relationshipofaxonalvoltage-gatedsodiumchannel1.8 (nav1.8 ... · 2012-04-20 · painful neuropathy...

Relationship of Axonal Voltage-gated Sodium Channel 1.8(NaV1.8) mRNA Accumulation to Sciatic Nerve Injury-inducedPainful Neuropathy in Rats*

Received for publication, May 17, 2011, and in revised form, September 27, 2011 Published, JBC Papers in Press, September 30, 2011, DOI 10.1074/jbc.M111.261701

Supanigar Ruangsri‡§¶, Audrey Lin‡, Yatendra Mulpuri§, Kyung Lee‡, Igor Spigelman§�, and Ichiro Nishimura‡§1

From the ‡Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials,and Hospital Dentistry, §Division of Oral Biology & Medicine, School of Dentistry, and �Brain Research Institute, UCLA, Los Angeles,California 90095 and the ¶Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand

Background: Painful neuropathy is an unsolved disease with increased voltage-gated sodium channel (NaV) activities.Results: NaV1.8 shRNA treatment attenuated injury-induced pain behavior and normalized the NaV1.8 mRNA levels in theaffected axons but not in somata of sensory neurons.Conclusion: Painful neuropathy may causally involve axonal NaV1.8 mRNA.Significance: Nerve injury may induce axonal NaV1.8 mRNA accumulation, which may offer a novel therapeutic target.

Painful peripheral neuropathy is a significant clinical prob-lem; however, its pathological mechanism and effective treat-ments remain elusive. Increased peripheral expression of tetro-dotoxin-resistant voltage-gated sodium channel 1.8 (NaV1.8)has been shown to associate with chronic pain symptoms inhumans and experimental animals. Sciatic nerve entrapment(SNE) injury was used to develop neuropathic pain symptoms inrats, resulting in increased NaV1.8 mRNA in the injured nervebut not in dorsal root ganglia (DRG).To study the role ofNaV1.8mRNA in the pathogenesis of SNE-induced painful neuropathy,NaV1.8 shRNA vector was delivered by subcutaneous injectionof cationized gelatin/plasmid DNA polyplex into the rat hind-paw and its subsequent retrograde transport via sciatic nerve toDRG. This in vivo NaV1.8 shRNA treatment reversibly andrepeatedly attenuated the SNE-induced pain symptoms, aneffect that became apparent following a distinct lag period of3–4 days and lasted for 4–6 days before returning to pretreat-ment levels. Surprisingly, apparent knockdown of NaV1.8mRNA occurred only in the injured nerve, not in the DRG, dur-ing the pain alleviation period. Levels of heteronuclear NaV1.8RNA were unaffected by SNE or shRNA treatments, suggestingthat transcription of the Scn10a gene encoding NaV1.8 wasunchanged. Based on these data, we postulate that increasedaxonal mRNA transport results in accumulation of functionalNaV1.8 protein in the injured nerve and the development ofpainful neuropathy symptoms. Thus, targeted delivery of agentsthat interfere with axonal NaV1.8 mRNA may represent effec-tive neuropathic pain treatments.

Injuries to peripheral nerves often cause chronic pain mani-festing as post-herpetic neuralgia, painful diabetic neuropathy,and painful post-traumatic neuroma. Chronic peripheral neu-ropathy affects �1.5% of the general population (1) and greatlyimpairs quality of life, because current treatments remainunsatisfactory for most patients. Painful neuropathies com-monly share clinical features such as light touch-evoked pain(allodynia) (2–5), burning sensation, exaggerated responses tonoxious stimuli (hyperalgesia), and either spontaneous orevoked unpleasant abnormal sensations (dysesthesia) (6).Although several pathologicalmechanismshave beenproposed(7–9), the precise molecular mechanisms contributing to thedevelopment and maintenance of peripheral neuropathy arestill elusive.Because the generation and propagation of action potentials

in sensory neurons depend on the activity of voltage-gatedsodium channels (VGSCs),2 it has been postulated that abnor-mal neuronal hyperexcitability caused by altered regulation ofVGSCs may play a key role in the molecular pathogenesis ofpainful neuropathy (10, 11). Altered expression of VGSCs thatare found in primary sensory neurons, e.g. NaV1.3, NaV1.7,NaV1.8, and NaV1.9, has been shown to associate at variousdegrees with human neuropathies (12–17) and animal neurop-athy models (18–21). Among the peripheral VGSC isoforms,NaV1.8 is the predominant tetrodotoxin-resistant sodiumchannel expressed exclusively in primary sensory neurons withparticularly high levels of expression in nociceptive neurons ofsmall- and medium-sized soma diameters (22). NaV1.8 isinvolved in nociceptive signaling through up-regulation ofchannel expression and kinetics after tissue inflammation andits contribution to action potential propagation in nociceptiveneurons (23, 24). After peripheral nerve injury, NaV1.8 appears

* This work was supported, in whole or in part, by National Institutes of HealthGrants NS049137 and DA023163. This work was also supported by a RoyalThai Government scholarship from Thailand, a UCLA School of Dentistryseed grant, and Research Facilities Improvement Program Grant C06RR014529 from NCRR, National Institutes of Health.

1 To whom correspondence should be addressed: The Weintraub Center forReconstructive Biotechnology, UCLA School of Dentistry, Box 951668, CHSB3-087 Los Angeles, CA 90095-1668. Tel.: 310-794-7612; Fax: 310-825-6345; E-mail: [email protected].

2 The abbreviations used are: VGSC, voltage-gated sodium channel; NaV, volt-age-gated sodium channel; SNE, sciatic nerve entrapment; DRG, dorsalroot ganglia; SNL, spinal nerve ligation; HBSS, Hanks’ balanced salt solu-tion; IB4, isolectin B4; CG, cationized gelatin; ANOVA, analysis of variance;LSD, least significant difference; hnRNA, heterogeneous nuclear RNA; CCI,chronic constriction injury.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 46, pp. 39836 –39847, November 18, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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to be redistributed preferentially to nerve axons (9, 25). Selec-tive pharmacological inhibition of NaV1.8 function (26) orNaV1.8 expression in DRG using intrathecal administration ofantisense oligodeoxynucleotides (27) has been shown todecrease mechanical allodynia and thermal hyperalgesia in ratswith spinal nerve ligation (SNL). These reports underscore therole of NaV1.8 in the pathogenesis; however, the molecularmechanism of how NaV1.8 develops and maintains painfulneuropathy is not yet fully established. Recently, we reportedthat not only NaV1.8 protein but also NaV1.8 mRNA was up-regulated in nerve axons after sciatic nerve entrapment (SNE)injury in rats (28). We have hypothesized that the NaV1.8mRNA in the injured axon may play an important role in thepathogenesis of pain behaviors. Here we tested this hypothesisby targeted suppression of NaV1.8 mRNA using shRNA deliv-ery to lumbar dorsal root ganglion (DRG) neurons in vivo. Wereport that shRNA-derived NaV1.8 knockdown attenuatedSNE-induced pain behaviors in rats after a distinct lag period. Aseries of mechanistic experiments suggested that the axonalaccumulation of NaV1.8 mRNA, possibly through an injury-activated subcellular transport system, plays a causal role in thedevelopment of painful neuropathy.

