Roles of ASIC3, TRPV1, and NaV1.8 in the transition from acute to chronic pain in a mouse model of fibromyalgia

Wei-Nan Chen, Cheng-Han Lee, Shing-Hong Lin, Chia-Wen Wong, Wei-Hsin Sun, John N Wood, Chih-Cheng Chen, Wei-Nan Chen, Cheng-Han Lee, Shing-Hong Lin, Chia-Wen Wong, Wei-Hsin Sun, John N Wood, Chih-Cheng Chen

Abstract

Background: Tissue acidosis is effective in causing chronic muscle pain. However, how muscle nociceptors contribute to the transition from acute to chronic pain is largely unknown.

Results: Here we showed that a single intramuscular acid injection induced a priming effect on muscle nociceptors of mice. The primed muscle nociceptors were plastic and permitted the development of long-lasting chronic hyperalgesia induced by a second acid insult. The plastic changes of muscle nociceptors were modality-specific and required the activation of acid-sensing ion channel 3 (ASIC3) or transient receptor potential cation channel V1 (TRPV1). Activation of ASIC3 was associated with increased activity of tetrodotoxin (TTX)-sensitive voltage-gated sodium channels but not protein kinase Cϵ (PKCϵ) in isolectin B4 (IB4)-negative muscle nociceptors. In contrast, increased activity of TTX-resistant voltage-gated sodium channels with ASIC3 or TRPV1 activation in NaV1.8-positive muscle nociceptors was required for the development of chronic hyperalgesia. Accordingly, compared to wild type mice, NaV1.8-null mice showed briefer acid-induced hyperalgesia (5 days vs. >27 days).

Conclusion: ASIC3 activation may manifest a new type of nociceptor priming in IB4-negative muscle nociceptors. The activation of ASIC3 and TRPV1 as well as enhanced NaV1.8 activity are essential for the development of long-lasting hyperalgesia in acid-induced, chronic, widespread muscle pain.

