ASIC3 Is Required for Development of Fatigue-Induced Hyperalgesia

Abstract

An acute bout of exercise can exacerbate pain, hindering participation in regular exercise and daily activities. The mechanisms underlying pain in response to acute exercise are poorly understood. We hypothesized that proton accumulation during muscle fatigue activates ASIC3 on muscle nociceptors to produce hyperalgesia. We investigated the role of ASIC3 using genetic and pharmacological approaches in a model of fatigue-enhanced hyperalgesia. This model uses two injections of pH 5.0 saline into muscle in combination with an electrically-induced fatigue of the same muscle just prior to the second injection of acid to induce mechanical hyperalgesia. We show a significant decrease in muscle force and decrease in muscle pH after 6 minutes of electrical stimulation. Genetic deletion of ASIC3 using knockout mice and pharmacological blockade of ASIC3 with APETx2 in muscle prevents the fatigue-enhanced hyperalgesia. However, ASIC3−/− mice and APETx2 have no effect on the fatigue response. Genetic deletion of ASIC3 in primary afferents innervating muscle using an HSV-1 expressing miRNA to ASIC3 surprisingly had no effect on the development of the hyperalgesia. Muscle fatigue increased the number of macrophages in muscle, and removal of macrophages from muscle with clodronate liposomes prevented the development of fatigue-enhanced hyperalgesia. Thus, these data suggest that fatigue reduces pH in muscle that subsequently activates ASIC3 on macrophages to enhance hyperalgesia to muscle insult.

Resident Macrophages in Muscle Contribute to Development of Hyperalgesia in a Mouse Model of Noninflammatory Muscle Pain

“…Quantitation of F4/80-stained muscle macrophages showed that the average number of macrophages was approximately 20.83 +/− 2.80 in 5 sections in gastrocnemius muscles treated with Clod and was significantly less than the number from PBS-Lipo which averaged 82.33 +/− 9.79 (P<0.0001), similar to prior data by us [17]. Twenty-four hours after the second injection of acidic saline, responses to repeated mechanical stimulation of the paw increased and muscle withdrawal thresholds significantly decreased bilaterally in control animals treated with PBS-Lipo (p<0.0001).…”

“…Similarly, in human subjects infusion of acidic solutions into muscle produces pain and hyperalgesia both locally at the site of infusion but also in a referred pain site [15]. It is well known that ASICs are sensitive to H + and involved in acute and chronic muscle hyperalgesia [1;17;52;61]. In general, they are widely expressed nervous systems, including sensory nerves that innervate the skeletal muscle (muscle afferents)[1;35].…”

“…Immunohistochemistry for macrophages was performed on cryopreserved sections of mouse muscle using rat monoclonal antibody recognizing F4/80 antigen (AbDSerotec, Raleigh, North Carolina), a glycoprotein expressed by mature murine macrophages using techniques we previously published [17;30]. Male and female C57BL/6J mice were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg) and transcardially perfused with heparinized saline followed by either 1) 4% paraformaldehyde (clodronate quantification), or 2) PLP fixative (2% paraformaldehyde; 75mM Lysine; 10mM periodate; 0.05M Phosphate Buffer) (Fig.…”

Neurobiology of fibromyalgia and chronic widespread pain

“…As outlined above, two injections of acidic saline into a single gastrocnemius muscle produce hyperalgesia not only at the site of injection but also in the contralateral muscle and viscera. Pharmacological blockade of ASIC3, with APETx2, at the first or second injection, prevents the development of the widespread long-lasting hyperalgesia (Karczewski et al, 2010, Chen and Chen, 2014, Gregory et al, 2015b), suggesting activation of ASIC3 on nociceptors innervating muscle is important for development of widespread hyperalgesia (Figure 3). Similarly, the hyperalgesia in the repeated acid model, or the fatigue-induced model, requires activation of acid sensing ion channel 3 (ASIC3) since the hyperalgesia does not in ASIC3 knockout mice (Sluka et al, 2003, Gregory et al, 2015a)(Figure 3).…”

“…Pharmacological blockade of ASIC3, with APETx2, at the first or second injection, prevents the development of the widespread long-lasting hyperalgesia (Karczewski et al, 2010, Chen and Chen, 2014, Gregory et al, 2015b), suggesting activation of ASIC3 on nociceptors innervating muscle is important for development of widespread hyperalgesia (Figure 3). Similarly, the hyperalgesia in the repeated acid model, or the fatigue-induced model, requires activation of acid sensing ion channel 3 (ASIC3) since the hyperalgesia does not in ASIC3 knockout mice (Sluka et al, 2003, Gregory et al, 2015a)(Figure 3). However, when an ASIC antagonist is given after the development of hyperalgesia, there is no effect on the hyperalgesia, suggesting that once developed the hyperalgesia is independent of nociceptor activation by acidic pH (Karczewski et al, 2010, Gautam et al, 2012).…”