Scholar
- Pongs O: Regulation of excitability by potassium channels. Results Probl Cell Differ 2008, 44: 145–161. 10.1007/400_2007_032PubMedView ArticleGoogle Scholar
- Yang EK, Takimoto K, Hayashi Y, de Groat WC, Yoshimura N: Altered expression of potassium channel subunit mRNA and alpha-dendrotoxin sensitivity of potassium currents in rat dorsal root ganglion neurons after axotomy. Neuroscience 2004, 123: 867–874. 10.1016/j.neuroscience.2003.11.014PubMedView ArticleGoogle Scholar
- Ishikawa K, Tanaka M, Black JA, Waxman SG: Changes in expression of voltage-gated potassium channels in dorsal root ganglion neurons following axotomy. Muscle Nerve 1999, 22: 502–507. 10.1002/(SICI)1097-4598(199904)22:4<502::AID-MUS12>3.0.CO;2-KPubMedView ArticleGoogle Scholar
- Rasband MN, Park EW, Vanderah TW, Lai J, Porreca F, Trimmer JS: Distinct potassium channels on pain-sensing neurons. Proc Natl Acad Sci USA 2001, 98: 13373–13378. 10.1073/pnas.231376298PubMed CentralPubMedView ArticleGoogle Scholar
- Kim DS, Choi JO, Rim HD, Cho HJ: Downregulation of voltage-gated potassium channel alpha gene expression in dorsal root ganglia following chronic constriction injury of the rat sciatic nerve. Brain Res Mol Brain Res 2002, 105: 146–152. 10.1016/S0169-328X(02)00388-1PubMedView ArticleGoogle Scholar
- Zhao X, Tang Z, Zhang H, Atianjoh FE, Zhao JY, Liang L, Wang W, Guan X, Kao SC, Tiwari V, Gao YJ, Hoffman PN, Cui H, Li M, Dong X, Tao YX: A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons. Nat Neurosci 2013, 16: 1024–1031. 10.1038/nn.3438PubMed CentralPubMedView ArticleGoogle Scholar
- Han PJ, Shukla S, Subramanian PS, Hoffman PN: Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity. Exp Neurol 2004, 189: 293–302. 10.1016/j.expneurol.2004.03.010PubMedView ArticleGoogle Scholar
- Xu Y, Gu Y, Wu P, Li GW, Huang LY: Efficiencies of transgene expression in nociceptive neurons through different routes of delivery of adeno-associated viral vectors. Hum Gene Ther 2003, 14: 897–906. 10.1089/104303403765701187PubMedView ArticleGoogle Scholar
- Xu Y, Gu Y, Xu GY, Wu P, Li GW, Huang LY: Adeno-associated viral transfer of opioid receptor gene to primary sensory neurons: a strategy to increase opioid antinociception. Proc Natl Acad Sci USA 2003, 100: 6204–6209. 10.1073/pnas.0930324100PubMed CentralPubMedView ArticleGoogle Scholar
- Guan X, Zhu X, Tao YX: Peripheral nerve injury up-regulates expression of interactor protein for cytohesin exchange factor 1 (IPCEF1) mRNA in rat dorsal root ganglion. Naunyn Schmiedebergs Arch Pharmacol 2009, 380: 459–463. 10.1007/s00210-009-0451-7PubMed CentralPubMedView ArticleGoogle Scholar
- Lee CY, Perez FM, Wang W, Guan X, Zhao X, Fisher JL, Guan Y, Sweitzer SM, Raja SN, Tao YX: Dynamic temporal and spatial regulation of mu opioid receptor expression in primary afferent neurons following spinal nerve injury. Eur J Pain 2011, 15: 669–675. 10.1016/j.ejpain.2010.11.018PubMed CentralPubMedView ArticleGoogle Scholar
- Singh OV, Yaster M, Xu JT, Guan Y, Guan X, Dharmarajan AM, Raja SN, Zeitlin PL, Tao YX: Proteome of synaptosome-associated proteins in spinal cord dorsal horn after peripheral nerve injury. Proteomics 2009, 9: 1241–1253. 10.1002/pmic.200800636PubMed CentralPubMedView ArticleGoogle Scholar
- Zhang B, Tao F, Liaw WJ, Bredt DS, Johns RA, Tao YX: Effect of knock down of spinal cord PSD-93/chapsin-110 on persistent pain induced by complete Freund’s adjuvant and peripheral nerve injury. Pain 2003, 106: 187–196. 10.1016/j.pain.2003.08.003PubMedView ArticleGoogle Scholar
- Lu YC, Chen CW, Wang SY, Wu FS: 17Beta-estradiol mediates the sex difference in capsaicin-induced nociception in rats. J Pharmacol Exp Ther 2009, 331: 1104–1110. 10.1124/jpet.109.158402PubMedView ArticleGoogle Scholar
- Jankowski MP, Lawson JJ, McIlwrath SL, Rau KK, Anderson CE, Albers KM, Koerber HR: Sensitization of cutaneous nociceptors after nerve transection and regeneration: possible role of target-derived neurotrophic factor signaling. J Neurosci 2009, 29: 1636–1647. 10.1523/JNEUROSCI.3474-08.2009PubMed CentralPubMedView ArticleGoogle Scholar
- Park JS, Voitenko N, Petralia RS, Guan X, Xu JT, Steinberg JP, Takamiya K, Sotnik A, Kopach O, Huganir RL, Tao YX: Persistent inflammation induces GluR2 internalization via NMDA receptor-triggered PKC activation in dorsal horn neurons. J Neurosci 2009, 29: 3206–3219. 10.1523/JNEUROSCI.4514-08.2009PubMed CentralPubMedView ArticleGoogle Scholar
