Nociceptor Overexpression of Na_V1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Animals

Behavioral experiments were performed on male and female adult Sprague-Dawley (Crl:CD, Charles River, Hollister, CA) rats, weighing 250–350 g. For the neonatal limited bedding (NLB) protocol, primiparous pregnant Sprague-Dawley (Crl:CD) female rats were purchased from the same provider. After delivery, dams were housed with their litter in standard cages on postnatal days 0–1. On postnatal day 2, some litters were assigned to the NLB protocol (see below). All animals were housed in the Laboratory Animal Resource Center of UCSF, under a 12-hours light/dark cycle (lights on 7 am–7 pm) and environmentally controlled conditions; ambient room temperature (21–23 °C), with food and water available ad libitum. Animal care and use in experiments conformed to National Institutes of Health guidelines. The UCSF Institutional Animal Care and Use Committee approved the experimental protocol. All efforts were made to minimize the number of animals used and their suffering.

Neonatal limited bedding (NLB) stress protocol

The details of our use of the NLB model have been described previously.^{9, 26, 32} Briefly, beginning on postnatal day 2, dams and their pups were placed in cages fitted with a stainless steel mesh platform that was raised ~2.5 cm from the floor of the home cage, to allow collection of urine and feces. The nesting/bedding material provided consisted of one sheet of paper towel (~13 × 23 cm). Litters were otherwise undisturbed during postnatal days 2–9.

Antisense oligodeoxynucleotides

The contribution of Na_V1.7 and ERK to muscle mechanical nociceptive threshold in rats submitted to the NLB model was evaluated using intrathecal injections of antisense oligodeoxynucleotides (AS ODN). This method has been shown to inhibit the expression of specific proteins that contribute to a number of signaling pathways in nociceptors (for a review, see Stone and Vulchanova⁶²).

An AS ODN sequence, 5′- TGT TGG TCA GTA TGC TCG C −3′, was directed against a unique sequence of Rattus norvegicus Na_V1.7 alpha subunit mRNA. The corresponding GenBank accession number and ODN position within the cDNA sequence are NM_133289 and 2074 to 2092, respectively. The mismatch (MM) ODN sequence, 5′- TAT TCG TCG GTC TGT TCA C −3′, corresponds to the Na_V1.7 antisense sequence, with 6 bases mismatched (denoted by bold type). The AS ODN sequence, 5′- GCC GCC GCC GCC GCC AT −3′, was directed against a unique sequence of R. norvegicus ERK (MAPK) mRNA. A sense (SE) ODN sequence, 5′- ATG GCG GCG GCG GCG GC −3′, was used as a control. This AS ODN sequence is directed against the initiation codon of the translation start site of rat ERK1 and ERK2 (44/42 MAPK) mRNAs, which are conserved in humans, mice, and rats.⁵⁵ This AS ODN has been previously shown to produce selective knockdown of the expression of ERK1/2 (44/42 MAPK) protein in the spinal dorsal horn and to reduce nociceptive behavior in the rat.^{16, 60}

A nucleotide BLAST search was performed to confirm that the mRNA sequences targeted by the AS ODN, or their MM, and SE controls, were not homologous to any other sequences in the rat database. The ODNs were synthesized and lyophilized by Invitrogen (Carlsbad, CA). Before use, ODNs were reconstituted in sterile 0.9% NaCl to a concentration of 10 μg/μl, aliquoted and stored at −20°C.

Intrathecal injections

Immediately before injection, AS, MM, or SE ODNs were diluted in sterile 0.9% NaCl and administered by intrathecal injection (120 μg/20 μl) once daily, for 3 days. ProTxII (Abcam, Cambridge, MA, USA), a toxin from the Peruvian green velvet tarantula (Thrixopelma pruriens) that selectively inhibits Na_V1.7⁵⁸, was diluted in 0.1% bovine serum albumin (BSA)-Dulbecco’s phosphate buffer (DPBS), and a dose of 1 μg/20 μl was injected i.t., 30 min prior to nociceptive testing. Rats were briefly anesthetized with 2.5% isoflurane (Phoenix Pharmaceuticals, St. Joseph, MO) to facilitate i.t. injections, and a 29 G × ½” hypodermic needle was inserted into the subarachnoid space on the midline, between the L4 and L5 vertebrae. Proper intrathecal injections were confirmed by a sudden flicking of the rat’s tail.⁴³

Tissue harvesting and protein extraction

Control and NLB rats were euthanized by exsanguination while under deep isoflurane anesthesia. L4–L5 DRGs, which contain the somas of the sensory neurons innervating the gastrocnemius muscle^{12, 30, 54}, were quickly removed, snap frozen on dry ice, and stored at −80°C until further processing. DRGs were transferred into homogenization buffer [150 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA, 2% sodium dodecylsulfate (SDS)] supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and homogenized with a sonicator. Proteins were solubilized by incubating the homogenate for 2 h at 25°C at 1400 r/min and then extracted by a 15 min centrifugation at 14,000 r/min. Protein concentration of the samples was determined using the micro bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) with BSA as a standard.

