Reduction of voltage gated sodium channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic neuropathy

A series of miRNA sequences targeting common rat Na_V α subunits were constructed and inserted into the nonreplicating HSV recombinant UL41E1G6-M [12]. The resulting series of vectors were used to transfect primary DRG neurons in culture at a multiplicity of infection (MOI) of 1 for 2 hours, and 48 hours later the amount of Na_V1.7 mRNA determined by RT-PCR. The most effective vector construct, designated QHmiNa_V (Figure 1) was used in the experiments reported. Control vector QHmiSc was identical to QHmiNa_V but contained a scrambled sequence in place of Na_V miRNA sequence.

The sequence inserted into QHmiNa_V was a perfect match for Na_V1.7 and for Na_V1.3, but an imperfect match for other Na_Vs (Figure 2a). We examined the effect of QHmiNa_V infection on expression of DRG Na_V isoforms Na_V 1.3, 1.6, 1.7 and 1.8 by examining isoform- specific mRNA levels 3 days after infection of primary DRG neurons in culture at an MOI of 1. Infection with QHmiNa_V produced a substantial reduction in the levels of Na_V1.6 and 1.7 mRNAs, and about a 50% reduction in the amount of Na_V1.8 mRNA in infected DRG neurons (Figure 2b). Infection with QHmiSc at an MOI of 1 resulted in no change in Na_V α subunit mRNA levels. The change in Na_V protein levels assessed using a well characterized anti-Na_V1.7 antibody, lagged several days behind the reduction in RNA reaching 80% of basal levels at 10 days after infection reflecting the half-life of already synthesized protein (data not shown).

Na_Vs are crucial determinants of neuronal excitability [13, 14], and the Na_V isoforms Na_V1.3, Na_V1.6, Na_V1.7, Na_V1.8, and Na_V1.9 are all expressed in DRG neurons. Transgenic mouse and knockdown studies have principally implicated the isoforms Na_V1.3, Na_V1.8 and Na_V1.9 in inflammatory and nerve injury related pain [15‐19]. Na_V1.8-null mutant mice lack slowly inactivating sodium currents and unable to sense cold pain or mechanical pressure [20]. These animals also exhibit deficits in inflammatory pain behavior, yet they respond normally to heat. In carrageenan and complete Freund's adjuvant-induced hyperalgesia there is an increase in Na_V1.8 and Na_V1.9 currents [21, 22] and an increase in the expression of Na_V1.8 in DRG has been reported [23]. Knockdown of Na_V1.8 by intrathecal delivery of antisense oligonucleotide against Na_V1.8 reduces CFA-induced hyperalgesia [24, 25].

There is a significant increase in Na_V1.7 protein in DRG of rats rendered diabetic by injection of streptozotocin (STZ) [2, 6] that correlates with thermal hyperalgesia, mechanical hyperalgesia and cold allodynia, characteristic of painful diabetic neuropathy in these animals. Subcutaneous inoculation of HSV vectors results in transduction of ipsilateral DRG [26]. Two weeks after the onset of diabetes 30 μl containing either 3 × 10⁹ pfu of QHmiNa_V or QHmiSc was injected into the plantar surface of both hind feet. Two weeks after the vector inoculation (4 weeks after onset of diabetes), the amount of Na_V1.7 protein was significantly increased in the diabetic animals (Figure 3a) and in diabetic animals inoculated with the QHmiSc, but there was a substantial reduction in the amount of Na_V1.7 in diabetic animals inoculated with QHmiNa_V (Figure 3a). QHmiNa_V brought the amount of Na_V1.7 protein in diabetic animals back to near normal but not completely normal levels. By in situ hybridization the reduction in Na_V1.7 RNA expression was found to be widely distributed in neurons in the DRG, and the in situ study suggested a reduction of about 50% in the amount of Na_V1.7 mRNA in the DRG compared to diabetic animals (Figure 3b). To separately estimate the number of DRG neurons transfected after footpad inoculation of the vector, we performed in situ hybridization for the reporter gene GFP which revealed 40-50% of the neurons in individual sections with GFP reporter RNA (Additional File 1).

