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. 2021 Jul;22(7):806-816.
doi: 10.1016/j.jpain.2021.02.003. Epub 2021 Feb 24.

Nociceptor Overexpression of NaV1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Pedro Alvarez  1 , Oliver Bogen  2 , Paul G Green  3 , Jon D Levine  4
Affiliations

Nociceptor Overexpression of NaV1.7 Contributes to Chronic Muscle Pain Induced by Early-Life Stress

Pedro Alvarez et al. J Pain. 2021 Jul.

Abstract

Adult rats previously submitted to neonatal limited bedding (NLB), a model of early-life stress, display muscle mechanical hyperalgesia and nociceptor hyperexcitability, the underlying mechanism for which is unknown. Since voltage-gated sodium channel subtype 7 (NaV1.7) contributes to mechanical hyperalgesia in several preclinical pain models and is critical for nociceptor excitability, we explored its role in the muscle hyperalgesia exhibited by adult NLB rats. Western blot analyses demonstrated increased NaV1.7 protein expression in L4-L5 dorsal root ganglia (DRG) from adult NLB rats, and antisense oligodeoxynucleotide (AS ODN) targeting NaV1.7 alpha subunit mRNA attenuated the expression of NaV1.7 in DRG extracts. While this AS ODN did not affect nociceptive threshold in normal rats it significantly attenuated hyperalgesia in NLB rats. The selective NaV1.7 activator OD1 produced dose-dependent mechanical hyperalgesia that was enhanced in NLB rats, whereas the NaV1.7 blocker ProTx-II prevented OD1-induced hyperalgesia in control rats and ongoing hyperalgesia in NLB rats. AS ODN knockdown of extracellular signal-regulated kinase 1/2, which enhances NaV1.7 function, also inhibited mechanical hyperalgesia in NLB rats. Our results support the hypothesis that overexpression of NaV1.7 in muscle nociceptors play a role in chronic muscle pain induced by early-life stress, suggesting that NaV1.7 is a target for the treatment of chronic muscle pain. PERSPECTIVE: We demonstrate that early-life adversity, induced by exposure to inconsistent maternal care, produces chronic muscle hyperalgesia, which depends, at least in part, on increased expression of NaV1.7 in nociceptors.

Keywords: ERK1/2; Maternal neglect; muscle hyperalgesia; neonatal limited bedding; nociceptor hyperexcitability.