Western blot analysis

Changes in Na_V1.7 expression in lumbar DRGs were evaluated by Western blot analysis as previously described.^6–8 Briefly, 40 μg of protein per sample were mixed with 4X sample buffer (62.5 mM Tris-HCl pH 6.8, 3% SDS, 10% glycerol, 0.025% bromphenol blue), denatured by shaking for 10 min at 500 r/min at 90°C and electrophoresed on a 4%–15% precast polyacrylamide gel (Biorad, Hercules, CA) in 25 mM Tris buffer containing 192 mM glycine, and 0.1% SDS. Proteins were transferred onto a nitrocellulose (NC) membrane using the semidry method (transfer time 2 h at 1.5 mA*cm−2 with 47.9 mM Tris, 38.9 mM glycine, 0.038% SDS, and 20% methanol). The blotting membrane was saturated by shaking in Tris-buffered saline (pH 7.4) containing 5% BSA and 0.1% Tween 20 (antibody dilution buffer) for 1 h at room temperature (RT). The blot was probed with a rabbit anti-Na_V1.7 antibody (Cell signaling technology, #14573; 1:500 in antibody dilution buffer) or a rabbit anti- β-actin antibody (Abcam, #ab8227, 1:1000 in antibody dilution buffer), at 4°C overnight, rinsed with TBST (3 times at RT, 15 min each) and probed with a donkey anti-rabbit horseradish peroxidase conjugated antibody (GE healthcare, #NA934V; 1:2500 in antibody dilution buffer) for 2 h at RT. The Western blot was rinsed with TBST (3 × at RT, 15 min each), the Na_V1.7 immunoreactivity visualized with the enhanced chemiluminescence (ECL) detection kit (Pierce) and expressed as arbitrary units (a.u.) of immunoreactivity using β-actin as the reference housekeeping protein.

Intramuscular injections

We performed intramuscular (i.m.) injections of OD1 (Tocris Bioscience, Minneapolis, MN) a toxin from the Iranian yellow scorpion (Odonthobuthus doriae) that selectively activates Na_V1.7.³⁸ Rats were briefly anesthetized with 2.5% isoflurane to facilitate the injection (50 μl) of OD1 (dissolved in 0.1% BSA in DPBS [10–300 nM]) or vehicle, into the belly of the gastrocnemius muscle. The skin overlying the injection site was previously shaved and scrubbed with alcohol. Immediately after i.m. injections, the site at which the skin was punctured was marked with a fine-tip indelible ink pen, so that the mechanical nociceptive threshold at the underlying i.m. injection site in the muscle could be measured over time.

Measurement of muscle mechanical nociceptive threshold

Mechanical nociceptive threshold in the gastrocnemius muscle was quantified using a digital force transducer (Chatillon DFI2; Amtek Inc., Largo, FL) with a 7 mm-diameter probe.^{9, 10, 26} Adult rats from litters previously submitted to the NLB model or kept undisturbed (naïve controls) were lightly restrained in an acrylic holder that allows for easy access to the hind limb and application of the transducer probe to the belly of the gastrocnemius muscle. The nociceptive threshold was defined as the force, in mN, required to elicit a flexion reflex. Baseline limb-withdrawal threshold was defined as the mean of 2–3 readings taken at 5-min intervals and one gastrocnemius muscle per rat was used in each experiment. Behavioral testing was performed blind to treatments.

Statistical analysis

Group data are expressed as mean ± SEM of n independent observations. Comparable numbers of male and female rats were used in each experiment and all data were combined and analyzed by condition. Statistical comparisons were made using Prism 9.0 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons between treatments were made by means of Student’s t-test, one, two or three-way repeated measures analysis of variance (ANOVA) followed by Bonferroni, Šídák’s or Dunnet’s multiple comparisons tests. P values < 0.05 were considered statistically significant.