Diabetic rats demonstrate thermal hyperalgesia, cold allodynia and mechanical hyperalgesia [6, 8]; all of these signs of neuropathic pain were improved by inoculation with QHmiNa_V. Thermal hyperalgesia was manifested by a decrease in withdrawal latency in response to noxious thermal stimuli (control 8.62 ± 1.3 sec; diabetic 4.89 ± 0.5 sec; p < 0.005). Two weeks after inoculation (4 weeks after diabetes) animals inoculated with QHmiNa_V showed a statistically significant increase in thermal latency (QHmiNa_V 7.92 ± 0.7 sec compared to diabetic; p < 0.005) and control vector QHmiSc inoculated diabetic animals (QHmiSc 5.5 ± 0.8 sec; p < 0.01 compared to QHmiNa_V) (Figure 4). Cold allodynia was manifested by a decreased latency to withdraw from a cold acetone spray in diabetic compared to control animals (diabetic 3.1 ± 0.6 sec; control 18.1 ± 3.9 sec p < 0.001). Diabetic animals inoculated with QHmiNa_V showed an increased latency to withdraw from this stimulus compared to diabetic control vector inoculated animals (QHmiNa_V 16.9 ± 2.1 sec; QHmiSc 6.2 ± 2.1 sec; p < 0.001). Mechanical hyperalgesia was tested using the method described by Randall and Sellito. Inoculation of QHmiNa_V significantly increased the pressure threshold (QHmiNa_V 105.9 ± 4.9 gm) compared to diabetic animals (55.5 ± 3.4 gm; p < 0.001) and QHmiSc-inoculated animals (62.7 ± 2.9 gm; p < 0.001) measured 4 week after inoculation (Figure 4).

In STZ-induced PDN there is a marked increase in Na_V 1.7 protein in DRG [2, 27, 28], an increase that is mediated by phosphorylation of PKC [6, 8]. While the pathogenesis of pain in PDN is complex and many molecular entities may be involved [29], in studies using HSV-based vectors to transfer genes to the DRG in vivo, we observed that vector-mediated release of gamma aminobutyric acid (GABA) from the DRG of diabetic animals results in a reduction in pain-related behaviors, coincident with a reduction in Na_V1.7 protein in the DRG achieved through activation of presynaptic GABA_B receptors [8]. We also found that gene transfer-mediated release of the inhibitory neurotransmitter enkephalin resulted in a greater reduction of neuropathic pain-related behaviors in animals with neuropathic pain resulting from diabetic neuropathy [6] compared to animals with neuropathic pain resulting from spinal nerve ligation [7], and that continuous activation of presynaptic delta opioid receptors by vector-produced enkephalin in the diabetic animals also resulted in a reduction in the amount of Na_V1.7 protein in diabetic DRG in vivo.

These results support the interpretation that increased Na_V1.7 protein in DRG plays in the pathogenesis of pain in this model of PDN. The potential role of Na_V1.7 in the pathogenesis of pain in PDN is also supported by the observation that gain-of-function mutations in SCN9A, the gene encoding Na_V1.7 result in the spontaneous pain syndromes primary erythermalgia and paroxysmal extreme pain disorder [30, 31], and that loss of function mutations in Na_V1.7 result in an inherited channelopathy characterized by total insensitivity to pain [32].

Na_V1.7 is the principal TTX-sensitive channel in small DRG neurons, and is responsible for almost half of the current in those neurons, and the increase in Na_V1.7 protein is accompanied by an increase in TTX-sensitive current in those cells [2]. There is also an increase in TTX-resistant currents in diabetic rats [27, 33] that is likely related to modulation of individual channel properties related to phosphorylation [2]. Waxman and colleagues have suggested that Na_V1.7, which opens in response to slow ramp depolarization may serve to "set the gain" for repetitive firing [14]. If that is the case, an increase in the number of Na_V1.7 channels can serve to lower the gain allowing for altered thresholds and spontaneous pain. There is one early published report in which an increase in Na_V1.7 in the DRG of STZ-diabetic rats was not observed using immunocytochemistry [27], but several subsequent studies using Western blot found an increase in Na_V1.7 [2, 6, 8]. The results of the current study provide support for the interpretation that an increase in the amount of Na_V1.7 plays a role in the pathogenesis of pain in this model of PDN.

