Why it hurts to exercise: a study of sex, acid sensing ion channels, and fatigue metabolites in the onset of muscle pain

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1 University of Iowa Iowa Research Online Theses and Dissertations Spring 2015 Why it hurts to exercise: a study of sex, acid sensing ion channels, and fatigue metabolites in the onset of muscle pain Nicholas Scott Gregory University of Iowa Copyright 2015 Nicholas S Gregory This dissertation is available at Iowa Research Online: Recommended Citation Gregory, Nicholas Scott. "Why it hurts to exercise: a study of sex, acid sensing ion channels, and fatigue metabolites in the onset of muscle pain." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Neuroscience and Neurobiology Commons

2 WHY IT HURTS TO EXERCISE: A STUDY OF SEX, ACID SENSING ION CHANNELS, AND FATIGUE METABOLITES IN THE ONSET OF MUSCLE PAIN by Nicholas Scott Gregory A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Neuroscience in the Graduate College of The University of Iowa May 2015 Thesis Supervisor: Professor Kathleen A. Sluka

3 Copyright by NICHOLAS SCOTT GREGORY 2015 All Rights Reserved

4 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL PH.D. THESIS This is to certify that the Ph.D. thesis of Nicholas Scott Gregory has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Neuroscience at the May 2015 graduation. Thesis Committee: Kathleen A. Sluka, Thesis Supervisor Christopher Benson Tim Brennan Laura Frey-Law Donna Hammond

5 To my mother and father ii

6 All finite things reveal infinitude: The mountain with its singular bright shade Like the blue shine on freshly frozen snow, The after-light upon ice-burdened pines; Odor of basswood on a mountain-slope, A scent beloved of bees; Silence of water above a sunken tree: The pure serene of memory in one man, -- A ripple widening from a single stone Winding around the waters of the world. Theodore Roethke The Far Field iii

7 ACKNOWLEDGMENTS Thank you to the University of Iowa Medical Scientist Training Program, Interdisciplinary Training Program in Pain Research, and National Institutes of Health for funding. Thank you to my thesis committee for their timely wisdom, invaluable criticism, and generosity. Thank you to my mentor, Kathleen Sluka, for her support and guidance throughout my training. iv

8 ABSTRACT Exercise has numerous health benefits. Yet, exercise can exacerbate pain for individuals with chronic musculoskeletal pain conditions such as myofascial pain syndrome (MPS) and fibromyalgia (FM). The exacerbation is out of proportion to the activity performed and lasts for long periods of time even after the cessation of activity. This pain acts as a barrier to healthy exercise and physical rehabilitation, which, when applied consistently, are effective treatments for MPS and FM two diseases that produce substantial suffering and disability. The goal of the proposed studies is to determine the underlying peripheral mechanisms that contribute to enhanced pain following exercise. A better understanding of these mechanisms will lead to better pain management and prevention for these diseases. Previous data show that two hours of running wheel activity lowers the threshold necessary to induce muscle pain by acidic saline injection, producing robust pain behaviors to normally innocuous stimuli. Muscle activity that produces fatigue is associated with extracellular increases in protons, lactate, and ATP. These fatigue metabolites can directly activate muscle nociceptors and, when combined, produce a potentiated effect. Acid sensing ion channels (ASICs) are non-selective cation channels that open in response to increased proton concentrations, a response that is enhanced when lactate binds at a separate location. Ionotropic purinergic receptors (P2X) similarly produce an inward current in response to elevated ATP. Evidence suggests certain ASIC and P2X subtypes are capable of a physical interaction that allows ASIC activation at lower proton concentrations in the presence of ATP. This suggests that ATP, lactate, and protons released during exercise could activate ASIC and P2X receptors on muscle nociceptors, exciting the nociceptors and sensitizing them to subsequent muscle insult. v

9 However, the limitations of these experiments leave several gaps. First, the running wheel task fails to produce measurable increases in fatigue metabolites, possibly due to the fact that there was minimal fatigue (10%) or that their levels quickly return to baseline. Further, the running wheel task depends on central nervous system (CNS) activity and volitional running, which may introduce confounding factors upstream of muscle activation and result in large variation in the rate and duration of running. Second, it is unclear whether ASICs are necessary for the development of mechanical hyperalgesia induced by muscle activity, nor is it understood which ASIC subtypes might be required for such an effect. Finally, the molecules necessary for the induction of mechanical hyperalgesia after exercise are not known. Protons, lactate, and ATP have been suggested, but it is not known if these compounds are themselves sufficient or if they interact in an additive or synergistic manner. We address these concerns by developing an electrically-stimulated muscle fatigue paradigm that reliably fatigues a single muscle independent of the CNS, allowing for metabolite measurement during muscle activity and in vivo study of molecular mechanisms of muscle pain in the peripheral tissue. We then use genetic and pharmacologic approaches to test the role of ASIC subtypes in the development of mechanical hyperalgesia after exercise. Finally, we test the effectiveness of by-products of muscle activity in recapitulating the effects of the exercise-enhanced pain model.. vi

10 PUBLIC ABSTRACT Exercise has numerous health benefits. Yet, exercise can be unpleasant, which may prevent adherence to a fitness program. Further, for people with certain chronic conditions, exercise can make some of their symptoms worse. In these chronic conditions, which include myofascial pain syndrome and fibromyalgia, exercise results in unexpectedly severe muscle pain and fatigue. The goal of this research is to better understand how exercise produces muscle pain so that such pain can be treated or prevented. Treating this pain may, in turn, result in better adherence to fitness programs among people who find exercise-related pain intolerable. Exercise consists of repeated muscle contractions, which results in accumulation of substances in the fluid around muscle. These substances include ATP, lactate, and acid. When these substances reach high enough levels in the fluid around muscle, they can cause pain by activating specialized pain receptors on nerves. In fact, the combination of ATP, lactate, and acid produces enhanced pain that is greater than the sum of the effects of each individual molecule. This research has three main objectives. The first is to develop an animal model of muscle pain produced by exercise. The second is to identify receptors responsible for muscle pain after exercise. Finally, the research looks at the interaction between ATP, lactate, and acid to determine the levels of each compound, alone or in combination, that are necessary for the development of muscle pain. vii

11 TABLE OF CONTENTS LIST OF TABLES... ix LIST OF FIGURES...x CHAPTER I INTRODUCTION...1! 1.1 Introduction...1! 1.2 Anatomy...3! 1.3 Sensory Transduction...4! 1.4 Models of Muscle Pain...10! 1.5 Chronic Pain Mechanisms...13! 1.6 Sex Differences...18! CHAPTER II FATIGUE-ENHANCED HYPERALGESIA IN RESPONSE TO MUSCLE INSULT: INDUCTION AND DEVELOPMENT OCCUR IN A SEX-DEPENDENT MANNER ! 2.1 Abstract...22! 2.2 Introduction...23! 2.3 Materials and Methods...26! 2.4 Results...35! 2.5 Discussion...40! CHAPTER III CONTRIBUTION OF ACID SENSING ION CHANNELS TO MECHANICAL HYPERALGESIA AFTER EXERCISE...55! 3.1 Abstract...55! 3.2 Introduction...56! 3.3 Materials and Methods...57! 3.4. Results...64! 3.5 Discussion...67! CHAPTER IV EFFECT OF INTRAMUSCULAR PROTONS, LACTATE, AND ATP ON MECHANICAL HYPERALGESIA IN RATS...79! 4.1 Abstract...79! 4.2 Introduction...80! 4.3 Materials and Methods...82! 4.4 Results...84! 4.5 Discussion...86! CHAPTER V DISCUSSION AND CONCLUSION...98! 5.1 Discussion of Hypotheses...98! Clinical Significance and Conclusion...106! REFERENCES...110!! viii

12 LIST OF TABLES Table 2-1 pnr1 staining in the RVM of mice treated with repeated ph 5 injections and electrically stimulated muscle contractions. Total number of cells stained for pnr1 across 5 sections containing the RVM or facial nucleus. There were no significant differences by sex, treatments or location....54!! ix

13 LIST OF FIGURES Figure 2-1 Electrically stimulated muscle contractions result in muscle fatigue and enhance the response to ph 5 saline injections. (A) Force elicited by 100 Hz electrical stimuli before and after 6 m fatiguing contractions (males n=12, females n=13) * p < 0.05, difference from baseline. (B) Recovery of force after 6 m fatiguing contractions (n=3) * p < 0.05, difference from initial, p < 0.05, difference from start of recovery. (C) Behavioral measure of sensitivity to mechanical stimuli at baseline and after treatment with a single ph 5 saline injection with electrically stimulated muscle contractions(males, n=6), two ph 5 acidic saline injections alone (pooled, males & females n=6 each), two ph 7.2 saline injections with electrically stimulated muscle contractions (pooled, males & females n=6 each), two ph 5 saline injections with test contractions but not fatigue (pooled, males & females n = 6 each), or two ph 5 saline injections with electrically stimulated muscle contractions (pooled, males & females n=6 each) * p < 0.05, difference from baseline and control groups at 24h....49! Figure 2-2 Sex differences in mechanical hypersensitivity after repeated ph 5 saline injection and electrically stimulated muscle contractions. (A) Duration of mechanical hypersensitivity (n=6) * p < 0.05, difference from baseline, p < 0.05, difference from females.(b & C) The final ph 5 saline injection was delayed after electrically stimulated muscle contractions for 0, 2, or 24 h (n=6 per group) * p < 0.05, difference from baseline, p < 0.05 difference from intact and ovariectomized females. (D, E, F, G) A spatial relationship between ph 5 saline injection and electrically stimulated muscle contractions was tested by varying the location of acidic saline injection (n=6) * p < 0.05, difference from baseline, p < 0.05, difference from intact and ovariectomized females. (D & E) ph 5 saline injections and electrically stimulated muscle contractions were given into the same muscle and mechanical hypersensitivity was tested in the gastrocnemius muscles (D) ipsilateral and (E) contralateral to the site of treatment. (F & G) ph 5 saline injections were given to the gastrocnemius muscle contralateral to the site of electrically stimulated muscle contractions. Mechanical hypersensitivity was tested in the muscle (F) ipsilateral and (G) contralateral to the site of fatigue....51! Figure 2-3 Inflammation in muscle treated with repeated ph 5 saline injections and electrically stimulated muscle contractions. Representative hemotoxylin and eosin stained gastrocnemius muscle sections taken from (A) naive mice and 24h after treatment with (B) repeated ph 5 saline injections alone, (C) electrically stimulated muscle contractions with ph 5 saline injections, and (D) 3% carrageenan. Thin arrows indicate centralized rowing of myocyte nuceli, indicative of regeneration, thick arrows show sites of multifocal inflammatory cell infiltrates which are magnified within the insets where specific cell types are circled (in (C) it is lymphocytes, in (D) it is neutrophils) and asterisks indicate degenerative myocytes, scale bars = 20 µm (inset scale bars = 10 µm). (E) Colorimetric assay to quantify myeloperoxidase, a neutrophil marker, in whole, homogenized gastrocnemius muscle tissue in naive mice (n=7) and after treatment with 3% carrageenan (n=6), repeated ph 5 saline injections alone (n=7), repeated ph 5 injections with electrically stimulated muscle contractions (n=7), and repeated ph 7.2 x