EXPERIMENTAL PROCEDURES

Animals—Adult Sprague-Dawley rats weighing 200–300 gwere used throughout. The animal experiments were per-formed in accordance with the guidance of the National Insti-tutes of Health on animal care and University of California atLos Angeles Animal Research Committee.NaV1.8 shRNA Plasmid DNA and Lentiviral Vector

Construction—Four siRNA target sequences to NaV1.8 mRNA(accession number NM_017247) were designed: shRNA1(nucleotides 1203–1221); shRNA2 (nucleotides 3407–3425);shRNA3 (nucleotides 6009–6027); and shRNA4 (nucleotides6033–6051). NaV1.8 shRNA1, 2, and 4 were previously pub-lished (29, 30), and shRNA3 was newly designed (Dharmacon,Lafayette, CO). Nucleotide BLAST verified that siRNAsequences were complimentary only to the Nav1.8 mRNA. Arandom shRNA served as a negative control: GCAGCAACTG-GACACGTGA. To create the short hairpin constructs, sensesiRNA sequences were linked to their antisense sequences by astem loop (TTCAAGAGA) (31). The 55-nucleotide sense oli-gonucleotides and the 59 nucleotides of antisense oligonucleo-tides containing additional nucleotides at 5� end for XhoI over-hang were subcloned into pLL3.7 (Lentilox 3.7; MIT,Cambridge, MA) (see Fig. 1A). The shRNA-expressing vectorcontained mouse U6 promoter for shRNA expression andCMV promoter for enhanced GFP expression. The pLL3.7-NaV1.8shRNA constructs were large scale-amplified, and eachlentiviral vector was produced by a triple transfection of threeplasmids: pLL3.7-NaV1.8 shRNA, p�8.9, and pVSVG.Culture of DRG Neurons—DRG neuron cultures (32) were

used to determine the efficiency and specificity of shRNA con-structs. Harvested rat lumbar DRGwere transferred to ice-coldHanks’ balanced salt solution (HBSS) containing 20% FBS andcut into small pieces. Tissues were washed with cold 20% FBS-HBSS and then HBSS before incubating in collagenase solution(1.25 mg of collagenase P/5 ml of HBSS and 0.2 mg of DNase

I/ml of HBSS) for 75 min. Next, 1.25 mg/ml trypsin and 0.5 mgof DNase I/ml in HBSS were added and incubated for 5 min at37 °C followed by wash with 20% FBS-HBSS followed by HBSS.After washing, the pellet was resuspended in dissociation solu-tion (12 mM MgSO4 and 0.2 mg of DNase I/ml of HBSS) andtriturated with a silicone-coated Pasteur pipette until the solu-tion appeared uniformly cloudy. After centrifugation, the cellpellet was collected, resuspended with 1000 ml of DRG culturemedium/well (10% FBS, 1% antibiotic-antimycotics, 0.5% of 1.5mg/ml uridine and 0.5% of 3.5 mg/ml floxuridine in minimumessential medium), and plated on a 6-well plate precoated withMatrigel (BectonDickinson, Franklin Lakes, NJ). The cells weresubjected to experiments 24 h after seeding. The medium waschanged every 2 days.NaV1.8 shRNA lentiviral vectors (1 mg of p24 � 5 � 107

infectious units in 293T cells/ml ofminimumessentialmediumwithout FBS), polybrene (8mg/ml), 1.5mg/ml uridine (final v/v0.5%), and 3.5 mg/ml floxuridine (final v/v 0.5%) were added toDRG cells and incubated for 24 h. DRG cells were washed andsupplemented with DRG culture medium. The cells were col-lected 48 h after transduction for FACS, RT-PCR, and immu-nocytochemistry analyses (see below).FACS Analysis—DRG cells were dissociated from the plate

with 2ml of trypsin solution. The cellswere collected and resus-pended with cold PBS. The cells were stained with 10 �g/ml ofisolectin B4 (IB4) conjugatedwithAlexa Fluor 647 (Invitrogen).Enhanced GFP and Alexa Fluor 647 were measured by FACS(EPICS Elite ESP; Beckman Coulter, Fullerton, CA).Quantitative Real Time PCR—Total RNA from DRG cul-

tures was isolated and treated with DNase I (Ambion, Austin,TX). The steady statemRNA levels ofNaV1.3, NaV1.6, NaV1.7,NaV1.8, and NaV1.9 were determined by TaqMan-based realtime PCR (RT-PCR): Nav1.3-Rn00565438_m1; Nav1.6-Rn00570506_m1, Nav1.7-Rn00581647_m1; NaV1.8-Rn_00568393_m1; and Nav1.9-Rn00570487_m1. The mRNAexpression levels were normalized using the comparative CTmethod.DRG Culture Immunocytochemistry—DRG culture medium

was decanted, and cultures were rinsed with PBS at 37 °C for 1min and fixed with 3.7% formaldehyde for 10 min. Next, thecells were treated with 0.2% Triton X-100 for 5 min and back-ground masking solution (ImageIT FX; Invitrogen) for 30 min,followed by incubationwith primary antibody against ratC-ter-minal NaV1.8 peptide at 4 °C overnight (1:200 dilution; Sigma)and followed by secondary antibody-conjugated with AlexaFluor 647 (Invitrogen) at room temperature for 1 h. After stain-ing with 10 �g/ml Alexa Fluor 594 conjugated with IB4 (Invit-rogen) at room temperature for 30 min, the slides were cover-slipped with mounting medium (ProLong Gold; Invitrogen). Aconfocal laser-scanning microscope (Carl Zeiss LSM 310) wasused to scan 1-�m focal layers of each specimen at 63� mag-nification. Individual sections were then digitally reconstructedand analyzed with digital software (ImageJ 1.43; National Insti-tutes of Health, Bethesda, MD).In Vivo Transfection with Cationized Gelatin (CG)/NaV1.8

shRNA Plasmid DNA Polyplexes—CG/shRNA plasmid DNApolyplex was prepared as previously described (32). Briefly,85–100 �l of CG/DNA polyplex injectates were prepared at

