P2X7-NLRP3-Caspase-1 signaling mediates activity-induced muscle pain in male but not female mice.

ABSTRACT: We developed an animal model of activity-induced muscle pain that is dependent on local macrophage activation and release of interleukin-1ß (IL-1ß). Activation of purinergic type 2X (P2X) 7 receptors recruits the NOD-like receptor protein (NLRP) 3 and activates Caspase-1 to release IL-1ß. We hypothesized that pharmacological blockade of P2X7, NLRP3, and Caspase-1 would prevent development of activity-induced muscle pain in vivo and release of IL-1ß from macrophages in vitro. The decrease in muscle withdrawal thresholds in male, but not female, mice was prevented by the administration of P2X7, NLRP3, and Caspase-1 inhibitors before induction of the model, whereas blockade of IL-1ß before induction prevented muscle hyperalgesia in both male and female mice. Blockade of P2X7, NLRP3, Capsase-1, or IL-1ß 24 hours, but not 1 week, after induction of the model alleviated muscle hyperalgesia in male, but not female, mice. mRNA expression of P2X7, NLRP3, Caspase-1, and IL-1ß from muscle was increased 24 hours after induction of the model in both male and female mice. Using multiplex, increases in IL-1ß induced by combining adenosine triphosphate with pH 6.5 in lipopolysaccharide-primed male and female macrophages were significantly lower with the presence of inhibitors of P2X7 (A740003), NLRP3 (MCC950), and Caspase-1 (Z-WEHD-FMK) when compared with the vehicle. The current data suggest the P2X7/NLRP3/Caspase-1 pathway contributed to activity-induced muscle pain initiation and early maintenance phases in male but not female, and not in late maintenance phases in male mice.

Short-duration physical activity prevents the development of activity-induced hyperalgesia through opioid and serotoninergic mechanisms.

Regular physical activity prevents the development of chronic muscle pain through the modulation of central mechanisms that involve rostral ventromedial medulla (RVM). We tested if pharmacological blockade or genetic deletion of mu-opioid receptors in physically active mice modulates excitatory and inhibitory systems in the RVM in an activity-induced hyperalgesia model. We examined response frequency to mechanical stimulation of the paw, muscle withdrawal thresholds, and expression of phosphorylation of the NR1 subunit of the N-methyl-D-aspartate receptor (p-NR1) and serotonin transporter (SERT) in the RVM. Mice that had performed 5 days of voluntary wheel running prior to the induction of the model were compared with sedentary mice. Sedentary mice showed significant increases in mechanical paw withdrawal frequency and a reduction in muscle withdrawal threshold; wheel running prevented the increase in paw withdrawal frequency. Naloxone-treated and MOR mice had increases in withdrawal frequency that were significantly greater than that in physically active control mice and similar to sedentary mice. Immunohistochemistry in the RVM showed increases in p-NR1 and SERT expression in sedentary mice 24 hours after the induction of the model. Wheel running prevented the increase in SERT, but not p-NR1. Physically active, naloxone-treated, and MOR mice showed significant increases in SERT immunoreactivity when compared with wild-type physically active control mice. Blockade of SERT in the RVM in sedentary mice reversed the activity-induced hyperalgesia of the paw and muscle. These results suggest that analgesia induced by 5 days of wheel running is mediated by mu-opioid receptors through the modulation of SERT, but not p-NR1, in RVM.

Regular physical activity prevents development of chronic muscle pain through modulation of supraspinal opioid and serotonergic mechanisms.

INTRODUCTION: It is generally believed that exercise produces its effects by activating central opioid receptors; there are little data that support this claim. The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) are key nuclei in opioid-induced analgesia, and opioids interact with serotonin to produce analgesia. OBJECTIVES: The purpose was to examine central inhibitory mechanisms involved in analgesia produced by wheel running. METHODS: C57/Black6 mice were given access to running wheels in their home cages before induction of chronic muscle hyperalgesia and compared with those without running wheels. Systemic, intra-PAG, and intra-RVM naloxone tested the role of central opioid receptors in the antinociceptive effects of wheel running in animals with muscle insult. Immunohistochemistry for the serotonin transporter (SERT) in the spinal cord and RVM, and pharmacological blockade of SERT, tested whether the serotonin system was modulated by muscle insult and wheel running. RESULTS: Wheel running prevented the development of muscle hyperalgesia. Systemic naloxone, intra-PAG naloxone, and intra-RVM naloxone reversed the antinociceptive effect of wheel running in animals that had received muscle insult. Induction of chronic muscle hyperalgesia increased SERT in the RVM, and blockade of SERT reversed the hyperalgesia in sedentary animals. Wheel running reduced SERT expression in animals with muscle insult. The serotonin transporter in the superficial dorsal horn of the spinal cord was unchanged after muscle insult, but increased after wheel running. CONCLUSION: These data support the hypothesis that wheel running produced analgesia through central inhibitory mechanisms involving opioidergic and serotonergic systems.