14 saline injections with electrically stimulated muscle contractions (n=7). * p < ! Figure 3-1 Measurement of force during fatiguing muscle contractions. No significant differences were observed between ASIC1-/- (n=7), ASIC3-/- (n=7), and wild type mice (n=7) (p = 0.116) (A). No significant differences were observed between mice pre-treated with the ASIC1 antagonist psalmotoxin-1(12 nm n = 4, 120 nm = 8, vehicle n = 9), ASIC3 antagonist ApeTx2 (20 µm n = 7, 200 µm n = 5, vehicle n = 9), or saline (psalmotoxin- 1, p = and ApeTx2, p =0.115) (B & C). Mean + SEM....75! Figure 3-2 ph of Muscle Before and After Fatiguing Muscle Contractions. Individual replicates for each ph measurement in before and after fatigue (n=5), and in inactive control (n=4) are shown in (A). The ph of fatigued muscle was significantly lower than baseline and the inactive group (B). *, p < , difference from baseline and inactive post treatment. Mean + SEM....76! Figure 3-3 Comparison of muscle withdrawal threshold in ASIC1-/-, ASIC3-/-, and wild type mice in the exercise enhanced pain model. At baseline there were no significant differences in muscle withdrawal threshold between ASIC1a (n=7), ASIC3-/- (n=8), and wild type animals (n=5). The muscle withdrawal threshold of ASIC3-/- mice was significantly higher than both ASIC1-/- and wild type after treatment with the exercise enhanced pain model. No sex differences were observed within ASIC1a-/- and ASIC3-/- mice. There were no significant differences between ASIC1-/- and wild type mice. *, p < 0.05, difference from ASIC1-/- and wild type. Mean + SEM....77! Figure 3-4 Comparison of muscle withdrawal thresholds in mice pre-treated with ASIC3 (A) and ASIC1 (B) antagonists in the exercise enhanced pain model. All groups showed similar muscle withdrawal thresholds at baseline. Muscle withdrawal thresholds of animal pre-treated with moderate and high doses of the ASIC3 antagonist ApeTx2 (20 µm n = 14, 70 µm n = 8, 200 µm n = 7, vehicle n = 16, naïve n = 4) were significantly greater than vehicle or low dose ApeTx2 (A). *, p < 0.05, difference from saline controls. Muscle withdrawal thresholds of animals pre-treated with the ASIC1 antagonist Psalmotoxin-1 (12 nm n = 8, 40 nm n = 7, 120 nm n = 12, vehicle n = 16, naïve n =4) were significantly higher than the vehicle control(b). *, p < 0.05, difference from naive. Mean + SEM....77! Figure 3-5 Specificity of psalmotoxin effects to muscle ASIC1a. (A)Muscle withdrawal threshold in ASIC1a knockout mice pre-treated with psalmotoxin- 1 in the exercise enhanced pain model. ASIC1a knockout mice were pretreated with either psalmotoxin (n = 4) or saline (n = 4) prior to fatiguing muscle contractions. There were no significant differences between groups (p = 0.458). (B)Muscle withdrawal threshold after injecting psalmotoxin-1 into the treated versus untreated muscle in the exercise enhanced pain model. Wild type mice were pre-treated with psalmotoxin in either the ispsilateral (fatigued, acid injected; n = 4) or contralateral (untreated; n = 4) muscle prior to fatiguing muscle contractions. There were no significant differences between groups at baseline. Muscle withdrawal thresholds after exposure to the exercise enhanced pain model were significantly higher in mice treated with ipsilateral injection of psalmotoxin (*, p < 0.001)....78! xi

15 Figure 4-1 Effect of acidic saline intramuscular injection on muscle withdrawal thresholds. Rats were injected with ph 4 (n = 12), ph 4.5 (n = 6), ph 5.0 (n = 6), ph 6.0 (n = 6), or neutral (n = 6) saline. There were no significant differences between groups at baseline. Muscle withdrawal thresholds were significantly lower in rats treated with ph 4 saline compared to higher ph solutions, which showed no change in muscle withdrawal threshold. Data are mean + SEM. (*, p < 0.001)....93! Figure 2 Effect of intramuscular lactate injection on muscle withdrawal thresholds. Rats were injected with lactate 470 µm (n = 8), 1.5 mm (n = 8), 4.7 mm (n = 8), 15 mm (n = 9), 47 mm (n = 8), 150 mm (n = 6), 470 mm (n = 6), 1.5 M (n = 6), or ph 7.0 saline (n = 6). No significant differences were observed between rats treated with saline and those treated with lactate (p > 0.05). Data are mean + SEM....94! Figure 4-3 Effect of ATP intramuscular injection on muscle withdrawal threshold. Rats were injected with ATP 760 nm (n = 6), 7.6 µm (n = 6), 76 µm (n = 6), 760 µm (n = 12), 2.4 mm (n = 6), 7.6 mm (n = 6), 24 mm (n = 6), or ph 7.0 saline (n = 6). No significant differences were seen between animals treated with saline and those treated with ATP (p>0.05). Data are mean + SEM....95! Figure 4-4 Effect of combinations of ph, lactate, and ATP on muscle withdrawal threshold. Rats were injected with either a combination of ATP, lactate, and protons, neutral saline (n = 6), or 3% carrageenan (n = 4). Iso1 consisted of ph 6.0, 474 µm lactate, and 2.4µM ATP (n = 6). Iso2 consisted of ph 6.5, 150µM lactate, 760 nm ATP (n = 6). Iso3 consisted of ph 7.0, 47 µm lactate, 240 nm ATP (n = 5). Iso4 consisted of ph 7.5, 15 µm lactate, 76 nm ATP (n = 6). The group with the lowest combination of ph, lactate, and ATP (Iso4) demonstrated significantly lower muscle withdrawal thresholds than the saline control (*, p < 0.05).Data are mean + SEM....96! Figure 4-5 Effect of α,β-methylene ATP on muscle withdrawal threshold. Rats were injected with α,β-methylene ATP 10 nm (n = 4), 30 nm (n = 5), 100 nm (n = 5), or neutral saline (n = 7). Muscle withdrawal threshold decreased significantly in rats injected with either 10, 30, or 100 nm α,β-methylene ATP as compared to vehicle controls (*, p < 0.05) Data are mean + SEM....97!! xii

16 1 CHAPTER I INTRODUCTION Introduction 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(1). Older, sedentary, unemployed, less well educated individuals with anxiety are more likely to suffer from chronic muscle pain (2, 3). Those who suffer from chronic muscle pain often report decreased productivity and a significant portion have had to change jobs or quit working entirely as a result of their pain(4). In the U.S. alone, such chronic pain is estimated to have an economic burden of $500 billion dollars annually(5). The two primary disease of chronic muscle pain are myofascial pain syndrome (MPS) and fibromyalgia (FM). Myofascial pain syndrome is characterized by regional muscle pain with areas of focal tenderness with pressure. These trigger points are palpable, taut masses typically found within the muscle belly. Affected muscles are stiff and contracted, which can put stresses on 1 Manuscript published as: Gregory, N. S., & Sluka, K. A. (2014). Anatomical and Physiological Factors Contributing to Chronic Muscle Pain. Current Topics in Behavioral Neurosciences. doi: /7854_2014_294

17 2 adjacent or antagonist muscles that lead to the development of secondary trigger points. MPS is often associated with anxiety and depression(6, 7). Fibromyalgia is the most extreme example of chronic muscle pain. Pain is widespread and, like MPS, patients often report areas of local tenderness to palpation. Along with pain, FM patients also report fatigue, depression, insomnia, and cognitive impairment (8, 9). The combination of these symptoms, particularly pain and fatigue, leads FM patients to report significantly more disability and worse physical fitness than other chronic pain conditions (10, 11). However, many other conditions, such as whiplash injury, neck pain, and chronic low back pain, also have subpopulations that can be considered chronic muscle pain that is localized to one or two regions. Treatment of chronic muscle pain has only been partially effective. Pharmacologic approaches have shown some usefulness in treating the pain in these conditions, but the fatigue, cognitive, and affective components have shown little improvement (9). Non-steroidal anti-inflammatory drugs, certain antidepressants(tricyclic antidepressants and serotonin-norepinephrine reuptake inhibitors), and certain anti-epileptics (gabapentin, pregabalin) have some benefit for pain and mixed effects on the attendant depression, insomnia, cognitive impairment, and fatigue (6, 9). Non-pharmacological approaches have significant roles in treatment of both MPS and FM. MPS patients benefit from gentle stretching of muscles with trigger points to reduce stiffness and ergonomic adjustments to reduce muscle overuse(6, 12). In FM, regular moderate exercise

18 3 improves pain, fatigue, mood, and loss of sleep, though exercise can acutely exacerbate pain in patients that are unaccustomed to exercise(8, 12). Chronic muscle pain, with its substantial suffering and poor treatment options, remains a significant health burden worldwide. 1.2 Anatomy Sensory fibers innervating the muscle are classified into four groups based on size and myelination(13). Group I and II fibers are large, myelinated fibers and play a role in proprioception. Group III and IV fibers are small myelinated and unmyelinated fibers, respectively, corresponding to A and C fibers of the skin. Group III and IV, but not I and II, respond to application of noxious mechanical, thermal, and chemical stimuli to the muscle, indicating that they transmit nociceptive information from the muscle(14-16). The endings of group III and IV afferents are distributed throughout the muscle, terminating in extrafusal fibers, intrafusal fibers, connective tissue, fat, and the adventitia of both venules and arterioles. The majority of these nerves terminate as free endings in the adventitia of blood vessels, an ideal location for sampling blood for metabolites released as by-products of muscle contraction(17). Additionally, the fascia surrounding muscle may contribute to symptoms of muscle pain. Nociceptive fibers innervate the fascia(18), which are sensitive to both noxious chemical and mechanical stimuli(19). These fibers can be sensitized by muscle contractions(20, 21) and have been implicated in the development of low back pain associated with the erector spinae muscles(22, 23).

19 4 Upon leaving the muscle, nociceptive and proprioceptive muscle afferents project to different layers of the spinal cord. Group I and II afferents project to deep layers of the dorsal horn (layers III-IV), while group III and IV afferents project to superficial layers (I, II, and the edge of III)(24, 25). Nociceptive muscle afferents then project to the brain via the spinothalamic tract(26), where they terminate in the nucleus submedius, paraventricular nucleus anterior (PVA)(27, 28), and ventral posterolateral nucleus of the thalamus(29). Muscle pain activates multiple regions of the brain in common with skin pain anterior cingulate cortex, primary and secondary somatosensory cortex, dorsolateral prefrontal cortex, and the insula(30). However, muscle pain also activates regions of the brain associated with emotional processing: bilateral amygdala, caudate, orbitofrontal cortex, hippocampus, parahippocampus, and the superior temporal pole(31, 32). Further, muscle pain is associated with pathways in the periaqueductal grey and rostral ventromedial medulla, which have been implicated in bulbospinal modulation of pain(33, 34). 1.3 Sensory Transduction Sensory Transduction Muscle afferents are well suited for detecting the condition of muscle tissue. While skin afferents provide detailed information about the external environment and give painful feedback in response to external sources of injury, muscle afferents are sensitive to the capacity of the muscle to function as a forcegenerating organ with intermittent periods of high metabolic demand. Muscle