Axonal NaV1.8 mRNA and Painful Neuropathy

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7.5:1 CG-to-DNA mass ratio containing 17–25 �g/injection.The CG/DNA polyplex injectates were incubated at 37 °C for30 min prior to injection.The rats were anesthetized with isoflurane (2%), and the vec-

tor/DNA polyplex was slowly (�1 min) injected subcutane-ously into the center of the plantar surface of the left hindpawwith 27-gauge needles. The needle was removed, and the injec-tion site was immediately sealedwith liquid Band-Aid (Johnson& Johnson, New Brunswick, NJ).Detection of Synthesized NaV1.8 siRNA Molecules—DRG

and nerve tissues were harvested 2.5 and 7 days after in vivoinjection of CG/NaV1.8 shRNA polyplexes. Small-size RNAspecimens were prepared from DRG and nerves (mirVaNaTM

small RNA isolation kit; Ambion). Synthesized siRNA wasdetected by TaqMan based stem-loop RT-PCR (33). RNA (100ng) was used for reverse transcription with custom stem-loopprimer for siRNA3 with 100 mM dNTPs, 50 units/�l multi-scribe reverse transcriptase, 10� reverse transcriptase buffer,and 20 units/�l RNase inhibitor, followed by RT-PCR. 4.5 SRNA served as an endogenous control. Spiked samples ofsiRNA3molecules were obtained by adding known amounts ofsynthetic siRNA3 (500, 200, 100, 50, 10, and 1 pmol) to totalRNA of rat DRG and nerves. The amount of mature siRNA3from experimental tissues was based on a comparison of CTvalues between experimental and spiked samples using thestandard curve.In Vivo Efficiency of shRNA-derived NaV1.8 Knockdown—

DRG tissues were harvested 2.5 days after in vivo injection ofCG/shRNA3 polyplex and acutely dissociated as describedabove. DRG culture was subjected to immunostaining withNaV1.8 antibody andGFP antibody, as well as IB4 staining. TheNaV1.8 immunofluorescence intensity was determined inGFP�/IB4� and GFP�/IB4� neurons.Sciatic Nerve Entrapment—Surgical procedure for the SNE

modelwas described previously (34, 35). Briefly, in anesthetizedrats, the left sciatic nerve was surgically exposed, and three pol-yethylene cuffs (1 mm long, 2.28-mm outer diameter, and0.76-mm inner diameter) were loosely fitted to the sciatic nerveproximal to the trifurcation of common peroneal, tibial, andsural nerves (see Fig. 3A). Muscle and skin were separatelyclosed.Behavioral Testing—For a set of control and SNE rats, daily

measurements of hindpaw withdrawal thresholds to mechani-cal stimuli and withdrawal latencies to thermal stimuli wereobtained in both naïve controls and SNE rats as described indetail previously (35).In Vivo Transfection with CG/NaV1.8 shRNA Plasmid DNA

Polyplexes—After the stable pain behavior was established;CG/shRNA plasmid DNA polyplex was injected to the ipsilat-eral hindpaw as described above.DRG, Nerve, and Hindpaw Skin Harvested from SNE Rats—

After completion of behavioral testing, a set of SNE andshRNA-treated rats were anesthetized with pentobarbital (80mg/kg) and perfusion-fixed for histological processing of tis-sues. A different set of rats were anesthetized with isoflurane(3%) and tissues (DRG, sciatic nerve, and hindpaw skin) col-lected for molecular studies.

Immunohistochemistry of Nerve and DRG Tissue—The ratswere anesthetized with pentobarbital (80 mg/kg) and perfusedthrough the ascending aortawith 300ml of 0.9%NaCl, followedby 300 ml of ice-cold freshly prepared 4% (w/v) paraformalde-hyde in 0.1 M phosphate buffer, pH 7.4. The L4/L5 DRG andsciatic nerves proximal to the injury were harvested and post-fixed at 4 °C for 2–4 h. All DRG and sciatic nerves were cryo-protected through sucrose gradient concentrations in 0.1 M

phosphate buffer at 4 °C overnight and then embedded in thefrozen mold using tissue freezing medium (TFMTM; TriangleBiomedical Sciences, Durham, NC). Specimens were cryosec-tioned at 25 �m (DRG) or 20 �m (sciatic nerve) and mountedon gelatin-coated precleaned microscopic slides. To minimizevariability between specimens, ipsilateral and contralateral tis-sues were processed simultaneously. Four specimens from ipsi-lateral and contralateral tissues were mounted on the sameslide. For immunostaining, tissue sections were fixed in coldacetone (�20 °C) for 10 min, rinsed with Tris-buffered saline(TBS) four times (3, 5, 7, and 7min, respectively), and air-dried.The sections were incubated (30 min) in blocking solution (1%BSA � 2% normal donkey serum � 0.2% Tween) and thenincubated inmousemonoclonal antiserum targeting the C-ter-minal residues (positions 1724–1956) of NaV1.8 (1:500 dilu-tion; Neuromab, Davis, CA) at 4 °C overnight. Then sectionswere washed and incubated in secondary antibody (Alexa Fluor488-conjugated donkey anti-mouse antiserum; Invitrogen)(1:500 dilution) for 1 h at room temperature and then in IB4-AlexaFluor 594, 1:250 dilution) for 30 min at room tempera-ture. All of the sections were coverslipped with mountingmedium (Vectashield; Vector Laboratories, Burlingame, CA).The imageswere acquired using a Leica confocal SP2 1P-FCS

microscope (Leica Microsystems, Bannockburn, IL) and cap-tured using the same parameter settings (i.e. gain, pinhole,thickness of scanned images). For each DRG and nerve, foursections were used for confocal scanning. Captured imageswere 512 � 512 pixels. ImageJ software was used for quantita-tive analysis. DRG neurons positive to IB4 and with clearly vis-ible nuclei were included for analysis. Only cytoplasmic immu-noreactivity of NaV1.8 in IB4� DRG neurons was outlined forquantification, making each region of interest correspond tothe cytoplasmic profile of a singleDRGneuron. Pixel intensitiesof image NaV1.8 immunoreactivity ranged from 0 (darkest) to255 (lightest). The mean immunoreactivity for each cytoplas-mic profile of DRG cells and nerves was converted to relativeimmunoreactivity using the formula (36); [(mean immunoreac-tivity value � MIN)/(MAX � MIN)]�100, where MAX andMIN are the maximum andminimummean immunoreactivityvalues, respectively. Relative immunoreactivity values wereused to generate scatter plots. In addition, the mean immuno-reactivity from DRG neuron and nerve sections was used forplotting bar graphs. DRG neurons were sorted based on size�700�m2 and 700–1200�m2.DRGneurons1200�m2werenot included in the analyses because of the generally low levelsof NaV1.8 expression.Scn10a Gene Transcription—Total RNA was extracted from

collected tissues (DRG, sciatic nerve, and skin). To evaluate thetranscriptional activity of Scn10A gene (encoding NaV1.8), theNaV1.8 heteronuclear RNA (hnRNA, also called pre-mRNA)