Acid-sensing ion channel 3 deficiency increases inflammation but decreases pain behavior in murine arthritis.

OBJECTIVE: Through its location on nociceptors, acid-sensing ion channel 3 (ASIC-3) is activated by decreases in pH and plays a significant role in musculoskeletal pain. We recently showed that decreases in pH activate ASIC-3 located on fibroblast-like synoviocytes (FLS), which are key cells in the inflammatory process. The purpose of this study was to test whether ASIC-3-deficient mice with arthritis have altered inflammation and pain relative to controls. METHODS: Collagen antibody-induced arthritis (CAIA) was generated by injection of an anti-type II collagen antibody cocktail. Inflammation and pain parameters in ASIC-3(-/-) and ASIC-3(+/+) mice were assessed. Disease severity was assessed by determining clinical arthritis scores, measuring joint diameters, analyzing joint histology, and assessing synovial gene expression by quantitative polymerase chain reaction analysis. Cell death was assessed with a Live/Dead assay of FLS in response to decreases in pH. Pain behaviors in the mice were measured by examining withdrawal thresholds in the joints and paws and by measuring their physical activity levels. RESULTS: Surprisingly, ASIC-3(-/-) mice with CAIA demonstrated significantly increased joint inflammation, joint destruction, and expression of interleukin-6 (IL-6), matrix metalloproteinase 3 (MMP-3), and MMP-13 in joint tissue as compared to ASIC-3(+/+) mice. ASIC-3(+/+) FLS showed enhanced cell death when exposed to pH 6.0 in the presence of IL-1ß, which was abolished in ASIC-3(-/-) FLS. Despite enhanced disease severity, ASIC-3(-/-) mice did not develop mechanical hypersensitivity of the paw and showed greater levels of physical activity. CONCLUSION: Our findings are consistent with the hypothesis that ASIC-3 plays a protective role in the inflammatory arthritides by limiting inflammation through enhanced synoviocyte cell death, which reduces disease severity, and through the production of pain, which reduces joint use.

Regular physical activity prevents development of chronic pain and activation of central neurons.

Chronic musculoskeletal pain is a significant health problem and is associated with increases in pain during acute physical activity. Regular physical activity is protective against many chronic diseases; however, it is unknown if it plays a role in development of chronic pain. The current study induced physical activity by placing running wheels in home cages of mice for 5 days or 8 wk and compared these to sedentary mice without running wheels in their home cages. Chronic muscle pain was induced by repeated intramuscular injection of pH 4.0 saline, exercise-enhanced pain was induced by combining a 2-h fatiguing exercise task with a low-dose muscle inflammation (0.03% carrageenan), and acute muscle inflammation was induced by 3% carrageenan. We tested the responses of the paw (response frequency) and muscle (withdrawal threshold) to nociceptive stimuli. Because the rostral ventromedial medulla (RVM) is involved in exercise-induced analgesia and chronic muscle pain, we tested for changes in phosphorylation of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor in the RVM. We demonstrate that regular physical activity prevents the development of chronic muscle pain and exercise-induced muscle pain by reducing phosphorylation of the NR1 subunit of the NMDA receptor in the central nervous system. However, regular physical activity has no effect on development of acute pain. Thus physical inactivity is a risk factor for development of chronic pain and may set the nervous system to respond in an exaggerated way to low-intensity muscle insults.

TRPV1 is important for mechanical and heat sensitivity in uninjured animals and development of heat hypersensitivity after muscle inflammation.