20 5 afferents have poor spatial resolution as compared to skin, with single mechanically sensitive fibers innervating multiple receptive fields as far as 2 cm apart(35). This fits with the clinical description of muscle pain as diffuse and hard to localize. Yet, muscles are equipped with mechanically sensitive fibers specialized to detect innocuous pressure, noxious pressure, and graded forces of contraction(36). Patients often report mild muscle pain at rest that is acutely worsened by either pressure or use of injured muscle. In addition to mechanical forces, muscle afferents appear to be attuned to byproducts released during muscle contraction or accumulated under ischemic conditions, as ischemia itself is sufficient to elicit pain(37). Thermal sensitive fibers, activated by cool or warm temperatures, are also present in muscle(35, 38), but increases in temperature due to physical activity are relatively small and do not approach the range that would activate heat nociceptors (39, 40). Multiple changes occur in muscle during fatigue. The fibers of the muscle themselves are placed under significant strain, which correlates with the release of interstitial ATP (41, 42). As the metabolic demands of muscle contraction increase, elevated oxygen uptake for aerobic respiration stimulates the production of reactive oxygen species(43) until the capacity for aerobic respiration is exceeded, at which point muscle begins accumulating lactic acid(44). Norepinephrine concentration is also elevated during exercise(45). Damage to muscle tissue during use plays an important role in muscle pain, as activity that

21 6 result in greater tissue damage produces significantly more pain than nondamaging muscle activity(46-49). This damage results in recruitment of neutrophils and macrophages(50, 51) and the release of a wide range of factors including prostaglandin E2(52, 53), bradykinin (54), serotonin (55, 56), and NGF (54, 57). Multiple targets for these molecules have been studied both in isolated cells and whole animals Acid Sensing Ion Channels. Acid sensing ion channels (ASICs) are non-selective cation channels activated by increases in extracellular proton concentrations(58). Four genes code for six subunit types (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) with distributions across the central and peripheral nervous systems depending on the subunit(59-62). It is particularly interesting that ASICs are found on fibers terminating in the tunica adventitia of muscular arteries, an ideal location for detecting protons released during muscle contraction(63). A functional channel is composed of three subunits. In the case of muscle afferents, ASICs form as heterotrimers of the ASIC1a, ASIC2, and ASIC3 subunits. In DRG innervating muscle ASICs are activated by ph and demonstrate a rapidly desensitizing peak current followed by a smaller sustained current (64). Application of acidic solutions activates group IV afferents(65) and produces enhanced pain behavior in animals(66) and pain in humans(67). In animal models, ASIC3 -/- mice do not

22 7 develop hyperalgesia after repeated intramuscular acidic saline injection and show reduced secondary hyperalgesia after injection with an inflammatory compound(68, 69). ASIC1a -/- mice, on the other hand, do develop hyperalgesia with intramuscular acidic saline injections but show reduced primary hyperalgesia in animals with muscle inflammation(68, 70). In contrast, downregulation of ASIC3 in adult mice prevents the development of both primary and secondary hyperalgesia after muscle inflammation(69). A number of factors can alter the function of ASICs. Expression of ASIC3 is enhanced under ischemic conditions and inflammation (71, 72) (73). Further, fatiguing muscle prior to treatment with acid or inflammatory compounds results in enhanced pain behavior in animals(74, 75). Electrophysiological studies have shown lactate increases the sensitivity and magnitude of currents in ASIC1a and ASIC3 in response to decreases in ph(76). Finally, release of ATP can enhance ASIC current through an interaction between ASIC3 and P2X5(77). Thus, ASICs appear to play a critical role in the development of muscle hyperalgesia as they can be activated under a number of conditions that are related to inflammation and fatigue and their selective knockdown or antagonism significantly reduces mechanical sensitivity in conditions known to produce muscle hyperalgesia.

23 Ionotropic Purine Receptors. The P2X family of ligand-gated ion channels is large and plays a role in diverse functions. Notably, P2X channels have been implicated in detecting stretch in the bladder(78, 79) and muscle afferents(80) as well as detection of hypoxia in the carotid bodies(81, 82) and lungs (83-85). P2X receptors also contribute to a wide range of pain conditions including cardiac(86, 87), visceral(88), inflammatory(89-91), neuropathic(92, 93), and cancer pain(94, 95). Application of P2X receptor agonists in the gastrocnemius(96, 97) and masseter(98) muscles activates group III and IV muscle afferents; similar treatments in animals(99) and humans(100) is painful. The specific subtype of P2X receptor contributing to these effects is unclear. P2X3 is well studied and localizes to the DRG(101) as well as the terminals of nerve fibers innervating the tunica adventitia of muscle afferents(63). Eccentric muscle contractions, which are associated with muscle pain, increase P2X3 expression in muscle afferents(42). Similarly, P2X4 and P2X5 are also found in the DRG(102, 103) and studies of calcium imaging in cultured DRGs show that cells responding to physiologically relevant combinations of lactate, protons, and ATP are inhibited by an array of P2X antagonists in a pattern suggesting both P2X4 and P2X5 contribute to the response(104). Further, P2X5 is capable of physically interacting with and enhancing the proton-gated current through ASIC3, which is expressed in muscle afferents(63, 77). The contribution of these specific P2X subtypes to muscle pain have not yet been studied in behavioral experiments. Thus, purinergic

24 9 receptors appear to be sensitive to the ATP released during muscle activity and may contribute to fatigue-related muscle pain either directly by generating an inward current through P2X3 or P2X4, and/or by enhancing the proton-gated current via P2X5-ASIC3 interactions Transient Receptor Potential Channels. The Transient Receptor Potential (TRP) family of ligand-gated ion channels contributes to many sensory modalities including cool (M8), noxious cold (A1), warm (TRPV1), noxious heat (TRPV2), acidic (TRPV1), inflammatory (TRPV1), and mechanical (TRPV4) stimuli. Among the TRP channels, TRPV1 is particularly well studied and has been implicated in detection of tissue ischemia ( ) and contributes to the response of mechanically sensitive fibers, though TRPV1 itself does not appear to mediate mechanical transduction(109, 110). Outside the muscle, TRPV1 also plays a role in a wide range of pain conditions, including neuropathic (111, 112), visceral (113, 114), skin (115), and orofacial(116) pain. Within muscle, TRPV1 is important for mediating the exercise pressor reflex( ). TRPV1 is expressed on muscle cells themselves and contributes to adaptive responses to exercise, such has muscle hypertrophy(120) and improved endurance(121). Skeletal muscle afferents also express TRPV1(63, 98, 122) and are excited by TRPV1 activation(104, 123). TRPV1 is activated by many, diverse ligands including naturally occurring

25 10 vanilloids (eg, capsaicin, capisate, piperine), endogenous lipids (e.g., oxidized lineoleic acid metabolites, anandamide, N-oleoyldopamine, and N- arachidonoyildopamine)(124, 125), inflammatory mediators(126), protons(127), and purine triphosphates(128). TRP channels, however, have not been well studied in muscle pain. Administration of either TRPV1 or TRPA1 agonists produces mechanical hyperalgesia in animals(129). In animal models of muscle pain, antagonism of TRPV1 prevents mechanical hyperalgesia caused by eccentric contractions, but not intramuscular injection of the inflammatory agent carrageenan(130). Studies of knockout animals confirm the role of TRPV1 in mechanical hyperalgesia caused by lengthening muscle contractions and also suggest TRPV4 plays an important role(131). TRPV1 knockouts also show decreased thermal hyperalgesia, but not mechanical hyperalgesia in the carrageen muscle inflammation model(132). Thus, several members of TRP family contribute to muscle hyperalgesia, likely by responding to distinct stimuli produced by tissue damage, inflammation, and muscle activity. 1.4 Models of Muscle Pain Muscle pain has been studied using multiple approaches, both in terms of measuring and inducing pain. Muscle pain is commonly studied in the gastrocnemius(66, 133) and masseter(42, 134) muscles. Assessment of muscle pain is typically done by applying force-sensitive instruments to the muscle belly

26 11 and observing the threshold at which a nocifensive behavior is triggered(135, 136). Induction of muscle pain can be grouped into three broad categories methods that rely on application of noxious compounds to muscle tissue, methods that use muscle activity, and methods that use stressful conditions to produce pain. Commonly, noxious substances including, but not limited to, acidic saline(66), hypertonic saline(137), carrageenan(138, 139), mustard oil(140), complete freund s adjuvant(cfa)(141, 142), and prostaglandin E2 (PGE2) (143, 144)are used to produce muscle hypersensitivity. Of these, repeated acidic saline (ph 4 in 0.9% saline)(66), CFA (141, 142), carrageenan (3% in 0.9% saline)(138, 139), and PGE2(143, 144) injections produce long lasting enhanced pain behavior in animals. Notably, in each of these paradigms, the enhanced pain behaviors last substantially longer than inflammatory or histological changes in the affected muscle. Further, both the repeated acidic saline injections and single injection of carrageenan produced widespread muscle hypersensitivity in the untreated contralateral muscle, indicating that these methods of pain induction may recruit central mechanisms of sensitization(145, 146). Fatiguing exercise has good face validity for the induction of muscle pain as many painful muscle conditions either result from damage during use (48) or demonstrate exacerbation of pain with activity(8). Eccentric contractions, in which a muscle is made to lengthen during contraction, produce measurable tissue damage and inflammation and are associated with delayed onset muscle soreness

27 12 (DOMS)(47). Animal models consist of stimulating muscle contractions with electrical pulses while applying a stronger mechanical force to the limb in the opposite direction, thus forcing the muscle to lengthen(147). This reflects the reallife condition of being handed an object and, finding that it is too heavy, easing the object to the ground. In this sense, eccentric contractions may serve as a model for muscle over-use or high intensity effort, which often results in temporary soreness. Another approach uses muscle activity to enhance muscle hypersensitivity to lowthreshold insults. In this method, a sub-threshold stimulus like 0.03% carrageenan or ph 5 saline is given to the muscle in combination with an exercise task. No changes are seen pain behavior when these are given alone, but when combined with muscle activity, animals show robust, long lasting and wide-spread mechanical hypersensitivity(74, 75, 148). Further, a prior inflammatory stimulus, such as carraggenan, can prime the muscle to respond in an exaggerated way to a subsequent stimulus, such as PGE2(143, 149, 150). Thus, multiple muscle insults can be combined to produce a long-lasting and widespread hyperalgesia that persists despite minimal tissue damage. Finally, stress has been used to induce widespread muscle pain. Diseases of chronic muscle pain, including FM and MPS, are associated with anxiety and disturbances in stress response(151, 152). Common methods for inducing stress are exposure to water(153), unexpected blasts of noise (136, 154, 155), and cooling of ambient temperature(135). These approaches produce long lasting

28 13 mechanical hypersensitivity in the muscle and exaggerated responses to subsequent mild muscle insults(156). 1.5 Chronic Pain Mechanisms Chronic Pain Mechanisms Chronic pain has been defined in two ways the duration of the pain and the underlying mechanisms for the pain. In the first case, any pain that exceeds some duration can be chronic pain and no assertions about the mechanisms of that pain are made. For the second case it is implied that pain becomes chronic after plastic changes in the nociceptive system. From this perspective, long lasting pain can be broken down into two types. The first type of long lasting pain is the result of a repeated or persistent acute injury. When the source of acute pain is removed, the pain rapidly subsides(157). The second type may be initiated by some acute injury, but the pain continues even after the injury is resolved. This second type of long lasting pain includes diseases like fibromyalgia and are the most difficult to treat Peripheral Mechanisms of Chronic Pain Peripheral sensitization involves changes in the function of the afferent terminals of nociceptive neurons. These changes can occur at the level of the receptor, intracellular signaling, or excitability of the cell. Likely, peripheral sensitization involves a synergistic combination of all three. Peripheral sensitization can occur after either inflammatory or non-inflammatory events.