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assay was performed as a surrogate measurement (37). Taq-Man-based primers were designed targeting the intronbetween exons 27 and 28 (5�-TCATGGCTTGAGACACT-GATTAGAC-3�) and exon 28 (5�-CAGTGACTTAAGGATT-GCAGAAAACA-3�). Total RNA (1 �g) from DRG of SNE-injured and shRNA-treated rats were reverse transcribed usinga random hexamer primer and subjected to RT-PCR analysisusing the hnRNA primer/probe set. The initial validation ofhnRNA RT-PCR assay was performed in vitro using acutelydissociatedDRGneuron cultures treatedwith different doses ofNGF (0, 10, 50, or 100 ng/ml) for 48 h.Quantitative Real Time PCR—The steady state NaV1.8

mRNA levels of DRG, sciatic nerve, and skin were determinedusing TaqMan-based RT-PCR. GAPDH served as internal con-trol. The data were expressed as a ratio of ipsilateral mRNAlevel over contralateralmRNA level within the same animals. Ina separate experiment, the steady statemRNA levels of NaV1.6,1.7, 1.8, and 1.9 were determined in DRG and nerve contralat-eral and ipsilateral to SNE.Statistical Analyses—Student’s t test for two-group compar-

ison and one- or two-way analysis of variance (ANOVA) withTukey’s multiple comparisons for multi-group comparisonwere used to analyze RT-PCR and immunohistochemistry datawith p � 0.05 accepted as statistically significant. Repeatedmeasures ANOVA analysis was used to compare hindpaw sen-sitivity to mechanical and thermal stimuli in naïve and SNE-injured rats. Ipsilateral and contralateral hindpaw data werestandardized by subtracting the ipsilateral and contralateraldata points to get absolute difference. For each animal, thebaseline value (i.e. average standardized score, post-SNE, or

preinjection) was subtracted from the corresponding value(absolute difference value) at each subsequent time point (post-injection) to adjust for any potential baseline differencebetween groups. The change from baseline means was com-pared parametrically using repeated measures ANOVA. Foreach outcome, estimated mean, S.E., and p value for betweengroup comparisons across time were reported. 5% least signif-icant difference (LSD) for within group comparison was calcu-lated. The mean difference between any two-time points mustbe larger than LSD.

RESULTS

shRNA-mediated NaV1.8 Knockdown in Vitro—We de-signed four RNA interference knockdown vectors containingU6 promoter-driven shRNA and CMV promoter-drivenenhancedGFP reporter (Fig. 1A). The efficacy and selectivity ofNaV1.8 mRNA knockdown was determined in cultured DRGneurons after lentivirus-assisted transduction of the shRNAvectors. Transduction efficiency assessed by detection ofenhanced GFP in IB4-positive neurons using FACS was deter-mined to be 69.5% (data not shown). SignificantNaV1.8mRNAknockdown was achieved by shRNA3 and shRNA4 (Fig. 1B).The effect of all shRNAs on other NaV isoforms (NaV1.3,NaV1.5, NaV1.6, NaV1.7, and NaV1.9) that share somesequence similarities with NaV1.8 was also examined. Off tar-get knockdown of NaV1.6 was observed with shRNA4, whereasshRNA1 decreased NaV1.3 mRNA (Fig. 1B). Thus, effectiveand specific NaV1.8 knockdownwas achieved only by shRNA3,which contained the 19-bp target sequence for siRNA withinthe coding region of NaV1.8 mRNA corresponding to nucleo-

FIGURE 1. Efficiency and specificity of RNA interference-derived knockdown of NaV1.8. A, third generation self-inactivating long terminal repeat (SIN-LTR)vector construct (Lentilox 3.7) carrying shRNA3 driven by the U6 promoter and the GFP reporter gene driven by the CMV promoter. B, RT-PCR for the steadystate mRNA levels of NaV1.3, NaV1.6, NaV1.7, NaV1.8, and NaV1.9 in acutely dissociated DRG neurons in vitro after transduction of lentiviral vectors carryingNaV1.8 shRNA 1, 2, 3, 4 or control random shRNA. *, p � 0.05 against random shRNA. C, NaV1.8 immunoreactivity of DRG neurons after shRNA3 and randomshRNA transduction. *, p � 0.05.




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tide positions 6009–6027 (CCATCTAGCTCAATGCAAA;accession number NM_017247). The reduction of NaV1.8 pro-tein expression by shRNA3 was measured and confirmed (p �0.05 from random shRNA) by immunohistochemistry of trans-fected IB4� DRG neurons in culture (Fig. 1C).In Vivo Administration of shRNA3 in Naïve Rats—We have

previously developed an in vivo nonviral gene transfer protocolwhereby subcutaneous injection of CG/plasmid DNA polyplexin the hindpaw resulted in its uptake, retrograde transport, andsubsequent expression in L4/L5 DRG neurons (Fig. 2A) (32).

This protocol was used to test the effects of the selectedshRNA3 construct in vivo. The shRNA3 plasmid DNA waspolyplexed with CG and subcutaneously injected into the rathindpaw.Based on previous evidence of reporter gene expression in

DRG at 2.5 and 6 days after hindpaw injection (32), we wantedto determine whether synthesis of the mature form of siRNA3molecules (21 nucleotides with 3�overhangs) would follow asimilar pattern. To determine the levels of synthesized siRNAmolecules, we designed a set of stem-loop RT primer, forward/reverse primers, and probe for TaqMan-based RT-PCR. Stand-ards were obtained by supplementing the total RNA samples ofnaïve DRG with 10, 50, 100, 200, or 500 pmol of syntheticsiRNA3 and subjecting them to stem-loop RT-PCR. There wasa linear correlation between the concentration of syntheticsiRNA3 and RT-PCR readings (R2 � 0.99), resulting in the for-mulation of a simple conversion equation (Fig. 2B). Using thismethod, �120 pmol of mature siRNA3 was detected in DRG2.5 days after the shRNA3 polyplex injection in naïve rats. Theamount of mature siRNA3 increased 2-fold in DRG collected 7days after injection (Fig. 2B). The amount of mature siRNA3 inthe sciatic nerve was relatively low and did not significantlychange between 2.5 and 7 days after injections.We further examined the effect of shRNA3 injection on

NaV1.8 immunoreactivity. L4/L5 DRGwere harvested 2.5 daysafter injection of CG/NaV1.8 shRNA polyplex and acutely dis-sociated. DRGwere cultured onMatrigel-coated glass chamberslides and immunocytology with NaV1.8 antibody, GFP anti-body and IB4 staining was performed. Analysis indicated thatNaV1.8 immunoreactivity of GFP�/IB4� DRG neurons wassignificantly reduced by �40% as compared with GFP�/IB4�

DRG neurons (Fig. 2C).In Vivo Administration of shRNA3 Does Not Affect Mechan-

ical or Thermal Sensitivity in Naïve Rats—In separate sets ofnaïve rats, daily behavioral testing of mechanical withdrawalthresholds and thermal withdrawal latency showed that neithermechanical nor thermal sensitivity was affected by unilateralinjections of NaV1.8 shRNA or injections of the control ran-dom sequence shRNA (Fig. 3B).In Vivo Administration of shRNA3 or shRNA4 Reverses SNE-

inducedNeuropathy Symptoms—Rats destined for SNE surgerywere monitored daily for mechanical withdrawal thresholdsand thermal withdrawal latencies. Once stable neuropathicpain symptoms were established, CG/shRNA3 polyplex wassubcutaneously injected in the hindpaw of one randomlyselected group (n � 9) ipsilateral to SNE, and the other groupreceived the CG/random shRNA injection (n � 10). After a lagperiod of 3–4 days, the mechanical withdrawal thresholds andthermal withdrawal latencies in the shRNA3-treated grouprapidly improved and reached their maximum levels 4–5 daysafter the shRNA3 injection (Fig. 3C). The uninjured contralat-eral side was used to standardize the threshold and latency, andthe LSDs of themean changes of threshold and latency from thebaseline (prior to the first injection) were determined using themultiple comparisons one-way ANOVA test. The LSD ofthe mechanical withdrawal threshold was achieved during the4-day period from 5 to 8 days after the injection, whereas theLSD of the thermal withdrawal latencywas achieved earlier and

FIGURE 2. RNA interference-derived knockdown of NaV1. 8 in DRG neu-rons in vivo. A, diagram of the retrograde gene transfer protocol using CGand plasmid DNA polyplex via subcutaneous peripheral injection of the rathindpaw. This protocol was used to deliver shRNAs in vivo. B, quantitativemeasurement of the mature siRNA synthesized from shRNA plasmid DNAusing stem-loop primer. The standard curve of relative siRNA3 expressioncorresponded to the known amount of siRNA3 added in DRG total RNA (left).Shown are the amounts of synthesized mature siRNA3 in DRG and nerve 2.5and 7 days after shRNA3 injection in naïve rats (n � 4, right). *, p � 0.05. C,NaV1.8 immunocytology of IB4-labeled DRG neurons acutely dissociated 2.5days after injection of the CG/NaV1.8 shRNA3 polyplex. Significantly reducedNaV1.8 immunoreactivity was observed in GFP�/IB4� neurons, comparedwith that in GFP�/IB4� neurons. *, p � 0.05.




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lasted 6 days. After the return of pain behaviors to preinjectionlevels (23 days post-SNE), a second injection achieved a similarmagnitude of pain alleviation (Fig. 3C). Importantly, no signs ofanaphylactic reaction or inflammation at the injection site weredetected in any of the rats.The random shRNA-treated group, to our surprise, pro-

duced a short period of pain alleviation after the first and thesecond injections (Fig. 3C). However, repeated measuresANOVA for the period between the first and second shRNAinjections revealed that the effect of shRNA3 was significantlydifferent from the random shRNA for the mechanical with-drawal thresholds (group p� 0.0001; time p� 0.0001; group�time p � 0.0001) and the thermal withdrawal latencies (groupp � 0.0370; time p � 0.0002; group � time p � 0.0006).

In a separate experiment, SNE-treated rats (n � 8) wereinjected with shRNA4 polyplexed with CG. Similar to shRNA3,there was a significant attenuation of neuropathic pain symp-

toms after shRNA4 (Fig. 3D). However, the onset of analgesiceffects occurred faster with shRNA4. Maximum analgesiceffects were observed after 2–3 days and lasted for 6 days(mechanical withdrawal thresholds) or 10 days (thermal with-drawal latencies). In addition, peak relief of mechanical allo-dynia symptoms was greater with shRNA4 (�75%) comparedwith shRNA3 (�50%).Effect of NaV1.8 shRNA3 Injection on NaV1.8 Protein

Expression—DRG and sciatic nerves were harvested duringmaximal suppression of neuropathy symptoms 12days after thesecond NaV1.8 shRNA3 injection. Analysis of NaV1.8 immu-noreactivity (NaV1.8-ir) in DRG sections co-stained with IB4(38) revealed a reduction of NaV1.8-ir in clusters of IB4� neu-rons (Fig. 4A). Scatter plot of NaV1.8-ir intensity measuredfrom a total of 857 IB4� DRGneurons demonstrated that thereare essentially two clusters: a high intensity cluster between 45and 60% and a low intensity cluster less than 40% (Fig. 4B). The

FIGURE 3. Reversal of SNE-induced neuropathy symptoms after the injection of NaV1. 8 shRNA or random shRNA. A, diagram of SNE-induced neuro-pathic pain model representing location of polyethylene cuffs. In some experiments, L4/L5 DRG and sciatic nerve tissues proximal to the SNE site wereharvested. B, hindpaw withdrawal thresholds (mean S.E.) to mechanical stimuli and hindpaw withdrawal latencies (mean S.E.) to thermal stimuli for theipsilateral and contralateral sides of two groups of naïve rats (n � 8 each) injected (day 0) with shRNA3 or random shRNA. C, changes in hindpaw withdrawalthresholds to mechanical stimuli (left) and withdrawal latencies to thermal stimuli (right) after unilateral SNE surgery (day 0) and after shRNA3 or random shRNAinjections (days 8 and 23). The data are presented as the means S.E. (n � 9 shRNA3, n � 10 random shRNA). D, changes in hindpaw withdrawal thresholds tomechanical stimuli (left) and withdrawal latencies to thermal stimuli (right) after unilateral SNE surgery (day 0) and after shRNA4 injection (day 8). The data arepresented as the means S.E. (n � 8) of differences between ipsilateral and contralateral thresholds and latencies. *, p � 0.05 (repeated measures ANOVA).