- Park JS, Yaster M, Guan X, Xu JT, Shih MH, Guan Y, Raja SN, Tao YX: Role of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in complete Freund’s adjuvant-induced inflammatory pain. Mol Pain 2008, 4: 67. 10.1186/1744-8069-4-67PubMed CentralPubMedView ArticleGoogle Scholar
- Tao F, Liaw WJ, Zhang B, Yaster M, Rothstein JD, Johns RA, Tao YX: Evidence of neuronal excitatory amino acid carrier 1 expression in rat dorsal root ganglion neurons and their central terminals. Neuroscience 2004, 123: 1045–1051. 10.1016/j.neuroscience.2003.11.026PubMedView ArticleGoogle Scholar
- Xu JT, Zhao X, Yaster M, Tao YX: Expression and distribution of mTOR, p70S6K, 4E-BP1, and their phosphorylated counterparts in rat dorsal root ganglion and spinal cord dorsal horn. Brain Res 2010, 1336: 46–57.PubMedView ArticleGoogle Scholar
- Douglas CL, Vyazovskiy V, Southard T, Chiu SY, Messing A, Tononi G, Cirelli C: Sleep in Kcna2 knockout mice. BMC Biol 2007, 5: 42. 10.1186/1741-7007-5-42PubMed CentralPubMedView ArticleGoogle Scholar
- Castle NA, London DO, Creech C, Fajloun Z, Stocker JW, Sabatier JM: Maurotoxin: a potent inhibitor of intermediate conductance Ca2 + −activated potassium channels. Mol Pharmacol 2003, 63: 409–418. 10.1124/mol.63.2.409PubMedView ArticleGoogle Scholar
- Fulton S, Thibault D, Mendez JA, Lahaie N, Tirotta E, Borrelli E, Bouvier M, Tempel BL, Trudeau LE: Contribution of Kv1.2 voltage-gated potassium channel to D2 autoreceptor regulation of axonal dopamine overflow. J Biol Chem 2011, 286: 9360–9372. 10.1074/jbc.M110.153262PubMed CentralPubMedView ArticleGoogle Scholar
- Visan V, Fajloun Z, Sabatier JM, Grissmer S: Mapping of maurotoxin binding sites on hKv1.2, hKv1.3, and hIKCa1 channels. Mol Pharmacol 2004, 66: 1103–1112. 10.1124/mol.104.002774PubMedView ArticleGoogle Scholar
- Bradbury EJ, Burnstock G, McMahon SB: The expression of P2X3 purinoreceptors in sensory neurons: effects of axotomy and glial-derived neurotrophic factor. Mol Cell Neurosci 1998, 12: 256–268. 10.1006/mcne.1998.0719PubMedView ArticleGoogle Scholar
- Ji RR, Shi TJ, Xu ZQ, Zhang Q, Sakagami H, Tsubochi H, Kondo H, Hokfelt T: Ca2+/calmodulin-dependent protein kinase type IV in dorsal root ganglion: colocalization with peptides, axonal transport and effect of axotomy. Brain Res 1996, 721: 167–173. 10.1016/0006-8993(95)01316-4PubMedView ArticleGoogle Scholar
- Papka RE, McNeill DL: Coexistence of calcitonin gene-related peptide and galanin immunoreactivity in female rat pelvic and lumbosacral dorsal root ganglia. Peptides 1992, 13: 761–767. 10.1016/0196-9781(92)90184-5PubMedView ArticleGoogle Scholar
- Aoki Y, Takahashi Y, Ohtori S, Moriya H, Takahashi K: Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review. Life Sci 2004, 74: 2627–2642. 10.1016/j.lfs.2004.01.008PubMedView ArticleGoogle Scholar
- Kim DS, Lee SJ, Cho HJ: Differential usage of multiple brain-derived neurotrophic factor promoter in rat dorsal root ganglia following peripheral nerve injuries and inflammation. Brain Res Mol Brain Res 2001, 92: 167–171. 10.1016/S0169-328X(01)00154-1PubMedView ArticleGoogle Scholar
- Park SY, Choi JY, Kim RU, Lee YS, Cho HJ, Kim DS: Downregulation of voltage-gated potassium channel alpha gene expression by axotomy and neurotrophins in rat dorsal root ganglia. Mol Cells 2003, 16: 256–259.PubMedGoogle Scholar
- Liu CN, Michaelis M, Amir R, Devor M: Spinal nerve injury enhances subthreshold membrane potential oscillations in DRG neurons: relation to neuropathic pain. J Neurophysiol 2000, 84: 205–215.PubMedGoogle Scholar
- Liu CN, Wall PD, Ben-Dor E, Michaelis M, Amir R, Devor M: Tactile allodynia in the absence of C-fiber activation: altered firing properties of DRG neurons following spinal nerve injury. Pain 2000, 85: 503–521. 10.1016/S0304-3959(00)00251-7PubMedView ArticleGoogle Scholar
- Tal M, Wall PD, Devor M: Myelinated afferent fiber types that become spontaneously active and mechanosensitive following nerve transection in the rat. Brain Res 1999, 824: 218–223. 10.1016/S0006-8993(99)01190-7PubMedView ArticleGoogle Scholar
- Devor M: Ectopic discharge in Abeta afferents as a source of neuropathic pain. Exp Brain Res 2009, 196: 115–128. 10.1007/s00221-009-1724-6PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.