Pain is ultimately a perceptual phenomenon. It is built from information gathered by specialized pain receptors in tissue, modified by spinal and supraspinal mechanisms, and integrated into a discrete sensory experience with an emotional valence in the brain. Because of this, studying intact animals allows the multidimensional nature of pain to be examined. A number of animal models have been developed, reflecting observations that pain phenotypes are mediated by distinct mechanisms. Animal models of pain are designed to mimic distinct clinical diseases to better evaluate underlying mechanisms and potential treatments. Outcome measures are designed to measure multiple parts of the pain experience including reflexive hyperalgesia measures, sensory and affective dimensions of pain and impact of pain on function and quality of life. In this review we discuss the common methods used for inducing each of the pain phenotypes related to clinical pain syndromes, as well as the main behavioral tests for assessing pain in each model.

Regular physical activity in healthy individuals prevents development of chronic musculoskeletal pain; however, the mechanisms underlying this exercise-induced analgesia are not well understood. Interleukin-10(IL-10), an anti-inflammatory cytokine which can reduce nociceptor sensitization, increases during regular physical activity. Since macrophages play a major role in cytokine production and are present in muscle tissue, we propose that physical activity alters macrophage phenotype to increase IL-10 and prevent chronic pain. Physical activity was induced by allowing C57BL/6J mice free access to running wheels for 8 weeks and compared to sedentary mice with no running wheels. Using immunohistochemical staining of the gastrocnemius muscle to label regulatory (M2, secretes anti-inflammatory cytokines) and classical (M1, secretes proinflammatory cytokines) macrophages, the percentage of M2-macrophages increased significantly in physically active mice (68.5±4.6% of total) compared to sedentary mice (45.8±7.1% of total). Repeated acid injections into the muscle enhanced mechanical sensitivity of the muscle and paw in sedentary animals that does not occur in physically active mice; no sex differences occur in either sedentary or physically active mice. Blockade of IL-10 systemically or locally prevented the analgesia in physically active mice, i.e. mice developed hyperalgesia. Conversely, sedentary mice pretreated systemically or locally with IL-10 had reduced hyperalgesia after repeated acid injections. Thus, these results suggest that regular physical activity increases the percentage of regulatory macrophages in muscle and that IL-10 is an essential mediator in the analgesia produced by regular physical activity.

Chronic muscle pain affects 20–50% of the population, is more common in women than men, and is associated with increased pain during physical activity and exercise. Muscle fatigue is common in people with chronic muscle pain, occurs in response to exercise and is associated with release of fatigue metabolites. Fatigue metabolites can sensitize muscle nociceptors which could enhance pain with exercise. Using a mouse model we tested whether fatigue of a single muscle, induced by electrical stimulation, resulted in enhanced muscle hyperalgesia and if the enhanced hyperalgesia was more pronounced in female mice. Muscle fatigue was induced in combination with a sub-threshold muscle insult (2 injections of pH 5.0 saline) in male and female mice. We show that male and female mice, fatigued immediately prior to muscle insult in the same muscle, develop similar muscle hyperalgesia 24h later. However, female mice also develop hyperalgesia when muscle fatigue and muscle insult occur in different muscles, and when muscle insult is administered 24 hours after fatigue in the same muscle. Further, hyperalgesia lasts significantly longer in females. Finally, muscle insult with or without muscle fatigue results in minimal inflammatory changes in the muscle itself, and sex differences are not related to estradiol (ovariectomy) or changes in brainstem activity (pNR1). Thus, the current model mimics muscle fatigue-induced enhancement of pain observed in chronic muscle pain conditions in the human population. Interactions between fatigue and muscle insult may underlie the development of chronic widespread pain with an associated female predominance observed in human subjects.

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.

Chronic muscle pain affects between 11–24% of the world’s population with the majority of people experiencing musculoskeletal pain at some time in their life. Acid sensing ion channels (ASICs) are important sensors of modest decreases in extracellular pH that occur within the physiological range. These decreases in extracellular pH occur in response to inflammation, fatiguing exercise,, and ischemia. Further, injection of acidic saline into muscle produces enhanced nociceptive behaviors in animals and pain in human subjects. Of the different types of ASICs, ASIC3 and ASIC1 have been implicated in transmission of nociceptive information from the musculoskeletal system. The current review will provide an overview of the evidence for ASIC3 and ASIC1 in musculoskeletal pain in both inflammatory and non-inflammatory models.

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Chronic muscle pain is a significant health problem leading to disability[1]. Muscle fatigue can exacerbate muscle pain. Metabolites, including ATP, lactate, and protons, are released during fatiguing exercise and produce pain in humans. These substances directly activate purinergic (P2X) and acid sensing ion channels (ASICs) on muscle nociceptors, and when combined, produce a greater increase in neuron firing than when given alone. Whether the enhanced effect of combining protons, lactate, and ATP is the sum of individual effects (additive) or more than the sum of individual effects (synergistic) is unknown. Using a rat model of muscle nociceptive behavior, we tested each of these compounds individually over a range of physiologic and supra-physiologic concentrations. Further, we combined all three compounds in a series of dilutions and tested their effect on muscle nociceptive behavior. We also tested a non-hydrolyzable form of ATP (α,β-meATP) alone and in combination with lactate and acidic pH. Surprisingly, we found no dose-dependent effect on muscle nociceptive behavior for protons, lactate, or ATP when given alone. We similarly found no effect after application of each two-metabolite combination. Only pH 4 saline and α,β-meATP produced hyperalgesia when given alone. When all 3 substances were combined, however, ATP (2.4μm), lactate (10mM), and acidic pH (pH 6.0) produced an enhanced effect greater than the sum of the effects of the individual components, i.e. synergism. α,β me ATP (3nmol), on the other hand, showed no enhanced effects when combined with lactate (10mM) or acidic pH (pH 6.0), i.e. additive. These data suggest that combining fatigue metabolites in muscle produces a synergistic effect on muscle nociception.

Chronic muscle pain remains a significant source of suffering and disability despite the adoption of pharmacologic and physical therapies. Muscle pain is mediated by free nerve endings distributed through the muscle along arteries. These nerves project to the superficial dorsal horn and are transmitted primarily through the spinothalamic tract to several cortical and subcortical structures, some of which are more active during the processing of muscle pain than other painful conditions. Mechanical forces, ischemia, and inflammation are the primary stimuli for muscle pain, which is reflected in the array of peripheral receptors contributing to muscle pain-ASIC, P2X, and TRP channels. Sensitization of peripheral receptors and of central pain processing structures are both critical for the development and maintenance of chronic muscle pain. Further, variations in peripheral receptors and central structures contribute to the significantly greater prevalence of chronic muscle pain in females.