Increased expression of Na_V1.7 in NLB rats

Compared to control (naïve) rats, male (control: 2521 ± 4.1 mN [n=42] vs NLB: 2218.7 ± 4.8 mN [n=27], P < 0.001), and female (control: 2435 ± 5.6 mN [n=33] vs NLB: 2185.3 ± 6.4 mN [n=32], P < 0.001) NLB rats exhibited, as adults, a significantly lower mechanical nociceptive threshold (i.e., muscle hyperalgesia) (Fig. 1A). In control rats a lower mechanical nociceptive threshold was observed in females compared to males (P < 0.001, Fig. 1A). This sex difference, albeit of small magnitude, was also observed in NLB rats (P < 0.001, Fig. 1A).

To test the hypothesis that Na_V1.7 plays a role in the muscle hyperalgesia exhibited by NLB rats, we assessed its protein expression in L4–L5 DRG extracts from NLB and control rats. Western blot analysis of extracts from NLB rats demonstrated a marked increase (~29%) in Na_V1.7 immunoreactivity (7.4 ± 0.4 a.u., [n = 6]), compared to extracts from control rats (5.7 ± 0.6 a.u., [n = 6], Student’s t-test, P = 0.026, Fig. 1B,C).

Antisense knockdown of Na_V1.7

To evaluate the involvement of nociceptor Na_V1.7 in mechanical hyperalgesia displayed by NLB rats, an AS ODN targeting Na_V1.7 mRNA was designed and its effects on the respective protein expression assessed. Western blot analysis of L4–L5 DRG extracts from rats submitted to the AS treatment demonstrated a marked decrease (~44%) in Na_V1.7 expression (1.76 ± 0.18 a.u., [n = 6]), compared to the MM treated group (3.14 ± 0.46 a.u., [n = 6], Student’s t-test, P = 0.0129, Fig. 2A,B). We next determined whether Na_V1.7 expression plays a role in the baseline mechanical nociceptive threshold. Compared to baseline (i.e., pre Na_V1.7 ODN), no significant difference in nociceptive threshold was observed between control rats treated with AS (2506.2 ± 12.9 mN vs 2487.3 ± 19.2 mN; [n=9], P > 0.05, Fig. 2C), or MM (2506.4 ± 13.9 mN vs 2486 ± 14.2 mN; [n=6], P > 0.05, Fig. 2B) ODN. In contrast, NLB rats treated with AS (2180.1 ± 16.4 mN vs 2417.1 ± 10.5 mN; [n=8], P < 0.001), but not MM (2210.7 ± 11.3 mN vs 2193.5 ± 15.7 mN; [n=10], P > 0.05) ODN, exhibited a significant increase in nociceptive threshold (Fig. 2D). Three-way ANOVA was performed on data in C and D: Time (pre-post) × NLB × NaV1.7 antisense P<0.0001, indicating NLB rats are different from control due to over-expression of NaV1.7.

Na_V1.7 activator OD1

To assess the direct involvement of Na_V1.7 in nociceptor terminals in muscle nociception, we studied the effect of i.m. injections of OD1, a selective Na_V1.7 activator. The change in nociceptive threshold induced by the highest dose of OD1 studied reached a peak 20 min after injection (vehicle: 2424.8 ± 20.2 mN [−1.9 ± 0.7%, n=8] vs OD1: 1641.6 ± 32.4 mN [−33.7 ± 1.6%, n=7]; P < 0.001, Fig. 3A). This hyperalgesia was still significant 2 hours after injection (vehicle: 2445.3 ± 14.4 mN [−1 ± 0.8%, n=8] vs OD1: 1819.7 ± 38.7 mN [−26.6 ± 1.3%, n=7]; P < 0.001, Fig. 3A). Compared to vehicle, OD1 (10, 30, 100, 300 nM/50 μl) produced a significant (n=6/8 group, P < 0.001), dose-dependent (r2 = 0.7732, P < 0.001) decrease in mechanical nociceptive threshold (Fig. 3B).

Since NLB rats display Na_V1.7-dependent muscle hyperalgesia, we compared OD1-induced hyperalgesia in NLB and control rats. Measurements of nociceptive threshold taken 30 min after a 100 nM/50 μl injection show a significant enhancement in OD1-induced muscle hyperalgesia in NLB (baseline: 2219.7 ± 16.1 mN vs post OD1: 1555.3 ± 31.6 mN [−29.9 ± 1.1%, n=6]), compared to control (baseline: 2479.3 ± 17.6 vs post OD1: 1860.8 ± 91.9 mN [−22.6 ± 3.5%, n=8]; P = 0.0165, Fig. 3C) rats. Readings taken 60 min after OD1 also revealed a significant increase in OD1-induced muscle hyperalgesia in NLB (post OD1: 1612.3 ± 23.3 mN [−27.4 ± 0.9%, n=6]), compared to control (baseline: 2479.3 ± 17.6 vs post OD1: 1906.9 ± 28.7 mN [−23.1 ± 1.0%, n=8]; P = 0.021, Fig. 3C) rats. Three-way ANOVA for Time (pre-post) × NLB × OD1 shows that OD1 effect does not differ with NLB treatment over time, P=0.5630.