Twenty one days after subcutaneous inoculation of QHmiNa_V into the hind paw of normal rats, the amount of Na_V1.7 was reduced by 50% and the amount of Na_V1.8 by 40% compared to QHmiSc-inoculated animals (Figure 5a and 5b). Animals inoculated with QHmiNa_V showed a small but statistically significant reduction in formalin - induced flinching behavior in the delayed phase of the formalin test (Figure 5c).

There is always a possibility in experiments of this type that the directed siRNA may knock down the expression of proteins unrelated to the target gene. While this is an important consideration, the prior evidence indicating a role for Na_V in the pathogenesis of pain, coupled with the reduction in pain when Na_V expression is reduced to just that we are looking at on-target effects of Na_V knockdown. We do not interpret our observation that vector-mediated knock down of Na_V resulted in only a modest reduction in spontaneous pain in the formalin test to indicate that Na_V1.7 may not important in inflammatory pain. Yeomans and coworkers reported previously that a related HSV vector engineered to express an Na_V1.7 antisense construct reduced inflammatory hyperalgesia resulting from injection of complete Freund's adjuvant [34], and Woods and colleagues reported that mice with double knockouts for both Na_V1.7 and Na_V1.8 show a substantial reduction in pain related behavior in the delayed phase of the formalin test, in contrast to Na_V1.8 knockouts that demonstrate a normal behavior in the delayed phase [35]. The results of the current study however do suggest that a 40 to 50% reduction from normal amounts in the amount of Na_V1.7 and Na_V1.8 in the DRG does not impair the ability of nociceptors to respond in a functionally meaningful manner to the acute inflammatory stimulus.

Woods and colleagues also reported that the Na_V1.7 and Na_V1.8 double knockouts demonstrate no impairment in the development of pain in response to nerve injury (L5 spinal nerve ligation) [35]. Those results, as well as other experiments that demonstrate different changes in Na_V isoform levels in different models of neuropathic pain [36] are not incompatible with the current study. Rather, they point to what may be important differences in the pathogenesis of different forms of neuropathic pain, in particular the role played by the substantial increase in Na_V1.7 protein in PDN.

To construct miRNAs targeting rat Na_V α subunits present in DRG, we constructed a vector to express a miRNA targeted to a common sequence of Na_V1 α subunits. A3 top strand oligo 5'-TGC TGG GAA CAA GCA GAT CAT GCT GTG TTT TGG CCA CTG ACA CAG CAT GCT GCT TGT TCC-3' A3 bottom strand oligo 5'-CCT GGG AAC AAG CAG CAT GCT GTG TCA GTC AGT GGC CAA AAC ACA GCA TGA TCT GCT TGT TCC C -3'. Equal amounts of the single-stranded oligos were annealed to generate a double-stranded oligo that ligated with the linearized plasmid pcDNATM6.2-GW/EmGFP-miRNA (Invitrogen), amplified using the following PCR primers with mulI site at 5' end an ECORI site at 3' end, forward primer: 5'-ACG CGT GCT AGT TAA GCT ATC AAC AAG-3' and reverse primer: 5'-GAA TTC GTA CAA GAA AGC TGG GTC TAG-3'. The PCR fragment was extracted by Qia-quick Gel Extraction Kit (Qiagen, Valencia, CA), and 3 ul of the gel-extracted PCR fragment ligated into PGEM-T vector system I kit (Promega; Madison, WI) for expansion. The resulting PCR fragment was cut out from PGEM-T plasmid by MluI and EcoRI, purified by gel electrophoresis and cloned into MluI and EcoRI -cut shuttle plasmid SASB3-M4. The shuttle plasmid containing the insert was then cotransfected with the non-replicating HSV vector UL41E1G6-M in complementing 7B cells, and and a single green virus isolated through serial dilution. Every round of purification was confirmed by sequencing, and the construct designated QHmiNa_V. A control vector identical to QHmiNa_V, but containing a scrambled-miRNA sequence in place of Na_V α subunit miRNA was constructed and designated QHmiSc.