29 14 Prostaglandins are released in settings of tissue damage and can result in longlasting muscle pain. In naive mice, application of PGE2 results in a short lasting, acute pain; however, after treatment with carrageenan, PGE2 triggers a long lasting mechanical hypersensitivity. The shorter duration pain in naive animals is mediated by activation of protein kinase A by adenylyl cyclase and subsequent camp production(157). The longer duration pain in carrageenan-treated animals, on the other hand, appears to be mediated by a change from Gs to Gi/o signaling downstream of PGE2 that requires protein kinase C epsilon (PKCε)(158). In a process that requires the EP1 receptor, recruitment of PKCε leads to the sensitization of TRPV1 by activating phospholipase C and subsequently reducing concentrations of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that normally inhibits TRPV1 function(159). PGE2 binding to another receptor, EP3, results in activation of protein kinase A (PKA) which in turn phosphorylates members of the P2X receptor family, among other targets. This enhances their response to ATP(160). Further, PGE2 application can enhance the response to acidic solutions(161). Bradykinin is also released during inflammation and results in mechanical hypersensitivity(54). Bradykinin activates intracellular signaling pathways by binding to the B1 and B2 receptors. Several mitogen activated protein kinases (MAPKs) are activated by bradykinin binding to either of these receptors, including p38 and JNK. Further, B1 signaling can recruit PKC-ε, which may

30 15 result in a similar activation of TRPV1, though this has not been tested directly(162). Nerve growth factor (NGF) is also released in the muscle during exercise(54, 57) and muscle incision(163). It is painful in both humans(22, 164) and animals (165). NGF binds two receptors TrkA and p75 NTR (166). NGF binding to TrkA activates the tyrosine kinase domain of TrkA, which triggers pathways involving ERK1/2, Akt, and PLCγ(167). NGF-enhanced pain in eccentric muscle contractions and ischemia is dependent on TRPV1, consistent with NGF s recruitment of PLC(108, 131). Further, application of NGF to muscle can strengthen the synapses of primary afferent projections to the dorsal horn, though it is unclear if this is independent from or a consequence of enhanced sensitivity of receptors at the terminal(168). Non-inflammatory mechanisms of peripheral sensitization are less well understood. Repeated acidic saline injections result in long lasting muscle pain and are associated with lymphoplasmacytic infiltrates(66), though these infiltrates do not appear to be necessary for mechanical hypersensitivity(148). ASIC3 is necessary for long-lasting mechanical hypersensitivity in muscle (68), but the role of ASIC3 in mediating this effect is unclear. Repeated acidic saline injections reduce substance P (SP) release from peripheral terminals. SP is typically considered an algesic substance, but signaling through the NK1 receptor at the terminal activates M-type potassium channels that reduce primary afferent

31 16 excitability. Thus, by inhibiting SP, repeated acidic saline injections may enhance neuronal excitability through ASIC3(169). In the sound stress-induced muscle pain model, epinephrine is critical for maintaining the long-lasting mechanical hyperalgesia in rats(136). Sustained exposure to epinephrine, either from stress or exogenous sources, switches intracellular signaling via EP receptors from Gs to Gi/o, resulting in activation of PCKε(158). Prolong exposure to epinephrine also triggers elevated expression of IL-6 and TNFR1(156). Peripheral sensitization is an important component of chronic muscle pain. Enhanced sensitivity of peripheral receptors may account for pain sensation in response to normally non-painful stimuli. Further, the enhanced excitability may translate into more frequent action potential firing, which may set the stage for alterations in the central nervous system that can continue to enhance muscle pain and spread it beyond the affected tissue(170) Central Mechanisms of Chronic Pain Central sensitization refers to changes in the brain and spinal cord that result in enhanced nociceptive processing. The processes underlying central sensitization are not well understood, but central changes may begin with enhanced peripheral input(170). Manipulation of muscle with inflammatory substances or eccentric contractions not only produces peripheral sensitization, but also results in changes in the dorsal horn that correlate with nocifensive behavior(142, 147, 171). Noxious stimulation of the muscle results in expansion

32 17 of the receptive field for dorsal horn neurons(133, 172) and triggers microglial activation in both the ipsilateral and contralateral dorsal horn(142) which alters synaptic plasticity and enhances transmission of nociceptive signals(173). Several subcortical structures have been implicated in chronic muscle pain. Descending input from the vagus nucleus modifies baseline nociceptive thresholds and the effects of bradykinin on muscle pain(174, 175). The rostroventromedial medulla (RVM) shows increased staining of phosphorylated NR1 subunit of the NMDA receptor and either NMDA antagonists or ropivicaine can reverse the muscle pain induced by repeated acidic saline injection(176). Further, acidic saline injection into the muscle results in increased staining of phosphorylated ERK in the central nucleus of the amygdala, piriform cortex, paraventricular hypothalamic nucleus, and anterior nucleus of the paraventricular thalamus that correlates with muscle pain. Interestingly, inhibition of ERK in the anterior nucleus of the paraventricular thalamus was sufficient to prevent long lasting muscle pain(28, 177). The contribution of cortical structures to chronic muscle pain is less well understood. A functional MRI study found that female fibromyalgia patients activated more regions of the cortex as compared to healthy controls when given an intramuscular solution of protons and PGE2 specifically, the left anterior insula ipsilateral to treatment showed greater activity(178). A structural analysis of the brains of female fibromyalgia patients found a greater striatal grey matter volume in fibromyalgia patients as compared to healthy controls(179). The

33 18 significance of these patterns is unclear. More work must be done to understand potential contributions of cortical structures to chronic muscle pain. 1.6 Sex Differences Substantial sex differences have been observed in diseases of chronic muscle pain. In particular, fibromyalgia affect up to eight times more women than men, depending on the diagnostic criteria(180, 181). Yet, little work has been done to understand these differences. At the level of the muscle, a study of eccentric exercise in humans found no significant sex differences in the levels of myoglobin, TNFα, IL1β, and nitric oxide, suggesting that the muscles of males and females are equally prone to damage under strenuous conditions(182). Instead, sex differences appear to be mediated by variations in the sensitivity of peripheral terminals and processing in central structures. Injection of NMDA into the masseter muscle evokes greater and longer lasting pain in female subjects. In animals, this treatment evokes more intense nociceptor firing (134). This NMDA effect is modulated by peripheral treatment with estrogen, suggesting an activational effect of estrogen on NMDA receptors. A similar effect has been found in the epinephrine-induced muscle pain model. Estrogen contributes to sex differences in the signaling through PKCε and PKA in nociceptive neurons(183, 184). Thus, circulating estrogen may contribute to sex differences in pain by directly modifying the sensitivity of nociceptors through multiple mechanisms. Sexual dimorphism in central processing of pain has not been well studied. Such central processing may be important in chronic, wide spread muscle pain

34 19 because low-intensity muscle insults separated by time or distance enhanced pain behavior in female but not male mice, indicating there are sex differences in the way stimuli are integrated centrally. This difference in central processing is not explained by changes in the rostral ventromedial medulla, an area of the brainstem implicated in exercise-enhanced pain (34, 148). Interestingly, this difference in integration of low-intensity stimuli is not abolished by ovariectomy of sexually mature animals. Greater excitability of dorsal horn neurons in female rats may contribute to such sex differences(185). Further, sexual dimorphism in the autonomic system may also contribute as females differ substantially in their response to vagotomy and medullectomy in both bradykinin- and epinephrineevoked muscle pain(174). The source of this dimorphism remains unclear, as gonadectomy of juvenile animals does not reverse this effect(186). Whether sex differences in the exercise- or epinephrine-enhanced pain are due to organizational effects of sex hormones at earlier stages of life or some other mechanism requires further examination(187). To better understand the basis of exercise-enhanced pain, I developed an animal model of exercise-enhanced pain and characterized the effects of muscle fatigue and sub-threshold muscle insult on mechanical hyperalgesia. Then, using this model, I tested the role of ASICs using genetic and pharmacological approaches. Finally, I attemped to recapitulate the effects of exercise-enhanced pain by testing the effect of protons, lactate, and ATP on mechanical hyperalgesia in animals.

35 20 Specific Aim 1: develop and characterize an animal model of isolated muscle fatigue of the gastrocnemius using electrical-stimulation to sensitize mice to mildly acidic saline injections. Hypothesis 1: Electrically stimulated muscle contractions in the gastrocnemius will result in significant fatigue. Hypothesis 2: Combining muscle fatigue with sub-threshold acidic saline injection will produce greater mechanical hyperalgesia than either fatigue or sub-threshold acidic saline alone. Hypothesis 3: Compared to male mice, female mice treated with this model will develop worse hyperalgesia as measured by greater magnitude of decrease in muscle withdrawal threshold, spread of hyperalgesia beyond the treated muscle, and longer duration of mechanical hyperalgesia. Manuscript published: N. S. Gregory, K. Gibson-Corley, L. Frey-Law, K. A. Sluka, Fatigue-enhanced hyperalgesia in response to muscle insult: Induction and development occur in a sex-dependent manner, PAIN (2013), doi: /j.pain Specific aim 2: identify metabolites and receptors required for the development of exercise-enhanced muscle hyperalgesia using biochemical, pharmacological, and genetic approaches. Hypothesis 4: Electrically stimulated muscle contractions will result in significant decreases in intramuscular ph.

36 21 Hypothesis 5: Disruption of ASIC1a function, by either genetic deletion or pharmacological inhibition, will prevent the development of muscle hyperalgesia in mice exposed to the exercise-enhanced pain model. Hypothesis 6: Disruption of ASIC3 function, by either genetic deletion or pharmacological inhibition, will prevent the development of muscle hyperalgesia in mice exposed to the exercise-enhanced pain model. Specific Aim 3: determine if lactate, ph, and ATP act in a synergistic way to produce muscle hyperalgesia. Hypothesis 7: Over a physiologically relevant range of lactate, ph, and ATP will produce dose-dependent decreases in muscle withdrawal thresholds 30 minutes after intramuscular injection. Hypothesis 8: Combining lactate, ph, and ATP will have a synergistic effect, producing greater decreases in muscle withdrawal threshold than the simple sum of their individual effects.

37 22 CHAPTER II FATIGUE-ENHANCED HYPERALGESIA IN RESPONSE TO MUSCLE INSULT: INDUCTION AND DEVELOPMENT OCCUR IN A SEX-DEPENDENT MANNER Abstract 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 2 Manuscript published as: Gregory, N. S., Gibson-Corley, K., Frey-Law, L., & Sluka, K. A. (2013). Fatigue-enhanced hyperalgesia in response to muscle insult: Induction and development occur in a sex-dependent manner. Pain, 154(12), doi: /j.pain

38 23 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 fatigueinduced 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. 2.2 Introduction Chronic pain affects over 100 million Americans (5) and chronic musculoskeletal pain is the most common type of chronic pain affecting up to 47 percent of the population (1). Fibromyalgia (FM) and myofascial pain syndrome (MPS) are syndromes of chronic muscle pain, characterized by both somatic (focal tenderness, muscle pain, fatigue) and psychological (depression, anxiety, difficulty concentrating, insomnia) symptoms (9, 188). These diseases can be devastating: 25% of FM patients are unable to work and those who can work report significant pain- and fatigue-limited loss of productivity ( ), but can acutely exacerbate pain in this population (191, 192). This results in aversion to physical activity, sedentary lifestyle, and decreased functional capacity (5, 10). While these clinical observations suggest a link between fatiguing exercise and muscle pain, the pathophysiology remains elusive. Early research on FM

39 24 found evidence of mitochondrial and ultrastructural changes in muscle tissue of FM patients (1, 193), but no clear pathology or inflammation (6, 9, ). Findings of increased pain sensitivity (188, 189, 197), temporal summation (8, 190, 198, 199), and decreased pain inhibition (5, 191, 192, 200) characteristics of central sensitization have shifted the focus to central changes in the pain processing system (1, 201). It is, however, unclear what events initiate the sensitization. Recently our laboratory demonstrated that a running wheel task, which fatigued the whole-body, resulted in hyperalgesia of the paw after an otherwise innocuous muscle insult (6, 9, 74, 75), indicating generalized fatiguing exercise enhanced the nociceptive response to muscle insult. While muscle fatigue is associated with release of fatigue metabolites (H +, lactate, and ATP) that can sensitize nociceptors and produce hyperalgesia (65, 76, 77, 96, 104, 188, 189, 202, 203), the running wheel task was not associated with changes in fatigue metabolites (8, 74, 190). Rather, we found blockade of central pathways during the fatigue task prevented this enhanced hyperalgesia from occurring (176, 191, 192). Collectively, these data suggest that central factors are critical to the initiation of the fatigue-enhanced hyperalgesia. The whole body nature of the running wheel task makes it difficult to localize the mechanism by which muscle fatigue alters the response to muscle insult. For example, it is unclear if fatigue of the insulted muscle is necessary for induction of hyperalgesia, which would suggest fatigue metabolites bind to and

40 25 sensitize peripheral terminals of nociceptors to produce the enhanced hyperalgesia. Alternatively, muscle fatigue may enhance the response to the low-dose muscle insult even when the muscle insult and fatigue are applied to different muscles, indicating that central structures are involved in producing the enhanced hyperalgesia. Whether fatigue-related signals from a single muscle are sufficient, or if fatigue across multiple muscles is required for induction of the enhanced hyperalgesia is also unanswered by the prior running wheel studies. Therefore the purpose of this study was to determine if localized muscle fatigue is sufficient to produce hyperalgesia when combined with ph 5 saline injections (muscle insult) and to test for sex differences in the initiation and nature of muscle pain.

