Lynn A. Rasmussen

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.

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.

Fatiguing exercise enhances hyperalgesia to muscle inflammation

&NA; 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 2 h. Carrageenan (0.03%) was injected into the gastrocnemius muscle either 2 h before or 2 h 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 24 h 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.

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

Summary TRPV1−/− mice do not develop heat hyperalgesia after muscle inflammation; hyperalgesia is restored by reexpression of TRPV1 into both skin and muscle of TRPV1−/− mice. Abstract 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.

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-1β, 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.

Exercise-Induced Pain Requires NMDA Receptor Activation in the Medullary Raphe Nuclei

PURPOSE Pain in response to physical activity is common in people with chronic musculoskeletal pain and is likely a barrier to regular exercise, which would lead to a sedentary lifestyle. We recently developed a model of exercise-induced pain that is associated with increased activation of neurons in the medullary raphe nuclei, i.e., the nucleus raphe obscurus (NRO) and nucleus raphe pallidus (NRP). Because the NRO and NRP not only modulate motor output but also respond to noxious stimuli, we hypothesized that the NRO and NRP were key nuclei in the interaction between pain and exercise. We tested whether exercise enhances hyperalgesia through activation of N-methyl D-aspartate (NMDA) receptors in the NRO/NRP. METHODS Muscle insult was induced by two injections of pH 5.0 saline 5 d apart into one gastrocnemius muscle. We initially tested whether hyperalgesia developed in mice injected with acidic saline (pH 5.0) into the gastrocnemius muscle immediately after a 30-min or 2-h exercise task or 2 h after a 2-h exercise task. Next, we tested whether blockade of NMDA receptors in the NRO/NRP during the exercise task prevented the development of exercise-induced hyperalgesia. Finally, we evaluated changes in phosphorylation of the NR1 subunit of the NMDA receptor (pNR1) after the exercise task at times in which muscle insult was given in behavioral experiments, i.e., immediately after a 30-min or 2-h exercise task or 2 h after the 2-h exercise task. RESULTS All exercise conditions enhanced nociception (hyperalgesia) after combining with two injections of pH 5.0 saline. Microinjection of AP5 (1.0-0.1 nmol; 2-amino-5-phophonopenanoate) dose-dependently prevented the development of exercise-induced hyperalgesia. All exercise conditions increased pNR1 in the NRO and NRP. CONCLUSIONS Thus, exercise-induced pain in sedentary mice is associated with increased phosphorylation and activation of NMDA receptors in the NRO/NRP, suggesting that changes in central excitability mediate an interaction between unaccustomed exercise and pain.

Exercise prevents development of autonomic dysregulation and hyperalgesia in a mouse model of chronic muscle pain.

Abstract Chronic musculoskeletal pain (CMP) conditions, like fibromyalgia, are associated with widespread pain and alterations in autonomic functions. Regular physical activity prevents the development of CMP and can reduce autonomic dysfunction. We tested if there were alterations in autonomic function of sedentary mice with CMP, and whether exercise reduced the autonomic dysfunction and pain induced by CMP. Chronic musculoskeletal pain was induced by 2 intramuscular injections of pH 5.0 in combination with a single fatiguing exercise task. A running wheel was placed into cages so that the mouse had free access to it for either 5 days or 8 weeks (exercise groups) and these animals were compared to sedentary mice without running wheels. Autonomic function and nociceptive withdrawal thresholds of the paw and muscle were assessed before and after induction of CMP in exercised and sedentary mice. In sedentary mice, we show decreased baroreflex sensitivity, increased blood pressure variability, decreased heart rate variability, and decreased withdrawal thresholds of the paw and muscle 24 hours after induction of CMP. There were no sex differences after induction of the CMP in any outcome measure. We further show that both 5 days and 8 weeks of physical activity prevent the development of autonomic dysfunction and decreases in withdrawal threshold induced by CMP. Thus, this study uniquely shows the development of autonomic dysfunction in animals with chronic muscle hyperalgesia, which can be prevented with as little as 5 days of physical activity, and suggest that physical activity may prevent the development of pain and autonomic dysfunction in people with CMP.

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

Abstract 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.