Following an overnight fast, male Sprague Dawley rats weighing 225-250 gms were injected with streptozotocin (STZ, Sigma, USA) 50 mg/kg, i.p. in citrate buffer (pH 5.5). The development of diabetes was confirmed by measuring blood glucose and animals with blood glucose level ≥ 300 mg/dl included as diabetic. There were 8-10 animals per group in all these studies.

Diabetic animals were inoculated subcutaneously in the footpad of the both hind paws with 30 μl containing 1 × 10⁷ plaque forming units with either QHmiNa_V or the control vector QHmiSc 2 weeks after the onset diabetes. Control animals or diabetic only animals were injected PBS in the footpad. A separate cohort of diabetic animals were inoculated with the vector and euthanized 2 weeks later to evaluate the efficacy of knockdown of RNA.

DRG neurons from 17-day-old rat embryos were cultured in Neurobasal Medium containing B27, Glutamax I, Albumax I, and penicillin/streptomycin (Gibco-BRL). After 7 days in culture, the cells were transfected with either QHmiNa_V or QHmiSc at a multiplicity of infection (MOI) of 1 for 2 h. Fresh medium was replaced and collected 7 days later for determination of Na_Vs.

cDNA prepared from RNA isolated from DRG cells or rat L4-6 DRG were amplified using following primer sets: β-actin-F (5'-CAG TTC GCC ATG GAT GAC GAT ATC-3') and β-actin-R (5'-CAC GCT CGG TCA GGA TCT TCA TG-3') for β-actin, Na_V1.6-F (5'-GAC AAT GAT GGT GGA GAC AGA CAC-3') and Na_V1.6-R (5'-TTG GAG GCC ATC TTT CTG CAG-3') for Na_V1.6. Na_V1.7-F (5'-CCA TCA TGA ACG TGC TTC TCG TG-3') and Na_V1.7-R (5'-CAA AGC AAA GAG CAG AGT GCG GAT C-3') for Na_V1.7 and Na_V1.8-F (5'-AAC AGC ACC GGC CAC TTC TTC-3') and Na_V1.8-R (5'-CCG TTG CTG TTG GGC AGG TTG-3') for Na_V1.8. All reactions involved initial denaturation at 94°C for 5 min followed by 28 cycles (for β-actin and Na_V1.7) and 30 cycles (for Na_V1.6 and Na_V1.7) at 94°C for 30 sec, 68°C for 3 min, followed by 1 cycle at 68°C for 8 min using a GeneAmp PCR 2700 (Applied Biosystems, Foster City, CA).

Cells or pooled samples of L4-L6 DRG, were homogenized with lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2% SDS, 10% glycerol, and 1:100 dilution of protease inhibitor mixture and phosphatase inhibitor mixture (Sigma), the homogenized cells and tissues were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant was stored at -80°C. An aliquot of supernatant was taken for protein estimation using a protein assay kit (Bio-Rad Laboratories, Hercules, CA). Total cell extract or total protein from DRG (20 μg of protein per lane) was separated by PAGE, transferred to an Immobilon-P membrane (0.45 μm; Millipore), blocked with 5% nonfat milk, and then incubated with the primary antibody. Primary antibodies included an antibody against Na_V1.7, Na_V1.8 (Chemicon) and anti-SP-19 (Sigma Aldrich) followed by horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (1:5000; GE Healthcare) and visualized with ECL (Pierce) using a PC-based image analysis system (ChemiDoc XRS System; Bio-Rad Laboratories). The membranes were stripped and re-probed with mouse anti-β-actin (1: 2000; Sigma Aldrich) as a loading control. The intensity of each band was determined by quantitative chemiluminescence using a PC-based image analysis system (ChemiDoc XRS System, Bio-Rad Laboratories).