Na_V1.7 blocker ProTx-II

We next explored the effect of intrathecal administration of ProTx-II, a selective Na_V1.7 blocker. To evaluate the effect of ProTx-II on Na_V1.7-dependent mechanical hyperalgesia, we studied its effect on the mechanical hyperalgesia induced by i.m. injection of the Na_V1.7 activator OD1, in normal rats. Rats were injected i.t. with ProTx-II (1 μg/20 μl), or vehicle (0.1% BSA-DPBS), and i.m. with OD1 (100 nM, 50 μl) into the right gastrocnemius muscle. Intrathecal ProTx-II, but not its vehicle, significantly inhibited the reduction of mechanical nociceptive threshold (i.e., mechanical hyperalgesia) produced by i.m. OD1, measured 30 min (ProTx-II: 2383.5 ± 4.7 mN [-8.8 ± 0.5%] vs vehicle: 1926.6 ± 13.5 mN [−26 ± 1.3%]; n = 6/group, P < 0.001), or 60 min (ProTx-II: 2358.7 ± 3.7 mN [−3.7 ± 0.5%] vs 1827.7 ± 23.3 mN [−25.7 ± 0.8%]; n = 6/group, P < 0.001) after injection (Fig. 4A).

Next, we evaluated the effect of i.t. ProTx-II on the mechanical hyperalgesia exhibited by NLB rats. Intrathecal ProTx-II, but not vehicle, significantly increased muscle nociceptive threshold (i.e., inhibited the mechanical hyperalgesia) in NLB rats, at 30 min (ProTx-II: 2414.8 ± 7 mN [10.4 ± 0.4%, n=9] vs vehicle: 2195.3 ± 12.5 mN [0.9 ± 0.7%, n=8]; P < 0.001), or 60 min (ProTx-II: 2404.7 ± 15.4 mN [9.9 ± 0.7%, n=9] vs vehicle: 2193.9 ± 16.5 mN [0.8 ± 0.7%, n=8]; P < 0.001), after i.t. injection (Fig. 4B).

Antisense knockdown of ERK1/2

Evidence indicates that posttranscriptional modifications of Na_V1.7, such as phosphorylation by pERK1/2, enhance nociceptor excitability⁶¹, likely contributing to hyperalgesia. Thus, to determine if ERK phosphorylation of Na_V1.7 contributes to hyperalgesia in NLB rats, we knocked down nociceptor ERK1/2 expression by means of AS ODN directed against ERK1/2 mRNA. In control rats, ERK1/2 AS ODN treatment did not significantly modify the nociceptive threshold (post AS: 2475.5 ± 14.1 mN, [n=6] vs post SE: 2470.7 ± 14.1 mN, [n=6], P > 0.05, Fig. 5A). While there were no significant differences at baseline (pre AS: 2229.7 ± 9 mN vs pre SE: 2210.3 ± 7.5 mN), NLB rats that received i.t. AS (2430.2 ± 12.8 mN, [n=6]) exhibited a significant increase in nociceptive threshold compared to the SE treated (2207.2 ± 6.5 mN, [n=6], P < 0.001) group (Fig. 5B). Three-way ANOVA analysis shows a significant interaction of Time (pre-post) × NLB × MAPK antisense, P<0.0001, indicating a role for MAPK mediating hyperalgesia in NLB rats.

Conclusion

In summary, persistent muscle mechanical hyperalgesia induced by early-life stress is mediated by increased expression of nociceptor Na_V1.7. Antisense ODN attenuation of nociceptor expression of Na_V1.7 or pharmacological blockade of Na_V1.7 inhibited muscle hyperalgesia observed in NLB rats. A contribution of ERK1/2 to this mechanical hyperalgesia was also observed, likely due to its sensitizing effect on Na_V1.7. These observations provide a novel mechanism for musculoskeletal pain after exposure to early-life adversity, suggesting that targeting Na_V1.7 could be a useful strategy for the treatment of stress-induced muscle pain.