41 Materials and Methods The experiments outlined below induced localized muscle fatigue in male and female mice using electrically-induced isometric contractions of the gastrocnemius muscle which are expected to maximize accumulation of fatigue metabolites. We combined this localized muscle fatigue with a subthreshold muscle insult (two ph 5.0 saline intramuscular injections) neither of which produced muscle hyperalgesia when given alone (see below for more details). For comparison we combined the muscle fatigue with neutral intramuscular saline injection (ph 7.2). Nociceptive behaviors were assessed as muscle withdrawal thresholds to pressure applied over the belly of the gastrocnemius muscle and decreases in thresholds were considered hyperalgesia (see below). All experiments were approved by the Institutional Animal Care and Use Committee and performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and IASP Ethical Guidelines for the Use of Animals in Research. Both male and female C57BL6/J mice from Jackson Laboratories (n=215), age 4-8 weeks, were used in all studies Electrically Stimulated Fatiguing Muscle Contractions To induce muscle fatigue, mice were deeply anesthetized with 2-4% isoflurane. Fatiguing muscle contractions were produced by applying electrical pulses through needle electrodes implanted in the proximal portion of the

42 27 gastrocnemius muscle using a modified Burke protocol (10, ) that was confirmed empirically. The electrical pulses were generated by a Grass S88 solidstate square wave stimulator (Grass Technologies, West Warwick, RI). To test total force output before and after fatiguing contraction, maximum force contractions were elicited by applying high-frequency (100 Hz), supramaximal stimulus trains: 7 volts (V) pulses with 1 ms pulse duration. Each train lasted 500 ms with 3 second (s) between trains. To produce muscle fatigue, supra-maximal, moderate frequency stimulations (40 Hz) were applied to the muscle by applying trains consisting of 90, 1 ms pulses (7 V) (train duration 3.75 s) every 8 sec (rest intervals 4.25 s between trains). Correct electrode placement in the gastrocnemius was confirmed by plantarflexion of the ankle joint without activation of toe flexors, tibialis anterior muscle, or muscles above the knee. A trial consisted of three maximum contractions (100 Hz stimulation) to establish the baseline force, six minutes (m) of sub-maximal (40 Hz stimulation) fatiguing contractions, and three maximum contractions immediately after the fatiguing contractions. A subset of animals from the behavior experiments were analyzed for baseline force and decline in force (male n=12, female n=13). In a separate group of animals (male, n=3), force recovery was monitored at 2, 4, 6, 8, and 10 m following completion of the fatiguing task, again using 3 maximum contractions. Force was continuously measured by attaching the hindpaw to an iworx FT-302 force transducer (iworx, Dover, NH) and sampling the analog output at 100 Hz using LabVIEW software (National Instruments, Austin, TX).

43 28 All force measurements were then computed off-line using Freemat and Python scripts. Fatigue was operationally defined as the decline in force between baseline and post-fatigue maximum force contractions Low-Intensity Muscle Insult The low-intensity muscle insult consisted of two intramuscular (i.m.) injections of 20 µl normal saline into the gastrocnemius muscle 5 days apart. The ph of the normal saline was adjusted with HCl to ph Control injections consisted of two injections of normal saline (ph 7.2 ± 0.1) 5 days apart. The unbuffered ph 5 saline injections reduce muscle ph to approximately 6.9, which is comparable to decreases seen after intense exercise (44, 66, 207, 208). Neither two injections of ph 5.0 nor two injections of ph 7.2 produce hyperalgesia (74, ) Pain Behavior Muscle withdrawal thresholds (MWT) were measured by applying force sensitive tweezers to the belly of the gastrocnemius as previously described, where lower thresholds indicate greater sensitivity (197, 209). Mice were acclimated to this behavioral paradigm in two 5 minute sessions over a two day period prior to the first injection. Briefly, mice were placed in a gardener s glove, the hindlimb was held in extension, and the muscle was squeezed with force sensitive tweezers until the animal withdrew its hindlimb. An average of 3 trials per animal was

44 29 taken at each time period. A decrease in withdrawal thresholds was interpreted as muscle hyperalgesia Ovariectomy To test the role of estrogen on sex-dependent effects observed in the current study, female C57BL6/J mice were ovariectomized. Briefly, each animal was deeply anesthetized with 2-4% isoflurane and the ovaries were removed by an abdominal approach as we previously published (75). Animals were given 100 µl of 0.3mg/ml buprenorphine every 12h for 3 days and monitored daily for 5 days. One week after ovariectomy, behavioral experiments were done Experimental Protocol: Induction of Muscle Hyperalgesia Standard Protocol We performed a set of three experiments to address our aims, with slight variations in each. However, the common protocol across all experiments was as follows. On day 0, baseline muscle withdrawal thresholds were assessed and the first saline injection was administered. Fatiguing muscle contractions were induced prior to the second saline injection on day 5. On day 6, the muscle withdrawal thresholds were reassessed. See below for experiment-specific details.

45 Experiment 1: Necessity of Acidic Saline and Fatigue Parameters in Initiating Muscle Hyperalgesia This experiment tested if localized (i.e., single muscle) muscle fatigue combined with a low-intensity muscle insult (2 injections of ph 5.0) produced muscle hyperalgesia compared to four control conditions. On day 1, mice (males n=6, females n=6 for each condition, total of 60 mice) were tested for baseline pain behaviors and given the first saline injection. On day 5, the second saline injection was administered immediately after completing the set of fatiguing muscle contractions to the homonymous gastrocnemius muscle. Each cohort of 12 animals was exposed to one of the following conditions: (1) two ph 5 injections with fatiguing muscle contractions (experimental),;(2) two ph 5 saline injections without fatiguing muscle contractions (control); (3) two ph 5 saline injections with two sets of maximum contractions 6 minutes apart (without the fatiguing muscle contractions)(control); (4) fatiguing muscle contractions with two ph 7.2 saline injections (control); or (5) a single ph 5 saline injection immediately after the fatiguing muscle contractions (control). Muscle withdrawal thresholds were measured bilaterally 24h after the second (or single for condition 5 ) saline injection Experiment 2: Course of Muscle Hyperalgesia This experiment tested the duration of hyperalgesia induced by combining muscle fatigue with ph 5.0 injections (males n=6, females n=6). Muscle

46 31 withdrawal thresholds were assessed 24h, 3d, 5d, 7d, and weekly afterwards until the pain behavior returned to baseline (6 weeks) Experiment 3: Final Acidic Saline Injection after Muscle Fatigue To test for a time-dependent effect between ph 5 saline injections and muscle fatigue, we varied the time between the second acid injection and fatiguing muscle contractions (for each condition males n=6, females n=6, total of 36). For each condition, mice received the first ph 5 saline injection on day 0 and on day 5 received the second ph 5 saline injection. We applied the fatiguing muscle contractions to the homonymous muscle either (1) immediately, (2) 2h, or (3) 24h (i.e., day 4) before the second ph 5 saline injection. Muscle withdrawal thresholds were measured at baseline and 24h after the second ph 5 saline injection. Muscle withdrawal thresholds were measured bilaterally 24h after the second ph 5 saline injection Experiment 4: Separation of Acidic Saline Injections and Muscle Fatigue To test the spatial characteristics of the fatigue-enhanced hyperalgesia response, the ph 5.0 injections and muscle fatigue were applied to homonymous and heteronymous muscles (for each condition males n=6, females n=6, total of 24 animals) and mechanical hyperalgesia tested bilaterally. Two ph 5 saline

47 32 injections spaced 5 days apart were combined with the fatiguing muscle contractions immediately prior to the second injection. However, the two conditions varied by whether the pair of saline injections were given to the fatigued (ipsilateral) gastrocnemius muscle or the unfatigued (contralateral) gastrocnemius muscle. Muscle withdrawal thresholds were measured bilaterally 24h after the second ph 5 saline injection Experiment 5: Assessment of Inflammatory Changes in Treated Muscle Inflammatory changes were assessed by histopathologic evaluation of tissues as well as by measuring tissue levels of myeloperoxidase. Mice were given two ph 5.0 saline injections five days apart with muscle fatigue immediately before the second injection. Controls consisted of animals that received (1) no treatment, i.e. naïve, (2) two ph 5 saline injections without muscle fatigue, (3) two ph 7.2 saline injections with muscle fatigue, (4) or a single injection of 3% carrageenan (positive control). 24 h after the treatment, mice were euthanized by exposure to 100% CO 2 for 5 minutes followed by thoracotomy. For histological analysis, the gastrocnemius muscles (for each condition, males n=2, total of 10 mice) were fixed in 10% neutral buffered formalin for 72 hours. These specimens were then embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin (H&E) for histological examination. A board-certified veterinary pathologist, who was blinded to treatment group, assessed the slides.