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Figures

Figure 1.
Figure 1.. Enhanced NaV1.7 expression associated with muscle mechanical hyperalgesia in NLB rats.
A. Consistent with previous reports, , NLB rats exhibit, as adults, decreased mechanical nociceptive threshold in the gastrocnemius muscle (~20% lower) compared to naïve control rats. Two-way ANOVA showed significant effects for condition (i.e, control or NLB, F1,72 = 2682, P < 0.001), sex (F1,58 = 124.3, P< 0.001) and interaction (F1,58 = 22.36, P < 0.001). Bonferroni’s post hoc test revealed significant differences in baseline between control and NLB rats. Of note, NLB rats exhibited a reduced sex difference in muscle mechanical nociceptive threshold. Inset figures show estimation plots; B. Representative Western blot from L4–L5 DRG extracts from control and NLB rats. The calculated molecular weight of NaV1.7 is 226 kDa (according to UniProt KB database entry O08562). β-actin, was used as a loading control (molecular weight of 42 kDa according to UniProt KB database entry P60711); C. Semi-quantitative Western blot analysis demonstrates a significant increase in NaV1.7 immunoreactivity in protein extracts from NLB rats compared to control rats. Numbers in the bars indicate sample size for each group. ***P < 0.001
Figure 2.
Figure 2.. Effect of AS ODN directed against NaV1.7.
A. Representative Western blot analysis of L4–L5 DRG extracts from rats injected daily i.t. with 120 μg/20 μl of AS or MM ODNs for 3 consecutive days revealed significant down-regulation of NaV1.7 in the AS ODN compared to MM ODN group (see results section for details). β-actin was used as a housekeeping gene product; B. Comparison of the protein expression by Western blotting demonstrated a significant decrease in NaV1.7 in DRG extracts from rats treated with AS ODN, compared to the MM ODN treated group; C. AS and MM ODN treatments were devoid of effect on baseline muscle mechanical nociceptive threshold in control (naïve) rats. Two-way repeated measures ANOVA showed only significant effect for time (F1,16 = 9.103, P < 0.001), but not for treatment (F1,16 = 0.0007, P = 0.9788), or interaction (F1,16 = 0.0142, P = 0.9065); D. NaV1.7 AS ODN treatment inhibits muscle hyperalgesia in NLB rats. Two-way repeated measures ANOVA showed significant effects for treatment (F1,16 = 35.36, P < 0.001), time (F1,16 = 97.89, P < 0.001), and interaction (F1,16 = 130.9, P < 0.001). Bonferroni’s post hoc test revealed significant differences in baseline between AS and MM groups at ODN treatment (Post) time point. Three-way ANOVA was performed on data in C and D: Time (pre-post) × NLB × NaV1.7 antisense F1, 64 = 37.43 P<0.0001, indicating NLB rats are different from control due to over-expression of NaV1.7.
Figure 3.
Figure 3.. Effects of NaV1.7 activator OD1 on muscle mechanical nociceptive threshold.
A. Time course of changes in the local mechanical nociceptive threshold of control rats injected i.m. with different doses of OD1 or vehicle. Two-way repeated measures ANOVA showed significant effects for treatment (F4,28 = 72.41, P < 0.001), time (F7,196 = 396.6, P < 0.001), and interaction (F28,196 = 38.92, P < 0.001). Bonferroni’s post hoc test revealed significant differences between vehicle and OD1-treated rats over a period of 0.5 to 2 h after injection (solid symbols, P < 0.001); B. Injection of OD1 into the right gastrocnemius muscle produced dose-dependent decrease in mechanical nociceptive threshold as measured 20 min after injection. One-way repeated measures ANOVA showed significant effect for treatment (F4,28 = 83.09, P < 0.001). Dunnett’s multiple comparisons test revealed significant differences between vehicle (0) and OD1 doses; C. Comparison of nociceptive effects of OD1 (100 nM/50 μl) in control and NLB rats at baseline (0), 30 and 60 min after i.m. injection. Two-way repeated measures ANOVA showed significant effects for treatment (F1,10 = 10.12, P = 0.0097), time (F2,20 = 752, P < 0.001), and interaction (F2,20 = 5.382, P = 0.013). NLB rats displayed increased hyperalgesia to OD1 compared to control rats 30 min after the injection, as revealed by Bonferroni’s multiple comparisons test. *P < 0.05; ***P < 0.001. Three-way ANOVA for Time (pre-post) × NLB × OD1 shows that OD1 effect does not differ with NLB treatment over time (F1, 22 = 0.3449, P=0.5630).
Figure 4.
Figure 4.. Effects of NaV1.7 inhibitor ProTx-II on muscle mechanical hyperalgesia.
A. Control rats were injected i.t. with ProTx-II (1 μg/20 μl), or vehicle (20 μl), followed by i.m. OD1 (100 nM/50 μl), and readings taken 30 and 60 min after OD1 injection. Two-way repeated measures ANOVA showed significant effects for treatment (F1,10 = 287.5, P < 0.001), time (F2,20 = 502.2, P < 0.001), and interaction (F2,20 = 295.6, P < 0.001). Bonferroni’s post hoc test revealed significant differences in nociceptive threshold between vehicle and ProTx-II treated rats 30 and 60 min after OD1 injection; B. NLB rats were injected i.t. with ProTx-II (1 μg/20 μl), or vehicle (20 μl), and readings taken 30 and 60 min after injection. Two-way repeated measures ANOVA showed significant effects for treatment (F1,15 = 150.4, P < 0.001), time (F2,30 = 82.09, P < 0.001), and interaction (F2,30 = 59.93, P < 0.001). Compared to vehicle, ProTx-II significantly attenuated the mechanical hyperalgesia exhibited by NLB rats 30 and 60 min after i.t. injection of ProTx-II, as revealed by Bonferroni’s multiple comparisons test. Numbers in bars indicate number of females included in each group. ***P < 0.001
Figure 5.
Figure 5.. Contribution of ERK1/2 (44/42 MAPK) to early-life stress-induced muscle mechanical hyperalgesia.
(A) Effect of AS ODN targeting ERK1/2 mRNA on muscle mechanical nociceptive threshold did not produce significant changes in mechanical nociceptive threshold in control rats (two-way repeated measures ANOVA: treatment [F1,10 = 0.01497, P = 0.905], time [F1,10 = 8.757, P = 0.0143], interaction [F1,10 = 0.1709, P = 0.688]); (B) In contrast, the same AS ODN treatment in NLB rats. Two-way repeated measures ANOVA showed significant effects for treatment (F1,10 = 140.1, P < 0.001), time (F1,10 = 147, P < 0.001), and interaction (F1,10 = 156.6, P < 0.001). Bonferroni’s post hoc test revealed significant differences in baseline between AS and SE groups after ODN treatment (Post time point). Three-way ANOVA analysis shows a significant interaction of Time (pre-post) × NLB × MAPK antisense F1, 20 = 77.96, P<0.0001.
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