Inflammatory thermal hyperalgesia is principally mediated through transient receptor potential vanilloid 1 (TRPV1) channels, as demonstrated by prior studies using models of cutaneous inflammation. Muscle pain is significantly different from cutaneous pain, and the involvement of TRPV1 in hyperalgesia induced by muscle inflammation is unknown. We tested whether TRPV1 contributes to the development of mechanical and heat hypersensitivity of the paw in TRPV1(-/-) mice after muscle inflammation. Because TRPV1(-/-) mice lack TRPV1 at the site of inflammation (muscle) and at the testing site (paw), we do not know whether TRPV1 is important as a mediator of nociceptor sensitization in the muscle or as a heat sensor in the paw. Using recombinant herpesviruses, we reexpressed TRPV1 in TRPV1(-/-) mice in primary afferents innervating skin, muscle, or both to determine which sites were important for the behavioral deficits. Responses to repeated application of noxious mechanical stimuli to the hind paw were enhanced in TRPV1(-/-) mice; this was restored by reexpression of TRPV1 into skin. Withdrawal latencies to noxious heat were increased in TRPV1(-/-) mice; normal latencies were restored by reexpression of TRPV1 in both skin and muscle. Heat hypersensitivity induced by muscle inflammation did not develop in TRPV1(-/-) mice; mechanical hypersensitivity was similar between TRPV1(-/-) and TRPV1(+/+) mice. Heat hypersensitivity induced by muscle inflammation was restored by reexpression of TRPV1 into both muscle and skin of TRPV1(-/-) mice. These results suggest that TRPV1 serves as both a mediator of nociceptor sensitization at the site of inflammation and as a heat sensor at the paw.

Fatiguing exercise enhances hyperalgesia to muscle inflammation.

Since many people with chronic fatigue present with pain and many people with chronic pain present with fatigue, we tested if fatigue would enhance the response to pain in male and female mice. We further tested for the activation of brainstem nuclei by the fatigue task using c-fos as a marker. Fatigue was induced by having mice spontaneously run in running wheel for 2h. Carrageenan (0.03%) was injected into the gastrocnemius muscle either 2h before or 2h after the fatigue task. The mechanical sensitivity of the paw (von Frey filaments), muscle (tweezers), grip force and running wheel activity was assessed before and 24h after injection of carrageenan. Both male and female mice that performed the fatigue task, either before or after intramuscular injection of carrageenan, showed an enhanced mechanical sensitivity of the paw, but not the muscle. Ovariectomized mice showed a similar response to male mice. There was a decrease in running wheel activity after carrageenan injection, but no change in grip force suggesting that mice had no deficit in motor performance induced by the carrageenan. C-fos expression was observed in the nucleus raphe pallidus, obscurus, and magnus after the fatigue task suggesting an increased activity in the raphe nuclei in response to the fatigue task. Therefore, widespread hyperalgesia is enhanced by the fatigue response but not hyperalgesia at the site of insult. We suggest that this effect is sex-dependent and involves mechanisms in the brainstem to result in an enhanced hyperalgesia.

ASIC1 and ASIC3 play different roles in the development of Hyperalgesia after inflammatory muscle injury.

UNLABELLED: Acid-sensing ion channels (ASICs) respond to acidosis that normally occurs after inflammation. We examined the expression of ASIC1, ASIC2, and ASIC3 mRNAs in lumbar dorsal root ganglion neurons before and 24 hours after carrageenan-induced muscle inflammation. Muscle inflammation causes bilateral increases of ASIC2 and ASIC3 but not ASIC1 (neither ASIC1a nor ASIC1b) mRNA, suggesting differential regulation of ASIC1 versus ASIC2 and ASIC3 mRNA. Similar mRNA increases were observed after inflammation in knockout mice: ASIC2 mRNA increases in ASIC3-/- mice; ASIC2 and ASIC3 mRNAs increase in ASIC1-/- mice. Prior behavioral studies in ASIC3-/- mice showed deficits in secondary hyperalgesia (increased response to noxious stimuli outside the site of injury) but not primary hyperalgesia (increased response to noxious stimuli at the site of injury). In this study, we show that ASIC1-/- mice do not develop primary muscle hyperalgesia but develop secondary paw hyperalgesia. In contrast, and as expected, ASIC3-/- mice develop primary muscle hyperalgesia but do not develop secondary paw hyperalgesia. The pharmacological utility of the nonselective ASIC inhibitor A-317567, given locally, was tested. A-317567 reverses both the primary and the secondary hyperalgesia induced by carrageenan muscle inflammation. Thus, peripherally located ASIC1 and ASIC3 play different roles in the development of hyperalgesia after muscle inflammation. PERSPECTIVE: This study shows changes in ASIC mRNA expression and behavioral hyperalgesia of C57Bl/6 (wild type), ASIC1-/-, and ASIC3-/- mice before and after the induction of muscle inflammation. A-317567 was effective in reversing hyperalgesia in these animals, suggesting the potential of ASICs as therapeutic targets for muscle inflammatory pain.