Rats were perfused with 4% paraformaldehyde, the L4-6 segment of DRG postfixed and cryoprotected, and 20 μm cryostat sections incubated with anti-GFP (Abcam). The secondary antibodies utilized were fluorescent anti-rabbit IgG Alexa Fluor 488 (1:2000; Molecular Probes, Eugene, OR). Images were captured using a Zeiss LSM 510 Meta confocal microscope.

Rats were anesthetized with ketamine/xylazine (100/10 mg/kg, IP) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. L4-6 DRG were post-fixed and cryoprotected overnight at 4°C in 30% sucrose, and serial sections (10 μm) of DRG was cut onto slides and desiccated overnight. Sections from the different groups were processed for in situ hybridization for detection of Na_V1.7 mRNA or GFP mRNA with incubation in 4% paraformaldehyde for 12 min and permeabilization with proteinase K for 6 min followed by hybridization with digoxigenin-labeled probes for Na_V1.7 (5DigN/TTA CGT CGC CGT CCA GCT CG/3DigN) and GFP (5DigN/TTC TCA TCG TCA CCC TTT TCC T/3DigN) at 53⁰ C overnight, followed by 1 hr blocking, 2 hrs of Anti-dig-AP antibody incubation and 1 hr color reaction with NBT/BCIP phosphate. The slides were then dehydrated and mounted in Permount. Digitized images of immunostained sections were captured with a Nikon E1000 microscope, and analyzed using a PC-based image analysis program (MCID, Imaging Research, Brock, ON, USA) by a technician blinded to the treatment group. All the cells in the cross-section of the DRG from three animals in each group were analyzed.

The latency to hind paw withdrawal from a thermal stimulus was determined by exposing the plantar surface of the hind paw to radiant heat using a modified Hargreaves thermal testing device [41]. Rats were placed in individual enclosures on a glass plate maintained at 30°C, and after a 30 min habituation period the plantar surface of the paw exposed to a beam of radiant heat applied through the glass floor. Activation of the bulb simultaneously activated a timer, and both were immediately turned off by paw withdrawal or at the 20 sec cut-off time. Testing was performed by a blinded observer in triplicate at 5 min intervals.

Mechanical nociceptive threshold was assessed using an analgesimeter (Ugo Basile, Comerio, VA, Italy) as described by Randall and Selitto [42]. A linearly increasing force was applied through a cone-shaped plastic tip with a diameter of 1 mm onto the dorsal surface of the hindpaw between the third and fourth metatarsals until the rat attempted to withdraw its paw or a pressure of 200 gms reached. The pain threshold determined as the mean of three consecutive stable values expressed in grams was determined by a blinded observer.

Animals were placed on a mesh floor 18 inches above the table and after 20 min of acclimatization, 0.1 ml of acetone was sprayed onto the plantar surface of the hind paw using a 1 cc syringe. The latency of the response, measured as the delay to a withdrawal response of either flinching or licking was used as a measure of cold allodynia, with a cut off limit at 40 sec. A total of 3 responses from each animal were assessed at 5 min intervals by a blinded observer.

To test inflammatory pain-related behavior, 21 days after inoculation of the vector into one hind paw, normal male Sprague Dawley rats 225-250 grams were acclimated to the test setup and injected with 50 μl of 5% formalin into the plantar surface of the ipsilateral hind paw. The number of flinches over the subsequent 60 minutes counted using an automated device [43].

The statistical significance of the difference between groups was determined by ANOVA (Systat 9) using Bonferroni's correction for the multiple post hoc analyses. All results are expressed as mean ± SEM. All the tissue culture experiments were repeated 3 times. The animal experiments, with 8-10 animals per group, were repeated twice.