Targeting ASIC3 for Relieving Mice Fibromyalgia Pain: Roles of Electroacupuncture, Opioid, and Adenosine

Many scientists are seeking better therapies for treating fibromyalgia (FM) pain. We used a mouse model of FM to determine if ASIC3 and its relevant signaling pathway participated in FM pain. We demonstrated that FM-induced mechanical hyperalgesia was attenuated by electroacupuncture (EA). The decrease in fatigue-induced lower motor function in FM mice was also reversed by EA. These EA-based effects were abolished by the opioid receptor antagonist naloxone and the adenosine A1 receptor antagonist rolofylline. Administration of opioid receptor agonist endomorphin (EM) or adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) has similar results to EA. Similar results were also observed in ASIC3−/− or ASIC3 antagonist (APETx2) injected mice. Using western blotting, we determined that pPKA, pPI3K, and pERK were increased during a dual acidic injection priming period. Nociceptive receptors, such as ASIC3, Nav1.7, and Nav1.8, were upregulated in the dorsal root ganglion (DRG) and spinal cord (SC) of FM mice. Furthermore, pPKA, pPI3K, and pERK were increased in the central thalamus. These aforementioned mechanisms were completely abolished in ASIC3 knockout mice. Electrophysiological results also indicated that acid potentiated Nav currents through ASIC3 and ERK pathway. Our results highlight the crucial role of ASIC3-mediated mechanisms in the treatment of FM-induced mechanical hyperalgesia.


Voltage-gated sodium currents in DRG neurons.
To investigate whether voltage-gated sodium currents were affected by acid saline injection produced FM mice, we conducted whole-cell patch recording to measure the ASIC3 inward currents or voltage-gated sodium currents. In DRG neurons, acid-induced ASIC3 currents (Fig. 8A) or voltage-gated sodium currents (Fig. 8B) were significantly increased in DRG neurons 8 days after FM  induction and further reversed by EA and ASIC3 gene deletion ( Fig. 8A and B). Furthermore, acid saline (pH 5.0) significantly potentiated the voltage-gated sodium currents in control DRG (Fig. 8C) that can be abolished by ASIC3 blocker salicylic acid (SA) or ERK antagonist U0126 (Fig. 8C). All data were analyzed and plotted in Fig. 8A-C. These results provide evidences that acid could increase the amplitudes of voltage-gated sodium currents through ASIC3 and ERK pathways.

Discussion
ASIC3 is involved in several types of pain syndromes such as inflammation and FM, that are highly associated with lower local pH 13,15 . These conditions activate ASIC3 via low pH, which initiates a transient inward current followed by a sustained inward current. The sustained currents from ASIC3 significantly prolong the sensation of acidosis pain in FM, arthritis, and inflammatory pain 46,47 . In this study, dual acid injections significantly initiated mechanical hyperalgesia via ASIC3, Nav1.7, and Nav1.8 signaling in both the peripheral DRG and the central SC. The potentiated ASIC3 signaling may respond to mechanical hyperalgesia from the peripheral acidosis site and transduce the acid-related pain signaling. Jeong et al. reported that ASIC3 was increased in the dorsal horn of the spinal cord in a spinal nerve ligation model, and this effect was ameliorated by amiloride, an ASIC3 blocker 48 .
Injections of the non-specific ASIC blocker amiloride, specific ASIC3 blocker APETx2, and artificial miRNA attenuated mechanical hyperalgesia in mice [49][50][51] . Izuma et al. demonstrated that ASIC3 in knee joint afferents was dramatically increased in an osteoarthritic mouse model. Injection of APETx2 reliably inhibited ASIC3 potentiation and pain behaviors 52 . Furthermore, in ASIC3 −/− mice, mechanical hyperalgesia after the induction of muscle inflammation was abolished 53 . Our previous results showed that intraplantar inflammation-mediated mechanical hyperalgesia was attenuated in ASIC3 −/− mice 54 . Repeated acidic saline injection-induced FM pain was not observed in ASIC3 −/− but was found in ASIC1 −/− mice, highlighting the crucial role of ASIC3 in this model 21 . A recent study showed that both the mechanical and thermal hyperalgesia initiated by two acidic saline injections were significantly reversed by 15 and 100 Hz EA 55 . Here we further determined that the EA-mediated attenuation of mechanical hyperalgesia was caused by a reduction in ASIC3, Nav1.7, and Nav1.8 proteins in both the peripheral DRG and central SC.
The Nav1.8 sodium channel was increased in rat and mouse DRG neurons after carrageenan and CFA-induced inflammatory pain 24,56 . Laird et al. showed that visceral pain and referred hyperalgesia were abolished in Nav1.8-null mice 57 . Intrathecal Nav1.8 antisense injections blocked Nav1.8 currents and attenuated mechanical allodynia after an intraplantar CFA injection 58 . Recently, A-803467, a novel specific blocker for Nav1.8, reduced nociception in animal models of neuropathic and inflammatory pain 59 . Our previous study showed that EA attenuated inflammatory pain by reducing Nav1.8 protein expression and functional currents 60 . Nielsen et al. reported that the sodium channel blocker mexiletine reliably reduce nociception of repeated injections of acidic saline 38 . We determined that EA attenuated mechanical hyperalgesia in FM mice by reducing ASIC3, Nav1.7, and Nav1.8 protein overexpression.