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high intensity cluster was composed of all groups, whereas thelow intensity cluster contained predominantly the SNE/shRNA3 group. The low intensity cluster accounted for 9.8–18.2% of neurons in the SNE/NaV1.8 shRNA group comparedwith only 1.5%of neurons from the SNE/randomshRNAgroup.The average NaV1.8 immunoreactivity was significantlydecreased in the SNE/shRNA3 group compared with SNE/ran-dom shRNA group of small (�700 �m2) and medium (700–1200 �m2) size IB4� DRG neurons (Fig. 4D). The expression of

NaV1.8 in larger diameter neurons (1200 �m2) was notincluded in this analysis because of their generally low levelNaV1.8 expression that could confound the measurements ofdecreases in NaV1.8 protein from shRNA3 injection.Sciatic nerve sections revealed a robust increase of NaV1.8-ir

in the SNE/random shRNA group, confirming previous find-ings of SNE-induced increases in NaV1.8-ir (28). By contrast,NaV1.8-ir in SNE-injured nerve was markedly reduced by theshRNA3 injection (Fig. 4A). Scatter plot of a total of 261 ran-

FIGURE 4. NaV1.8 immunostaining in DRG and nerve tissues harvested from SNE rats injected with either shRNA3 or random shRNA. A, NaV1.8immunoreactivity and IB4 staining in representative ipsilateral/contralateral sections from L4/L5 DRG and sciatic nerves proximal to the SNE site. Note thedecreased NaV1.8 immunoreactivity in DRG and nerve after shRNA3 injections compared with random shRNA injections. B, scatter plot of relative NaV1.8immunofluorescence intensity of IB4� L4/L5 DRG neurons ipsilateral (Ipsi) to shRNA3 injection (red triangles, n � 297 cells, four rats), random shRNA injection(green circles, n � 258 cells, four rats), and contralateral (Contra) DRG (blue diamonds, n � 302 cells, eight rats). C, scatter plot of relative NaV1.8 immunofluo-rescence intensity of nerve sections ipsilateral to shRNA3 injection (red triangles, n � 81 areas, 27 sections), ipsilateral to random shRNA injection (green circles,n � 102 areas, 34 sections), and in contralateral nerve (blue diamonds, n � 78 areas, 26 sections). D, average NaV1.8 immunofluorescence intensity of IB4� DRGneurons sorted by cell area � 700 and 700 –1200 �m2 (mean S.E.). *, p � 0.05. E, average NaV1.8 immunofluorescence intensity of nerve sections. *, p � 0.05.




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domly selectedmicroscopic fields of sciatic nerve illustrates theuniform increases in ipsilateral NaV1.8-ir and its reduction bythe shRNA3 injection to the range comparable with that of thecontralateral (uninjured) sciatic nerve (Fig. 4C). The averageNaV1.8-ir was significantly higher in the SNE-injured/randomshRNA-injected group than in the uninjured contralateralgroup. Injection of shRNA3 effectively attenuated the increasedNaV1.8-ir, although falling short of completely normalizing theaverage NaV1.8-ir to the level of uninjured contralateral sciaticnerves (Fig. 4E).Effect of shRNA3 Injection on NaV1.8 Gene Expression—We

first determinedwhether SNE- and shRNA-induced changes in

NaV1.8 were due to altered transcription of the Scn10A gene,which encodesNaV1.8. Because of difficulties experiencedwitha conventional nuclear run-off assay, we used an alternativemethod (37) in which heterogeneous nuclear RNA (hnRNA) orpre-mRNA of NaV1.8 was measured using primers designedacross the intron-exon boundaries of the Scn10A gene (Fig.5A). Measurements in the DRG of SNE rats (n � 4) revealedsimilar levels of NaV1.8 hnRNA among all groups, suggestingthat neither SNE nor shRNA injections affected the Scn10Agene transcription (Fig. 5B). Because NaV1.8 hnRNA was notdetected in sciatic nerve (Fig. 5B), local non-neuronal cells wereunlikely to be the source of NaV1.8 mRNA in sciatic nerves. In

FIGURE 5. Effects of SNE and shRNA3 treatment on NaV1. 8 gene transcription and steady state mRNA levels in DRG and nerve. A, diagram of relevantexon structures Scn10A encoding NaV1.8 and the locations of PCR primers. targeting NaV1.8 mRNA (forward primer in exon 14 and reverse primer in exon 15)and NaV1.8 hnRNA (forward primer-intron, reverse primer-exon 28). B, Scn10A gene transcription was measured by NaV1.8 hnRNA in SNE-treated rats withNaV1.8 shRNA (n � 4) or random shRNA (n � 4) injection. The data are presented with the contralateral tissue to SNE of the random shRNA group as thestandard. The transcriptional activity of Scn10a in DRG was not affected by SNE injury and the injection of NaV1.8 shRNA. There was no NaV1.8 hnRNA detectedin the nerve tissues as well as in the food pad tissue where cationized gelatin/plasmid DNA polyplex was injected. C, mRNA levels of NaV1.6, 1.7, 1.8, and 1.9 inthe DRG and sciatic nerve of the SNE rat model. The relative mRNA level was normalized by the housekeeping gene expression in DRG. *, p � 0.05. D, steadystate NaV1.8 mRNA levels in DRG and sciatic nerve harvested from naïve or SNE-injured rats treated with shRNA3 (black bars) or random shRNA (white bars).RT-PCR data were compared using the untreated contralateral tissue as the standard in each group (n � 4). *, p � 0.05. ipsi, ipsilateral; contra, contralateral.




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a separate group (n � 4) of SNE-treated rats without shRNAinjections, the steady state NaV1.8 mRNA in DRG was notaffected by SNE, whereas NaV1.8 mRNA, but not NaV1.6,NaV1.7, or NaV1.9 mRNA, was significantly increased in theaffected nerve (Fig. 5C), confirming previous findings (28).Next, we addressed the effect of the shRNA3 treatment on

the steady state NaV1.8 mRNA. L4/L5 DRG and sciatic nervetissues were harvested from naïve (n � 4) or SNE-injured (n �4) rats after shRNA3 or random shRNA injections. Naïve rattissues were obtained 11 days after shRNA injections. Tissuesfrom SNE rats were harvested 11 days after the second shRNA3injection, when alleviation of SNE-induced pain symptoms wasstill evident (Fig. 5D). Comparedwith naïve rats, SNEdecreasedthe steady state NaV1.8 mRNA levels in the DRG. Surprisingly,NaV1.8 mRNA levels in the DRGwere not affected by shRNA3injection as compared with the random shRNA injection (Fig.5D). However, the increasedNaV1.8mRNA level by SNE injuryin the nerve was significantly attenuated by the shRNA3 treat-ment to the levels observed in the naïve nerve (Fig. 5D).