48 33 As a marker for neutrophilic infiltration, myeloperoxidase concentration was quantified by spectrophotometry. The gastrocnemius muscle (for each condition, males n=6, total of 30 mice) was harvested and weighed. The tissue was minced with scissors before being homogenized in 1 ml 0.5% hexadecyltrimethylammonium bromide (HTAB) on ice. The homogenate was then treated with 3 freeze-thaw cycles by immersion in 95% ethanol chilled by crushed dry ice followed by immersion in hot water. Lysates were centrifuged at 1,000 RPM for 15 minutes and 10 µl was plated in triplicate on 96 well flat bottom cell culture plates. 190 µl of freshly prepared assay reagent (10 mg o- dianisidine dihydrochloride with 1 µl 30% hydrogen peroxide dissolved in 50 ml 1M phosphate buffer) was then added to the lysate. Each plate also included a serial dilution of stock myeloperoxidase as a standard curve. Absorbance of light at 450 nm was measured using a microplate reader (SpectraMax Plus384) running SoftMax Experiment 6: Quantification of pnr1 Positive Cells in the RVM To test if sex differences were due to altered modulation of central facilitatory pathways, previously found to show sexual dimorphism in response to noxious stimuli (10, 210), we tested if there was increased phosphorylation of the NR1 (pnr1) subunit of the NMDA receptor (pnr1). We examined pnr1 in the rostral ventomedual medulla (RVM) since previous studies in our laboratory using animal

49 34 models of muscle pain show 1) increased pnr1 immunoreactivity, 2) blockade of NMDA receptors reduces hyperalgesia, 3) downregulation of NR1 reduces muscle hyperalgesia, and 4) over-expression of NR1 induces muscle hyperalgesia (34, 176, 211, 212). Male and female mice were given two ph 5.0 saline injections five days apart with muscle fatigue immediately before the second injection.. Comparison groups consisted of naïve mice for each sex. Twenty-four hours after the second injection, mice were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with 4% paraformaldehyde. Brains were removed, and the RVM was blocked, embedded in OCT, and frozen at -20 C until analysis. Sections were cut on a cryostat at 20 mm through the medulla and placed on slides. Serial sections containing the RVM were immunohistochemically stained as previously described (176, 212). Sections from all animals were stained simultaneously using a primary rabbit polyclonal antibody against phosphorylated serine 897 of the NMDA R1 subunit (pnr1) (Millipore, 1:500 dilution). Sections were incubated overnight in the primary antibody followed the next day by 1h incubation with biotinylated goat anti-rabbit (Life Technologies, 1:200) and then 1h incubation in streptavidin-alexa 568 conjugate (Life Technologies, 1:200). Images of the RVM were taken at 20x magnification on an Olympus BX-51 light microscope using the same settings for each section and between animals. Quantification was performed of line using Image J and counting the number of stained cells in the RVM in five sections per

50 35 animal as previously described (176, 212). As a control for staining, the facial nucleus, contained in the same sections as the RVM, was similarly quantified Statistical Analysis Data are reported as the mean + S.E.M. The effects of the fatiguing muscle contractions on force output were compared to baseline using student s t tests. Repeated measures ANOVA were used for assessing fatigue recovery and behavioral measurements, with Duncan s post-hoc tests for between group followup assessments. Student s t-tests with rank ordered Bonferroni corrections were used to test for differences between sexes across time. One-way ANOVA with Duncan s post-hoc test was used to analyze the myeloperoxidase assay, and immunohistochemistry data. For all experiments, p<0.05 was considered statistically significant. 2.4 Results Electrical stimulation of muscle produces shortlasting muscle fatigue To induce local muscle fatigue we electrically stimulated the gastrocnemius muscle for 6 minutes using a modified Burke protocol (198, 199, ) to produce isometric contractions that resulted in an approximately 50% decrease in force. Maximum intensity, high frequency electrical stimulation produced 6.2 (+/- 0.5 SEM) and 6.4 (+/- 0.5 SEM) grams (g) of force at baseline in male and female mice, respectively (fig 2-1A), and there was a similar decrease in force between the sexes. This decrease in force recovered rapidly, returning to 93% of baseline

51 36 by 10 minutes (fig 2-1B). There were no significant differences in force (ph 5 = g; ph 7.2 = g) or fatigue magnitude (ph 5 = %; ph 7.2 = %) between animals treated with ph 5.0 or ph 7.2 saline Muscle fatigue combined with sub-threshold muscle insult produces hyperalgesia in a sex-dependent manner Combination of ph 5.0 Saline and Muscle Fatigue is Necessary to Produce Muscle Hyperalgesia To determine if ph 5.0 saline must be combined with local muscle fatigue to produce hyperalgesia, we measured MWT in mice exposed to either ph 5.0 saline or local muscle fatigue, or both in combination. There was a significant decrease in muscle withdrawal thresholds in mice that received two ph 5.0 saline injections in combination with muscle fatigue (Fig 2-1C) when compared to baseline or controls. Mice that received two ph 5.0 saline injections without muscle fatigue, a single ph 5.0 saline injection with muscle fatigue, two ph 7.2 saline injections with muscle fatigue, or ph 5.0 with only the maximum contractions but no fatigue showed no change in their muscle withdrawal thresholds (F 4,51 = , p < 0.001) (fig 2-1C) Muscle Hyperalgesia Lasts Significantly Longer in Female Mice To examine the duration of the decreased muscle withdrawal thresholds we followed mice for up to 6 weeks after the second acidic saline injection. There was

52 37 a significant sex difference in the duration of the decreased muscle withdrawal thresholds after combining muscle fatigue with muscle insult (F 1,10 = , p=0.002). Muscle withdrawal thresholds returned to baseline by day 14 in males, but not until day 42 in females (fig 2-2A). Post-hoc tests confirmed significant differences in withdrawal thresholds between males and female mice after the 14 day period Delaying Final Acidic Saline Injection after Muscle Fatigue Results in Time-Dependent Decrease in Hyperalgesia for Male, but not Female Mice To determine if the muscle fatigue had to occur in close time proximity to the muscle insult, we performed the muscle fatigue task immediately, 2h, or 24h prior the muscle insult. Significant sex differences were observed between the time for induction of muscle fatigue and the muscle insult (F 6,37 = 3.208, p=0.012). For males the greatest decrease in withdrawal thresholds occurred when muscle fatigue was induced immediately before the second injection, an intermediate effect was seen for 2h delay, and no decrease in withdrawal thresholds occurred when the fatigue task was given 24h before the second injection (fig 2-2B). Surprisingly, in females withdrawal thresholds did not vary significantly between time intervals muscle fatigue induced immediately, 2h, or 24h before the second injection resulted in comparable decreases in withdrawal thresholds (fig 2-2C). Post-hoc tests revealed a significant difference between

53 38 males and females for the group in which muscle fatigue was induced 24h before the second injection Spatial Separation of Acidic Saline Injections and Muscle Fatigue Fails to Produce Hyperalgesia in Male, but not Female Mice To test the spatial characteristics of the fatigue-enhanced response to muscle insult, muscle fatigue and muscle insult were applied to different muscles and hyperalgesia was tested bilaterally. Again, significant sex differences were observed (F 11,64 = , p < 0.001). In males when muscle fatigue and muscle insult occurred in the same muscle there was a significant decrease in withdrawal thresholds ipsilaterally, but not contralaterally. When the muscle fatigue and muscle insult were given to heteronymous gastrocnemius muscles, male mice showed no change in withdrawal thresholds (figs 2-2D-G). In contrast, female mice showed decreased muscle withdrawal thresholds bilaterally regardless of whether the localized muscle fatigue and muscle insult occurred in the same or contralateral muscles (figs 2-2D & 2-2E) Muscle insult and fatigue is not associated with acute muscle inflammation To determine if there was muscle damage and inflammation the muscles were examined histologically. All groups, naïve controls, ph 7.2 controls, ph 4.0 injection, and fatigued cohorts demonstrated mild, multifocal myofiber

54 39 degeneration, as characterized by myocyte hypereosinophilia and loss of crossstriations. Similarly, all groups also exhibited signs of mild, multifocal regeneration as characterized by centralized rowing of nuclei. Compared to naïve controls, animals treated with acidic saline, with or without fatigue, had a mild, multifocal lymphoplasmacytic infiltrate, but notably lacked neutrophils (fig. 2-3A-C). In contrast, 3% carrageenan was used as a positive control and showed primarily a moderate to sometimes marked multifocal to coalescing neutrophilic infiltrate (fig. 3D). The absence of neutrophils in response to muscle fatigue and muscle insult was confirmed by assaying for myeloperoxidase from whole muscle tissue. Mice treated with muscle fatigue and ph 7.2 saline, ph 5 saline alone, or muscle fatigue with ph 5.0 saline showed quantities of myeloperoxidase comparable to the naïve control while those treated with 3% carrageenan had significantly greater concentrations (F 6,47 = , p < 0.001) (fig. 2-3E) Ovariectomy has no effect on sex differences in the induction of muscle hyperalgesia To determine if circulating estrogen levels were responsible for the sex differences observed in the behavioral experiments, we compared ovariectomized females to intact males and females in the acidic saline fatigue model. We found that ovariectomized mice behave no differently than gonadally intact females they develop similar bilateral hyperalgesia whether acidic saline is given to the

55 40 heteronymous or homonymous muscle (fig. 2-2D-G) and when acidic saline administration is delayed by 24 h (fig. 2B) No changes in pnr1 in the RVM after Treatment with Acidic Saline and Muscle Fatigue To examine if the sex differences were related to changes in central excitability, we examined if there was enhanced pnr1 in the RVM, previously shown to facilitate pain through NMDA receptors and the NR1 subunit (75, 176, 212). Sections of RVM stained for pnr1 were examined for the number of positively-staining cells. No significant differences in the number of pnr1 positive cells in the RVM were found between naïve and treated animals, or between sexes (see Table 2-1). The facial nucleus similarly showed no significant differences between sexes or treatment. 2.5 Discussion The current study shows that combining localized muscle fatigue with a low-intensity muscle insult results in long-lasting muscle hyperalgesia that parallels previous studies using whole-body fatiguing exercise (75). Accordingly, priming of innocuous muscle insult to produce hyperalgesia does not require widespread input from multiple fatiguing muscles. Initiation of this hyperalgesia is sex-dependent with female mice showing a wider window of time and a greater distance between muscle fatigue and muscle insult to produce hyperalgesia, and longer duration of hyperalgesia. Finally, this model does not produce acute

56 41 inflammation or tissue damage. These data suggest that combining two low intensity muscle insults with localized muscle fatigue produces sexually dimorphic patterns of muscle hyperalgesia. Chronic musculoskeletal pain is more prevalent among women (6, 9, ). Consistent with this, we found significant differences between sexes in both the requirements for initiation of hyperalgesia and the nature of hyperalgesia induced by combining muscle fatigue with a subthreshold muscle insult. Our preliminary experiment showed ovariectomy does not alter the hyperalgesia in female mice, suggesting release of estradiol from the ovaries at the time of induction is not necessary for the development of hyperalgesia and differs from our prior study showing ovariectomy reduces sex-differences in whole-body fatigue induced pain [67]. The mechanisms for the sex differences in the current study are unclear. Masculinization of the brain by testosterone or sex chromosome-linked genes remain potential mechanisms for these sex differences(216). For example, expression of sex hormone receptors within the PAG differs between sexes(217) which may contribute to the sexual dimorphism in µ-opioid expression in the PAG(218). Alternatively, differential processing of nociceptive input centrally could also contribute to sex differences. Previous studies show that contralateral hyperalgesia in uninjured tissue is mediated by central mechanisms, since removal of afferent fiber input from the site of insult has no effect on the contralateral hyperalgesia (5, 145, 146, ). Bilateral hyperalgesia develops in both sexes

57 42 after unilateral treatment with ph 4.0 saline or ph 5.0 saline (low-intensity insult) in combination with whole-body fatigue (1, 66, 74), suggesting central sensitization occurs in both sexes when the stimuli are high intensity (ph 4.0) or widespread (whole-body fatigue). However, the present study shows sex differences emerge when animals are treated with low intensity, focal stimuli. In human subjects, females have greater central excitability in a number of measures: temporal summation (6, 9, ), secondary hyperalgesia (188, 189, 226), referred pain (8, 67, 190), and decreased conditioned pain modulation (191, 192, 227, 228). The basis for greater central excitability is unknown, but sex differences occur in the connectivity and activity of the periaqueductal gray (PAG) and rostral ventromedial medulla (RVM), regions implicated in pain modulation (10, 210). While increased levels of pnr1 staining in the RVM has been associated with fatigue and pain (176), the current study found no differences between treated and naïve mice. Higher intensity stimuli may be necessary for elevations in pnr1. Whether this means widespread muscle hyperalgesia is mediated by different molecular mechanisms in the RVM, a different structure in the CNS, or some other process requires further study. The current study also shows that females develop hyperalgesia even when there is a substantial delay between muscle fatigue and the muscle insult. This suggests that females fail to attenuate an ongoing central response to muscle fatigue. This has significant consequences for the initiation of muscle pain, as seemingly innocuous stimuli ph 5.0 saline and isolated muscle fatigue given