Conclusion
In this study, EA at acupoint ST36 reliably reduced mechanical hyperalgesia and motor dysfunction in acidic saline injection-induced FM mice. Both nociceptive behavior and priming molecules were abolished in ASIC3-null mice, highlighting the crucial role of this protein in FM hyperalgesia. The pPKA, pPI3K, and pERK signaling pathway was potentiated in the DRG and SC during acid injection-induced hyperalgesia priming. ASIC3, Nav1.7, and Nav1.8 proteins were increased 8 days after FM modeling, and this effect was attenuated in the DRG and SC of FM mice by EA at acupoint ST36. Furthermore, the ASIC, pPKA, pPI3K, pERK, and Nav signaling pathway was increased in the thalamus, and this effect was attenuated by EA. A similar pattern was observed for pERK. We also provide physiological evidences that voltage-gated sodium currents were increased in FM DRGs and reduced in EA or ASIC3 −/− mice. Acid saline potentiated sodium currents through ASIC3 receptors and ERK pathway. Our results provide highly valuable data for the investigation of EA-related analgesic mechanisms and can be applied in clinical practice.

Methods
Animals. Experiments were conducted using C57/B6 mice (ages 8 to 12 weeks) purchased from BioLASCO Co. Ltd, Taipei EA treatment and pharmacological injection. EA was applied using stainless steel needles (0.5″ inch, 32 G, YU KUANG, Taiwan) that were inserted into the muscle layer to a depth of 2-3 mm at ST36 acupoint. EA was administered immediate after the second injection acid saline every day at the same time (10:00-12:00 AM).

FM induction and animal behavior of mechanical hyperalgesia.
We injected 20 μ L of pH 4.0 acid saline into gastrocnemius muscle (GM) while the mice were anesthetized with isoflurane (1%). The second acid saline injection was performed at day 5 after first injection to successfully induce FM mice. All experiments were performed at room temperature (approximately 25 °C) and the stimuli were applied only when the animals were calm but not sleeping or grooming. Mechanical sensitivity was measured by testing the force of responses to stimulation with three applications of electronic von Frey filaments (North Coast Medical, Gilroy, CA, USA). Mice were placed on a metal mesh and adapted to the new environment for at least 30 min. The mechanical hyperalgesia of the hindpaw was measured before, 4 h, 1, 5, 6, 8 days after modeling. The FM mice were further euthanized and the L3-L5 DRG neurons, lumbar SC, and brain thalamus were isolated for analysis.
Rotarod. The mice were put on a rotating machine with different speeds and durations can be tested. When mice fall down, the sensor can record the falling latency. Mice were placed on an accelerating rotarod apparatus (MK-660D, Muromachi Kikai, Tokyo, Japan) for 12 trials (4 trials per day on 3 consecutive days; D1-D3) with 5-min intervals between trials. Each trial lasted for 60 s with a steady speed of 4 rpm. The latency of each mouse falling from the rod was recorded for each trial. On day 4, mice underwent 4 rpm and increase the speeds with a 3 rpm increase with a 10 s duration.
Tissue sampling and western blot analysis. L3-L5 DRG, lumbar SC, and thalamus tissues were excised to extract proteins. The total proteins were prepared by homogenizing the DRG, SC, and thalamus in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 250 mM NaCl, 1% NP-40, 5 mM EDTA, 50 mM NaF, 1 mM Na 3 VO 4 , 0.02% NaNO 3 , and 1 × protease inhibitor cocktail (AMRESCO). The extracted proteins (30 μ g per sample according to the BCA protein assay) were subjected to 8% SDS-Tris glycine gel electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBS-T buffer (10 mM Tris pH 7.5, 100 mM NaCl, 0.1% Tween 20), incubated with the first antibody in TBS-T and 1% bovine serum albumin, and incubated for 1 h at room temperature. A peroxidase-conjugated anti-rabbit antibody (1:5000) was used as the secondary antibody. The bands were visualized using an enhanced chemiluminescencent substrate kit (PIERCE) with LAS-3000 Fujifilm (Fuji Photo Film Co. Ltd). If appropriate, the image intensities of specific bands were quantified with NIH ImageJ software (Bethesda, MD, USA). The protein ratios were obtained by dividing the target protein intensities by the intensity of α -tubulin in the same sample. The calculated ratios were then adjusted by dividing the ratios from the same comparison group relative to the control.
Immunohistochemical staining. Mice were anesthetized with isoflurane and then perfused transcardially with 4% paraformaldehyde. The tissue samples were cryprotected with 30% sucrose. The tissues were cut to a thickness of 15 μ m and were post-fixed briefly with 4% paraformaldehyde and then incubated with blocking solution containing 3% BSA, 0.1% Triton X-100, and 0.02% sodium azide in PBS for 2 h at room temperature. After blocking, the sections were incubated at 4 °C overnight with the primary antibodies prepared in blocking solution. The secondary antibody was goat anti-rabbit (1:500) antibody (Molecular Probes, Carlsbad, CA, USA). We incubated the slices with fluorescence-conjugated secondary antibodies or avidin-biotin horseradish peroxidase complex (1 h), washed them three times with 0.1 M Tris buffer (5 min each), and then developed them in diaminobenzidine tetrahydrochloride (1-2 min), before washing three times with 0.1 M Tris buffer (5 min each). Finally, the sections were incubated with 0.1 M Tris buffer to stop the reaction. The slides were mounted with cover slips and visualized by using a CKX41 microscope with an Olympus U-RFLT50 Power Supply Unit (Olympus, Tokyo, Japan).

Statistical analysis.
All of the data were expressed as the mean ± standard error. Significant differences between groups were tested using ANOVA, followed by a post hoc Tukey's test. p < 0.05 was considered significantly different.