DISCUSSION

The present study demonstrated that NaV1.8 knockdownusing peripheral administration of shRNA in the SNE-injuredrats resulted in significant reduction of neuropathic painbehaviors. This confirms and extends previous studies in whichpharmacological blockade of NaV1.8 function or expressionwas shown to decrease mechanical allodynia and thermalhyperalgesia in experimental neuropathies. The peripheralneuropathy induced by SNE is highly comparable with that ofchronic constriction injury (CCI), which uses chromic gutsuture materials instead of polyethylene cuffs (25, 39, 40). SNEwas demonstrated to produce consistent pain behaviors (34,41), a bona fide transient loss of varicosities in nociceptive fibers(42), and increases in evoked excitability of sciatic nerve com-pound action potentials (28). The increased excitability of theinjured sciatic nerve likely contributes to the ectopic activity inthe injured nerve and the exaggerated afterdischarge of widedynamic range neurons in the spinal cord evoked by mechani-cal cutaneous stimulation in this model (43). The hyperexcit-ability and ectopic burst discharge of primary sensory neuronsare widely considered to be the major contributors to painsymptomatology of peripheral neuropathy models.Although the behavioral symptoms of the SNE/CCI models

are superficially similar to the model of segmental deafferenta-tion induced by L5/L6 SNL (44), these models differ in manyimportant respects. In contrast to the SNE/CCI models, whereinjured and uninjured axons commingle in the sciatic nerve, thetight ligation (deafferentation) of L5/L6 spinal nerve segmentsin the SNL model results in complete segregation of the deaf-ferented neuronal somata in L5/L6 DRG and the “uninjured”somata and axons of L4 DRG neurons. The phenotypic altera-tions in SNL include large decreases inNaV1.8mRNA, protein,and function in the deafferented L5 DRG and large increases inNaV1.8 mRNA, protein, and function in the uninjured L4 DRG(45), concomitant with large increases in NaV1.8 protein, func-tion and resistance to tetrodotoxin blockade of action potentialconduction in the uninjured sciatic nerve axons (9, 27). By con-trast, the SNE/CCI produce modest decreases in NaV1.8

mRNAandproteinwithin L4/L5DRG (25, 28, 29) and increasesin immunohistochemical detection of axonal NaV1.8 protein(27, 28). Functionally, only modest increases in resistance totetrodotoxin conduction block are observed after SNE (28).Moreover, the demonstrated increases in sciatic nerve NaV1.8mRNA after SNE (28) and the lack of nerve NaV1.8 mRNAincreases after the L5 spinal nerve ligation3 suggest differencesin the molecular mechanisms of axonal NaV1.8 accumulationin the two models.Targeted Gene Delivery to the Sciatic Nerve via Subcutaneous

Injection of CG/DNAPolyplex—Weused a simple gene transferprotocol of subcutaneous CG/DNA polyplex injection in thehindpaw (32) to deliver shRNA plasmidDNA targeting NaV1.8mRNA to sciatic nerve, which resulted in retrograde transfec-tion and synthesis of mature siRNA molecules in L4/L5 DRG(Fig. 2B). Unlike other studies using intrathecal injections ofantisense oligodeoxynucleotides (9, 27) or epidural injections ofsiRNA targeting NaV1.8 (29), the expression of plasmid DNAby injection of CG/DNA polyplex is limited to the DRG andsciatic nerve innervating the subcutaneous injection site (32).In this study,�10% of small andmediumDRG cells were foundpositive for the reporter GFP. Puigdellívol-Sánchez et al. (46)estimated only 20–30% of DRG neurons to innervate the hind-paw. Therefore, the relevant transfection efficiency of the pres-ent method may exceed 40%.Significant alleviation of mechanical allodynia and thermal

hyperalgesia in SNE-injured rats was achieved with a distinctlatent period of 3–4 days after hindpaw injection of shRNA3specific to NaV1.8 (Fig. 3C). Following neuronal uptake, theinjected CG/DNA polyplex may undergo retrograde axonaltransport along the sciatic nerve (10–15 cm) before reachingthe soma in the DRG. The rate of microtubule-mediated retro-grade axonal transport along the sciatic and spinal nerves hasbeen estimated to be 10–25 cm/day (47, 48). Thus, CG/DNApolyplex should reach DRG neuronal nuclei within a day, andthe mature siRNA should be synthesized. Indeed, the synthesisof mature siRNA as well as measurable NaV1.8 knockdown inDRG neurons was evident 2.5 days after the NaV1.8 shRNAinjection but doubled by 7 days (Fig. 2B). Therefore, the latentperiod of 3–4 days is likely due to the continued accumulationof the siRNA and resultant NaV1.8 knockdown.In rats injected with shRNA4, the late onset pain alleviation

appeared to be consistent with the pain alleviation pattern ofshRNA3, but the effect was more significant (Fig. 3D). Becauseboth shRNA3 and shRNA4 were shown to decrease NaV1.8mRNA levels, the late onset pain alleviationmay likely be due toNaV1.8 knockdown, and the robust pain alleviation by shRNA4may directly relate to its larger knockdown efficiency (Fig. 1B).It was noted, however, that significant pain relief was fasterwithshRNA4. Because shRNA4 also affected NaV1.6 (Fig. 1B), it istempting to speculate that the knockdown of NaV1.6 couldcontribute to the earlier onset of pain relief. Further studies areneeded.The U6 promoter in our shRNA constructs is widely used in

mammalian expression systems; however, when applied in vivo,

3 D. K. Thakor, S. Mitrirattanakul, I. Spigelman, and I. Nishimura, unpublishedobservations.




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its transcriptional activity was shown to be silenced after 1�2weeks (49). Therefore, the gradual return of pain symptomsafter shRNA injection may primarily be due to the silencing ofpromoters in the expression plasmid.In the random shRNA-injected group, we unexpectedly

observed small decreases in neuropathy symptoms of relativelyshort duration (Fig. 3C). Suspecting an off target effect, werepeated a BLAST search using the 19 nucleotides of the ran-dom sequence siRNA; however, none of the mRNA species inthe database were detected. It is possible that the seed sequence(nucleotides 2–7) of random siRNA could behave as a miRNAand promote translational inhibition of the off target gene(s)(50). Within the scope of this study, possible off target mole-cules were not determined.NaV1.8Expression inDRGandSciaticNerve after SNE Injury—

Decreases in expression of NaV1.8 in the DRG were demon-strated after SNL (27), CCI (25, 29), as well as SNE (28), whereasexpression of NaV1.8 in peripheral nerve increases (9, 25, 28).The present study confirmed these previous reports (Fig. 4) andalso demonstrated the accumulation of NaV1.8 mRNA in sci-atic nerve ipsilateral to SNE injury (Fig. 5C).A recent study suggested that chronic nerve compression

resulted in the up-regulation of NaV1.8 immunoreactivity inSchwann cells (51). Notably, the chronic nerve compressionmodel does not induce mechanical allodynia or thermal hyper-algesia, instead resulting in progressive decreases inmechanicalsensitivity (52). In our study, the lack of detectable NaV1.8hnRNA in the SNE-injured nerve (Fig. 5B) strongly suggeststhat the source of NaV1.8 mRNA is not the non-neuronal cells,such as Schwann cells, but the axons themselves.The present study further addressed whether increases in