58 43 far apart in time and space, are able to converge on central structures such as the RVM(34) and produce widespread hyperalgesia in females. The prolonged nature of the hyperalgesia observed in females compared to males also points to greater central sensitivity as the hyperalgesia presumably exceeds the duration of muscle insult. These data indicate that females have less stringent requirements for the onset and significantly greater duration of muscle hyperalgesia, which could account for the greater probability of developing muscle pain observed in female patients. Chronic muscle pain syndromes, such FM and MPS, are characterized by constant pain at rest, enhanced pain in response to pressure applied to the muscle, and enhanced pain with acute exercise (6, 9, 192, , 229). We chose isometric contractions at a force sufficient to occlude muscle perfusion in order to maximize accumulation of fatigue metabolites while minimizing tissue damage (197, 225, 230). Fatigue metabolites, such as protons, lactate and ATP, contribute to loss of muscle force (198, 199, 231) and activate receptors located on nociceptors, acid sensing ion channels (ASICs) and purinergic receptors, respectively (63, 65, 96, 97, 200, 232). ASICs and purinergic receptors are wellestablished to play a role in nociceptive processing including that from the muscle (42, 69, 201, ). Further, acid, lactate and ATP can interact to produce a potentiated response. ASICs demonstrate enhanced sensitivity to ph changes when bound by lactate or in the presence of ATP (74-77). Similarly, dorsal root ganglia treated with acid, lactate and ATP show enhanced intracellular calcium

59 44 compared to those receiving each treatment alone (65, 76, 77, 96, 104, 202, 203). Primary afferent fibers show robust responses to both ATP and acid (65, 74, 96). Notably, these metabolites return to normal within minutes to hours after a fatiguing task with a concomitant recovery of muscle force (176, 231). A range of ph solutions and exercise durations have previously been examined in the acidic saline model of muscle pain. ph 4 saline injections produce bilateral hyperalgesia regardless of exposure to whole-body exercise and ph 5 saline injections produce hyperalgesia only in animals that perform fatiguing exercise. Higher ph solutions, 6 or 7.2, do not produce hyperalgesia even in exercising animals (74, ). Further, mice treated with 30 min or 2h of running wheel activity alone do not develop hyperalgesia (74, 176). While this data does not indicate if the interaction between fatigue and acidic saline is additive or synergistic, it does suggest that the threshold for muscle hyperalgesia is significantly lower when acidic saline injections are paired with even brief bouts of fatiguing exercise. Release of metabolites from fatiguing stimuli may sensitize and prime the nociceptors, resulting in enhanced response to a subsequent low-intensity muscle insult (76, 77, 209). Thus, fatigue metabolites likely contribute to the development of muscle pain by priming muscle nociceptors for a greater response to the subsequent muscle insult. Interestingly, delaying the final muscle insult after muscle fatigue resulted in a time-dependent decrease in hyperalgesia in male mice, but not female mice. Presumably, this delay reduced concentrations of fatigue

60 45 metabolites and allowed partial recovery from priming effects. When considered with the localized nature and shorter duration of hyperalgesia in male mice, these findings suggest that, in the absence of central sensitization, hyperalgesia in response to low-intensity muscle insult in male mice derives from these peripheral mechanisms. Acute exercise has profound effects on both the innate and acquired immune system, leading to catecholamine- and cortisol-related transient elevations of circulating leukocytes ( , 236). In the present study, no neutrophils or macrophages were observed in the tissue 24h after treatment, likely due to the fact that circulating levels of these cells return to normal before 24h (75, 236). Neutrophils are also recruited to sites of tissue damage and some types of acute exercise, such as eccentric contractions, result in damage and inflammation of muscle tissue with subsequent delayed onset muscle soreness (DOMS) (6, 9, 21, 46, ). In the current study, the absence of neutrophils in the muscle suggests that acute inflammation is not contributing to the hyperalgesia observed after isometric muscle contractions, distinguishing fatigue-enhanced hyperalgesia from DOMS. While we find no evidence of recruitment of these cells in the affected muscle, it is possible that release of cytokines from local or circulating inflammatory cells could contribute to muscle pain. Consistent with earlier work, we found mild lymphoplasmacytic infiltration in mice that received ph 5.0 injections, but not those receiving ph 7.2 with electrically stimulated muscle contractions(66). Lymphocytes are a functionally

61 46 diverse population and the exact nature of the lymphoplasmacytic infiltrate after injection of ph 5.0 is unknown. Lymphocytes have the potential to enhance pain states by releasing pro-algesic factors, e.g. TNF-α, IL-1, and prostaglandins (75, 237) and are present in chronically inflamed tissue (134, ). However cultured lymphocytes (cytotoxic T-cells) exposed to decreases in ph show phdependent decreases in inflammatory cytokine release evoked by specific antigens ( ). Further, the current study shows comparable lymphocyte infiltration between the group that developed hyperalgesia (ph 5.0 saline with muscle fatigue) and a control that failed to develop hyperalgesia (ph 5.0 saline alone), suggesting lymphocytes could contribute to but are not by themselves sufficient for the observed hyperalgesia. In summary, we show that localized fatiguing isometric contractions in a single muscle treated with acidic saline is sufficient for the development of longlasting muscle hyperalgesia. Taken together, these data suggest that seemingly mild muscle insults, when combined, are capable of producing long-lasting and widespread muscle pain out of proportion to the injury in a sex-dependent manner. Multiple low-intensity insults could be an underlying factor in the transition from acute to chronic pain and could underlie the female predominance of muscle pain observed clinically. Undertanding the mechanisms of exercise-induced pain could lead to treatments targeting the deleterious consequences of acute exercise resulting in improved compliance and a more active lifestyle in individuals with musculoskeletal pain. Therefore, this model could be valuable for exploring the

62 47 mechanisms underlying fatigue-enhanced muscle pain, the greater prevalence of chronic muscle pain among females, and the factors that influence the transition from acute to chronic pain.

63 48 Contraction Force (g) Contraction Force (g) MWT (g) A B C Initial Males Females * Baseline 0 * 2 * Baseline 4 6 Recovery (m) Final # # # 8 Single inj ph 7 stim + ph 5 stim - Max stim * ph 5 stim + 24h 10

64 Figure 2-1 Electrically stimulated muscle contractions result in muscle fatigue and enhance the response to ph 5 saline injections. (A) Force elicited by 100 Hz electrical stimuli before and after 6 m fatiguing contractions (males n=12, females n=13) * p < 0.05, difference from baseline. (B) Recovery of force after 6 m fatiguing contractions (n=3) * p < 0.05, difference from initial, p < 0.05, difference from start of recovery. (C) Behavioral measure of sensitivity to mechanical stimuli at baseline and after treatment with a single ph 5 saline injection with electrically stimulated muscle contractions(males, n=6), two ph 5 acidic saline injections alone (pooled, males & females n=6 each), two ph 7.2 saline injections with electrically stimulated muscle contractions (pooled, males & females n=6 each), two ph 5 saline injections with test contractions but not fatigue (pooled, males & females n = 6 each), or two ph 5 saline injections with electrically stimulated muscle contractions (pooled, males & females n=6 each) * p < 0.05, difference from baseline and control groups at 24h. 49

65 50 A B 1750 MWT (g) * * * * Males # * # # # * Day of Testing C 1750 * * Females Male s Females MWT (g) No delay 2h delay 24h delay Baseline # * * 24h MWT (g) No delay 2h delay 24h delay 24h delay OVX Baseline * 24h D Ipsilateral to Fatigue E Contralateral to Fatigue # MWT (g) Male Female OVX Baseline 24h * * * MWT (g) Male Femal OVX e Baseline * 24h Ipsilateral to Fatigue Contralateral to Fatigue F # G # MWT (g) Male Female OVX Baseline * 24h MWT (g) Mal Female OVX e Baseline * 24h

66 Figure 2-2 Sex differences in mechanical hypersensitivity after repeated ph 5 saline injection and electrically stimulated muscle contractions. (A) Duration of mechanical hypersensitivity (n=6) * p < 0.05, difference from baseline, p < 0.05, difference from females.(b & C) The final ph 5 saline injection was delayed after electrically stimulated muscle contractions for 0, 2, or 24 h (n=6 per group) * p < 0.05, difference from baseline, p < 0.05 difference from intact and ovariectomized females. (D, E, F, G) A spatial relationship between ph 5 saline injection and electrically stimulated muscle contractions was tested by varying the location of acidic saline injection (n=6) * p < 0.05, difference from baseline, p < 0.05, difference from intact and ovariectomized females. (D & E) ph 5 saline injections and electrically stimulated muscle contractions were given into the same muscle and mechanical hypersensitivity was tested in the gastrocnemius muscles (D) ipsilateral and (E) contralateral to the site of treatment. (F & G) ph 5 saline injections were given to the gastrocnemius muscle contralateral to the site of electrically stimulated muscle contractions. Mechanical hypersensitivity was tested in the muscle (F) ipsilateral and (G) contralateral to the site of fatigue. 51

67 ph 7. 2 ph + st st im 5 im ph 5+ ve 0.20 ai ca rr a E N 3% Absorbance (AU) 52 Myeloperoxidase *

68 Figure 2-3 Inflammation in muscle treated with repeated ph 5 saline injections and electrically stimulated muscle contractions. Representative hemotoxylin and eosin stained gastrocnemius muscle sections taken from (A) naive mice and 24h after treatment with (B) repeated ph 5 saline injections alone, (C) electrically stimulated muscle contractions with ph 5 saline injections, and (D) 3% carrageenan. Thin arrows indicate centralized rowing of myocyte nuceli, indicative of regeneration, thick arrows show sites of multifocal inflammatory cell infiltrates which are magnified within the insets where specific cell types are circled (in (C) it is lymphocytes, in (D) it is neutrophils) and asterisks indicate degenerative myocytes, scale bars = 20 µm (inset scale bars = 10 µm). (E) Colorimetric assay to quantify myeloperoxidase, a neutrophil marker, in whole, homogenized gastrocnemius muscle tissue in naive mice (n=7) and after treatment with 3% carrageenan (n=6), repeated ph 5 saline injections alone (n=7), repeated ph 5 injections with electrically stimulated muscle contractions (n=7), and repeated ph 7.2 saline injections with electrically stimulated muscle contractions (n=7). * p <

69 54 Group RVM Facial Nucleus Males- 24 +/- 1.5 N=3 Fatigue & Acid /- 2.0 Males / /- 3.6 No Treatment N=4 Females 27 +/ /- 5.2 Faitgue & Acid Females N= / /- 3.9 N=4 No Treatment Table 2-1 pnr1 staining in the RVM of mice treated with repeated ph 5 injections and electrically stimulated muscle contractions. Total number of cells stained for pnr1 across 5 sections containing the RVM or facial nucleus. There were no significant differences by sex, treatments or location.