Figures

Figure 1
Figure 1
Representative traces of 5 acid-evoked current types in small- to medium-sized muscle afferent dorsal root ganglion (DRG) neurons. (A) In acid-sensing ion channel 3 (ASIC3)-negative, transient receptor potential cation channel V1 (TRPV1)-negative neurons, acid-induced currents were resistant to 500 μM salicylic acid (SA, a selective blocker for ASIC3) and 10 μM capsazepine (CZP, a selective blocker for TRPV1). (B) In ASIC3-positive, TRPV1-negative neurons, acid-induced currents were inhibited by SA but not by CZP. (C) In ASIC3-negative, TRPV1-positive neurons, acid-induced currents were inhibited by CZP but not by SA. (D) In ASIC3-positive, TRPV1-positive neurons, acid-induced currents were inhibited by both SA and CZP. (E) In a small subset of neurons, no acid-induced current was obtained. Numbers are number of neurons to total recorded neurons.
Figure 2
Figure 2
Involvement of peripheral TRPV1 in intramuscular-acid–induced mechanical hyperalgesia. The withdrawal responses of mouse hind paws to a 0.2-mN bending force in Trpv1+/+ and Trpv1−/− mice before and after intramuscular acid injection. (A)Trpv1+/+ and (B)Trpv1−/− mice were injected with pH 4.0 saline on days 0 and 5. (C) Co-injection of acid with capsazepine (1 nmole) at the first injection did not affect the development of hyperalgesia to the repeated acid injection in wild-type (WT) mice. (D) Capsazepine (1 nmole) at the second acid injection did not affect the development of hyperalgesia. (E) Capsazepine (1 nmole) at both acid injections prevented the development of long-lasting hyperalgesia. (F) Dual acid injections induced long-lasting hyperalgesia more than 19 days. (G) Coinjection of acid with capsazepine (1 nmole) at day 0 resulted in short-lasting hyperalgesia, for 7 days. Black arrows indicate when mice received the intramuscular acid injection. Red arrows indicate when mice received the co-injection of acid with capsazepine. B, baseline on day 0; D, day. *P < 0.05 compared with the response before the second acid injection.
Figure 3
Figure 3
Contribution of ASIC3 and TRPV1 to hyperalgesic priming in muscle nociceptors. (A,B) Dual intramuscular acid injections induced chronic hyperalgesia in Asic3+/+ mice but did not induce hyperalgesia in Asic3−/− mice. (C) Co-injection of acid with APETx2 (20 pmole) abolished the acid-induced transient hyperalgesia and prevented the development of long-lasting hyperalgesia with the second acid injection on day 5 in wild-type mice. (D) APETx2 (20 pmole) at the second acid injection produced only transient hyperalgesia in wild-type mice. (E-I) Mice received dual acid injections 1 day apart. The hyperalgesia lasted more than 12 days (E). Mice developed shorter terms of hyperalgesia up to 7 or 3 days with the first acid injection combined with 20 pmole (F) or 200 pmole (G) APETx2, respectively. (H) Co-injection of 20 pmole APETx2 and 1 nmole capsazepine in the first acid injection abolished the development of long-lasting hyperalgesia with the second acid injection. (I) Co-injection of acid and 1 nmole capsazepine shortened the second acid-induced hyperalgesia to 9 days. (J-L) Mice received dual acid injections 2 days apart. No coinjection (J), co-injection of acid and 20 pmole APETx2 (K), and co-injection of acid and 1 nmole capsazepine (L) had different effects on hyperalgesia duration induced by the second acid injection. Black arrows indicate when mice received intramuscular acid injections. Green, red, and purple arrows indicate when mice received the co-injection of acid with APETx2, capsazepine, and APETx2 combined with capsazepine respectively. B, baseline on day 0; D, day. *P < 0.05 compared with the response before the second acid injection.
Figure 4
Figure 4
The effect of ASIC3 and TRPV1 signaling on voltage-gated sodium currents (INaV) in the acid-induced muscle pain model. (A) Representative current traces show tetrodotoxin (TTX)-sensitive and -resistant (TTXr) INaV evoked in medium-sized gastrocnemius muscle (GM) DRG neurons. (B) The experimental design of INaV analysis on muscle afferent DRG neurons. Mice were injected with 20 μL pH 7.4 saline, pH 4.0 saline, pH 4.0 saline with 20 pmole APETx2, or pH 4.0 saline with 1 nmole capsazepine. Effect of intramuscular acid injections on INaV in (C) non-TTXr and (D) TTXr GM DRG neurons isolated 2 days after acid injection. Effect of intramuscular acid injection on INaV in (E) non-TTXr and (F) TTXr GM DRG neurons isolated 5 days after acid injection. Data are mean ± SEM; *P < 0.05 and **P < 0.01 vs. pH 7.4; #P < 0.05 vs. pH4.0; $ P = 0.053 vs. pH4.0.
Figure 5
Figure 5
Expression of NaV1.8 and isolectin B4 (IB4) in muscle afferent DRG neurons labeled with retrograde tracing of flourogold in NaV1.8+/−-Cre mice carrying the CAG-STOPfloxed-EGFP allele. (A) NaV1.8-positive, IB4-negative neurons represented 11.5% of total muscle afferent DRG neurons. (B) NaV1.8-negtive, IB4-positive neurons represented 4.7% of total muscle afferent DRG neurons. (C) NaV1.8-positive, IB4-positive neurons represented 20.0% total muscle afferent DRG neurons. (D) NaV1.8-negative, IB4-negative neurons represented 63.7% of total muscle afferent DRG neurons. Arrows indicate the neurons of the cell type described at the right panel.
Figure 6
Figure 6
Involvement of NaV1.8 in acid-induced chronic muscle pain. (A, B) Dual acid injections spaced 2 (A) or 5 (B) days apart induced short-term hyperalgesia (2–4 days) in NaV1.8−/− mice. (C, D) Analgesic effect of NaV1.8-selective blocker A-803467 was tested at 3 days after mice have developed chronic hyperalgesia induced by dual intramuscular acid injection spaced 5 days apart. The A-803467 (70 mg/kg, i.p.) or vehicle was injected immediately after the baseline response (control) had been obtained. B, baseline on day 0; D, day. Blue and red arrows indicate the time mice receive intraperitoneal injection of vehicle or A-803467 respectively. *P < 0.05 compared with the response before the second acid injection or control.
Figure 7
Figure 7
Protein kinase Cϵ (PKCϵ) does not contribute to nociceptor priming in acid-induced muscle pain model. (A-D) Effect of PKCϵ inhibitor peptide (V1–2, EAVSLKPT) on acid-induced hyperalgesia. Intramuscular injection of neutral saline (A, C) or 1 nmole PKCϵ inhibitor peptide (B, D) 5 hr after the first acid injection (A, B) or 3 min before the second acid injection (C, D) did not affect the development of hyperalgesia. (E-H) Effect of a general PKC inhibitor (BIM) on acid-induced hyperalgesia. Intramuscular injection of neutral saline (E, G) or 9.7 nmole BIM (F, H) 5 hr after the first acid injection (E, F) or 3 min before the second acid injection (G, H) did not affect the development of hyperalgesia. Black arrows indicate when mice received the intramuscular acid injection. B, baseline on day 0; D, day. *P < 0.05 compared with the response before the second acid injection.
Figure 8
Figure 8
A schematic model of ion channel-mediated hyperalgesic priming in muscle nociceptors. ASIC3 and TRPV1 are expressed in different subsets of muscle nociceptors with or without NaV1.8 expression. In NaV1.8-negative muscle nociceptors, ASIC3 is the major acid sensor responsible for acid-induced transient hyperalgesia and the duration of hyperalgesic priming; TRPV1 may play a minor but essential role in the nociceptor priming. In NaV1.8-positive muscle nociceptors, both ASIC3 and TRPV1 contribute to the acid-enhanced TTXr INaV, which is required for the establishment of priming that permits the development and maintenance of long-term hyperalgesia induced by a second acid insult. ASIC3 and TRPV1 are expressed alone or together, but ASIC3 is exclusively expressed in non-IB4 muscle nociceptors.
Figure 9
Figure 9
The temporal sequence of five experimental groups in the dual acid injection model. (A) Experimental design to probe the role of ASIC3 and TRPV1 in acid-induced hyperalgesia in the dual acid-injection scheme spaced 5 days apart. (B,C) Experimental design to test the role of ASIC3 and TRPV1 in acid-induced hyperalgesic priming when the second acid injection was given at day 1 (B) or day 2 (C). (D) Experimental design to test the analgesic effect of NaV1.8 blocker on the acid-induced chronic widespread pain. (E) Experimental designs to test the role of PKCϵ on acid-induced hyperalgesic priming.

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