NaV1.8 transcription from the Scn10A gene in DRG contrib-uted to the axonal NaV1.8 mRNA accumulation. The NaV1.8hnRNA level was not influenced by SNE injury (Fig. 5B), andthus de novo Scn10A gene transcription should not have adirect mechanistic role in the accumulation of axonal NaV1.8mRNA. Also, we previously determined that the accumulationof axonal NaV1.8 was not due to the increase of the mRNAhalf-life evaluated by poly(A) tail elongation (28).It has been established that mRNAs are actively transported

to subcellular sites (53). For example, subcellular localization of�-actin mRNA occurs during neuronal regeneration facilitatedby the binding of zip code protein (ZBP1) to its 3�-UTR (54).Therefore, it is reasonable to postulate thatNaV1.8mRNAmaybe post-transcriptionally transported to the SNE-injured axonsfrom their DRG somata (Fig. 6). Increased transport of NaV1.8mRNA to axons in the absence of increased somatic mRNAproduction (Fig. 5B) might be expected to result in decreasedsteady state levels of somatic NaV1.8 mRNA ipsilateral to theSNE treatment. We have previously observed a trend ofdecreased somatic NaV1.8mRNA ipsilateral to SNE and signif-icant decreases in somatic NaV1.8 immunoreactivity (28). Asimilar trend of decreased somatic NaV1.8 mRNAmay be seenin DRG ipsilateral to SNE compared with contralateral or naïverat DRG in Fig. 5D. It must also be noted that the amount ofNaV1.8 mRNA in the axons is �1/10 to 1/50 of the DRGNaV1.8mRNA (Fig. 5C). Therefore, relatively large increases in

axonal NaV1.8 mRNA may result from relatively smalldecreases in the amount of somatic NaV1.8 mRNA.Because all of the necessary molecular components for pro-

tein translation exist in peripheral axons (55, 56), the accumu-lated axonal NaV1.8 mRNA may, in part, contribute to theincreased functional expression of NaV1.8 in the injured nerve.The present study further demonstrated that injection ofshRNA3 achieved a normalization of SNE-induced NaV1.8mRNA in the nerve during the time (Fig. 5D) when suppressionof pain symptoms was maximal (Fig. 3C). From these observa-tions, NaV1.8 mRNA in the nerve, not in the DRG, appears toplay a more relevant role in the pathogenesis of this painfulneuropathy.Mechanism of NaV1.8 Knockdown and Neuropathic Pain

Alleviation—Most of the mature siRNA3 was detected in theDRG (Fig. 2B), suggesting that the somata of sensory neurons intheDRGmay be themain site of siRNA-derivedNaV1.8mRNAdegradation. If so, the attenuation of pain symptoms wouldoccur after the depletion of axonally transported NaV1.8mRNA within the somata of DRG neurons and subsequentdecreases in axonal NaV1.8 synthesis (Fig. 6). However, it hasalso been well documented that RNAi machinery exists in theperipheral nerve axons (57, 58). Therefore, siRNA-mediateddegradation of NaV1.8 mRNA could also occur in peripheralaxons.Maximum relief of mechanical allodynia with shRNA3 in the

present study was �50% and was less effective than the relief of

FIGURE 6. A hypothetical molecular mechanism of painful neuropathyinvolving axonal NaV1. 8 mRNA. A, NaV1.8 mRNA is largely localized in DRGin the uninjured neurons. B, SNE injury induces axonal accumulation ofNaV1.8 mRNA, likely through active mRNA transport, not by the increase ofgene transcription or axonal mRNA half-life. C, retrograde transport of NaV1.8shRNA facilitates RNAi-derived NaV1.8 mRNA degradation in DRG, whicheventually normalizes axonal NaV1.8 mRNA, leading to the attenuation ofneuropathic pain symptoms after a distinct lag period.




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thermal hyperalgesia, which reached nearly 100% (Fig. 3C).Mechanical stimulation activates low threshold mechanore-ceptors (e.g. Meissner’s corpuscle and Merkel disk receptors)on the encapsulated terminals of A� or A� fibers (59). Thesefibers terminate subcutaneously at the epidermal-dermal junc-tion and are thickly myelinated. By contrast, thinly myelinatedA� fibers and unmyelinated C fibers have bare nerve endings,and both highly innervate the glabrous skin of hindpaw.Although A� fibers are high threshold mechanosensors andconstitute the afferent portion of the reflex arc that results inwithdrawal from noxious and mechanical stimuli, C fibers arepolymodal and respond to thermal (both heat and cold),mechanical, and chemical stimuli (59). Subcutaneously injectedCG/DNA polyplex may be better endocytosed into bare nerveendings of A� fiber and C fiber than into the encapsulated ter-minals of A� and A� fibers. Because NaV1.8 is primarily syn-thesized in A� and C nociceptors (60), the nearly completeNaV1.8 knockdown observed in the sciatic nerve tissue mayreflect the preferential CG/shRNA uptake by these neurons,reflected in the nearly complete alleviation of thermalhyperalgesia.Conclusions—Taken together, this study demonstrated that

NaV1.8 in the affected sciatic nerve, not in DRG, plays a signif-icant role in the development and maintenance of painful neu-ropathy symptoms.We propose that themolecularmechanismof SNE-inducedneuropathy includes injury-inducedpost-tran-scriptional NaV1.8 mRNA transport, axonal accumulation ofNaV1.8 mRNA, and its local protein translation leading to theincreased NaV1.8 functional expression in injured nerve. Thisstudy further suggests that axonal NaV1.8 mRNA may be anattractive therapeutic target for painful neuropathy, and thesubcutaneous injection of CG/DNA polyplex at the pain sitemay present a novel therapeutic modality.

Acknowledgments—We thank Prof. Yasuhiko Tabata (Institute forFrontier Medical Science, Kyoto University, Japan) for providing cat-ionized gelatin nanoparticles, Dr. Emmanuelle Faure-Kuma (VectorCore, UCLA) for lentivirus production, Dr.Mathew Schibler (Califor-nia NanoSystems Institute and Brain Research Institute microscopycore facility, UCLA) for confocal laser scanning micrography, Mari-ane Cilluffo (Brain Research Institute microscopy core facility) forimmunohistochemistry consultation, and (Daniela Markovic ofDepartment of Biomathematics), and David Geffen (School of Medi-cine at UCLA) for statistical consultation. We also thank Prof. DavidWong (Associate Dean of Research, UCLA School of Dentistry) forgenerous funding support for statistical consultations.

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Ichiro NishimuraSupanigar Ruangsri, Audrey Lin, Yatendra Mulpuri, Kyung Lee, Igor Spigelman and

Accumulation to Sciatic Nerve Injury-induced Painful Neuropathy in RatsRelationship of Axonal Voltage-gated Sodium Channel 1.8 (NaV1.8) mRNA

doi: 10.1074/jbc.M111.261701 originally published online September 30, 20112011, 286:39836-39847.J. Biol. Chem.

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relationshipofaxonalvoltage-gatedsodiumchannel1.8 (nav1.8 ... · 2012-04-20 · painful neuropathy...

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