70 55 CHAPTER III CONTRIBUTION OF ACID SENSING ION CHANNELS TO MECHANICAL HYPERALGESIA AFTER EXERCISE Abstract The mechanisms underlying chronic muscle pain are not well understood. A number of algesic agents have been identified including protons, lactate, ATP, NGF, and inflammatory cytokines. Previous experiments have shown that repeated acidic saline injection is sufficient to produce long-lasting muscle hyperalgesia in animals and that genetic deletion of acid sensing ion channels (ASICs) can prevent such effects, suggesting ASICs play a critical role in sensation in the muscle. The combination of fatiguing muscle contractions and sub-threshold muscle insult also produces long lasting muscle pain in mice. Using a mouse model, we tested whether ASICs contribute to fatigue and development of exercise-enhanced muscle pain. The development of fatigue was tested by electrically stimulated muscle contractions untreated wild type, ASIC knockout, and ASIC antagonist-treated wild-type mice. The development of mechanical hyperalgesia was tested by combining electrically stimulated muscle contractions with sub-threshold muscle insult (2 injections of ph 5.0) in untreated wild type, 3 Manuscript published as: Gregory, N. S., Brito, R. G., Fusaro, M. C. G. O., & Sluka, K. A. (2015). ASIC3 Is Required for Development of Fatigue-Induced Hyperalgesia. Molecular Neurobiology. doi: /s

71 56 ASIC knockout, and ASIC antagonist-treated wild-type mice. We show that neither genetic deletion of ASICs nor antagonism of ASICs have an effect on electrically stimulated muscle contractions. Further, we show wild type and ASIC1a knockout mice both develop hyperalgesia, but ASIC3 knockouts do not. In contrast, both the ASIC1a antagonist psalmotoxin-1 and ASIC3 antagonist ApeTx2 prevent the mechanical hyperalgesia. The effect of psalmotoxin-1, however, is lost ASIC1a knockout mice, indicating that ASIC1a is present in the wild type channel, but is not the critical subunit for the development of hyperalgesia in exercise-enhanced pain. ASIC3, on the other hand, is critical for the development of exercise-enhanced hyperalgesia. 3.2 Introduction Regular exercise remains one of the more effective treatments for chronic pain conditions such as fibromyalgia(244, 245), but an acute bout of exercise can exacerbate pain providing a barrier to participation in regular exercise (8, 191, 246, 247).The acute exacerbation of muscle pain with exercise in people with fibromyalgia is not well understood as it occurs with levels of activity that do not typically produce tissue damage or pain in healthy subjects (192, 248). Animal studies similarly show that fatiguing exercise in combination with a low-dose muscle insult enhances hyperalgesia (74, 75, 148). Whole-body muscle fatigue enhances hyperalgesia and activity in brainstem neurons without significant changes in the muscle metabolites (34, 74, 176). Recently we showed that fatiguing a single muscle enhanced the nociceptive response to a subsequent

72 57 injection of acidic saline(148). However this model was not associated with changes in brainstem neurons after induction of hyperalgesia. Muscle fatigue releases metabolites, including decreases in ph and lactate, which would subsequently activate nociceptors to produce pain. Decreasing ph in muscle produces pain and hyperalgesia in healthy adults(67, 249), and decreasing ph in muscle produces hyperalgesia in animals(66). Acid sensing ion channels (ASICs) are sensitive to the accumulation of protons and lactate (76, 104). Muscle nociceptors express ASIC1a and ASIC3, which form functional trimeric channels, and ASIC3 is found in higher quantities in muscle compared to skin (63, 64, 139, 250). Both ASIC1 and ASIC3 have been implicated in the development of hyperalgesia in models of muscle pain(68, 70, 74). Intramuscular ph is approximately 7.1 at rest and decreases to approximately 6.6 with moderate fatiguing exercise(251, 252), values that would activate ASICs in muscle(58). We therefore hypothesized that decreases in ph and increases in lactate occur during muscle fatigue that then activate ASICs to enhance the hyperalgesia to a low-dose muscle insult. We assessed the contribution of ASIC1 and ASIC3 in an animal model of isolated exercise-enhanced muscle pain using genetic and pharmacological approaches, and determined if muscle fatigue increases the accumulation of protons.

73 Materials and Methods Animals All experiments were approved by the Institutional Animal Care and Use Committee and performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals and IASP Ethical Guidelines for the Use of Animals in Research. Male and female mice (6-10 weeks old) were bred at the University of Iowa. Congenic mice on C57BL/6J background lacking the ASIC1 (ASIC1 -/-, n = 7 male, 6 female) or ASIC3 gene (ASIC3-/-, n= 7 male, 4 female) and C57BL/6J mice (n= 91 male, 6 female). Both strains of mice have been previously characterized (253, 254), and show similar results when compared against wild-type littermates and C57/BL6 in study of muscle pain (68, 233) Fatigue Paradigm Muscle fatigue was induced as previously described(148). Briefly, mice were deeply anesthetized using 2-4% isoflurane and needle electrodes connected to a Grass S88 solid-state square waveform generator (Grass Technologies, West Warwick, RI) were inserted into the belly of the gastrocnemius. Three maximum force contractions were elicited by applying 7 volts at 100 Hz for 500 ms to establish baseline force. Six minutes of sub-maximal contractions were stimulated using 7 volts at 40 Hz for 3.75 seconds with 4.25 seconds of rest between contractions. Three additional maximum force contractions were then elicited to determine the decline in force after fatiguing contractions. Force was measured by attaching the plantar surface of the foot to a force plate connected to an iworx

74 59 FT-302 force transducer (iworx, Dover, NH). Data was collected using LabVIEW software and analyzed using Freemat and Python scripts. Raw data was processed by subtracting the baseline force from each contraction, calculated by the average force on the plate at time points immediately before and after muscle contraction. The raw data was then multiplied by a conversion factor derived from a standard curve in 1 g increments to get grams of force. Fatigue was operationally defined as a decline in force between baseline and final maximum force contractions. Male ASIC1-/- (n=7) and ASIC3-/- (n=7) mice were compared to male wild type mice (n=6) for initial maximum contraction force, final maximum contraction force, and at each of the submaximal fatiguing contractions. Animals missing 20 or more data points (>45%) in a fatigue recording were excluded from the analysis (n=2 ASIC1-/-, n=2 ASIC3-/-, n=2 wild type) Metabolite Recording Muscle ph was measured in deeply anesthetized animals using a ph probe inserted into the belly of gastrocnemius before and after electrically stimulated muscle contractions. After shaving the overlying hair, an incision was made in the skin to open an 8 mm x 8 mm square over the center of the gastrocnemius muscle. Muscle fibers were gently teased apart and the tip of the probe was inserted into the muscle.

75 60 For the measurement of ph, a micro-ph probe (Lazar Research Laboratories, Los Angeles) connected to a JENCO 6230N (Jenco Instruments, San Diego) ph meter was calibrated at ph 7.0 and 4.0 before insertion into the muscle of male wild type mice. In the control group (n=4), the muscle was exposed and measurements were taken 6 m apart. For the experimental group (n=5), the muscle was exposed and then measurements were taken before and after the 6m fatigue treatment. The ph and force data were sampled continuously at 30 Hz using a Python script to record data from the JENCO 6230N ph meter. Though the sampling was continuous, the ph probe was applied to the muscle for only 30 s at a time to avoid loss of sensitivity. When not measuring muscle ph, the sensor was stored in a ph 6.8 phosphate buffer containing heparin, which served as both a reference to ensure sensitivity was maintained and as a cleaning solution. After each contact with the muscle, the tip was cleaned briefly with a cotton swab soaked in acetone. In order to avoid electrical interference caused by unwanted circuits forming between the mains power supply, electrical stimulator, and measurement probes, a wirelessly controlled battery powered electrical stimulator was developed. An Arduino Uno R3 microcontroller was programmed with the electrically stimulated muscle fatigue protocol. A digital output pin was used to control a transistor that gated a 7 volt current through needle electrodes. An RN-42 bluetooth antenna (Sparkfun Electronics, Boulder, CO) transmitted serial input to and from an Android device in order to receive and confirm instructions from the

76 61 user. This device was validated against the Grass S88 solid-state square waveform generator and produced identical contractions at 40 and 100 Hz Exercise Enhanced Pain Model The low-intensity muscle insult consisted of two intramuscular (i.m.) injections of 20 µl normal saline into the gastrocnemius muscle 5 days apart while the mouse was anesthetized with 4% isoflurane. The ph of the normal saline was adjusted with HCl to ph Control injections consisted of two injections of normal saline (ph 7.2 ± 0.1) 5 days apart. The unbuffered ph 5 saline injections reduce muscle ph to approximately 6.9 (66), which is comparable to decreases seen after intense exercise (6, 21). Neither 2 injections of ph 5.0 nor 2 injections of ph 7.2 produce hyperalgesia (74, 75, 148) Muscle withdrawal thresholds. Muscle withdrawal thresholds (MWT) were measured by applying force sensitive tweezers to the belly of the gastrocnemius muscle as previously described, where lower thresholds indicate greater sensitivity. Mice were acclimated to this behavioral paradigm in two 5 minute sessions over a two day period prior to the first injection. Briefly, mice were placed in a gardener s glove, the hindlimb was held in extension, and the muscle was squeezed with force sensitive tweezers until the animal withdrew its hindlimb. An average of 3 trials

77 62 per animal was taken at each time period. A decrease in withdrawal thresholds was interpreted as muscle hyperalgesia Genetic and Pharmacologic Studies of ASIC1 and ASIC3 in the Exercise Enhanced Pain Model The role of ASIC1 and ASIC3 were assessed using genetic and pharmacological approaches. ASIC1-/- (n = 3 male, 4 female) or ASIC3-/- (n= 4 male, 4 female) were compared side-by-side with C57BL/6 mice (n=5 male} in the exercise enhanced pain model. Previous work shows that male and female wild type mice develop similar mechanical hyperalgesia in the treated limb, but females also develop mechanical hyperalgesia in the contralateral limb(148). Sex differences in ASIC1-/- and ASIC3-/- mice in this model, however, have not been evaluated. Muscle withdrawal thresholds were measured at baseline and 24 h after the induction of the pain model, i.e. after the second injection of ph 5.0 saline. For the behavioral pharmacology studies, male C57BL/6 mice were were pre-treated with intramuscular injection of psalmotoxin-1 (12nM, n = 8; 40 nm, n = 7; 120 nm, n = 12), ApeTx2 (20µM, n = 14; 70µM, n = 8; 200 µm, n = 7) or vehicle (0.9% saline, n= 16) 5 minutes before muscle fatigue on day h after the second ph 5 saline injection, animals were tested for muscle withdrawal threshold. Animals were divided into multiple groups tested across several weeks. Each testing group consisted of vehicle control and multiple doses of both ApeTx2 and psalmotoxin-1. Though each group contained representative samples for each

78 63 compound and dose, for legibility, ApeTx2 and psalmotoxin-1 are plotted on separate graphs. Naive (n = 4) and vehicle controls are identical between graphs for comparison. Given the discrepancy in behavior between ASIC1a-/- mice and wild type mice treated with psalmotoxin 1 (fig. 3-3, fig. 3-4), specificity for psalmotoxin-1 to ASIC1a was tested in the same behavioral pharmacology protocol. ASIC1a-/- mice were subjected to the exercise-enhanced pain model and pre-treated with either 40 nm psalmotoxin (n = 2 male, 2 female) or saline (n = 2 male, 2 female) prior to fatigue. Muscle withdrawal thresholds were measured at baseline and 24 h after induction of the pain model. Because psalmotoxin-1 can trigger the release of enkephalin and reduce pain behaviors when present in the brain(255), systemic effects of psalmotoxin-1 were tested by exposing wildtype mice to the exercise-enhanced pain model and then pre-treating with intramuscular injection of either the ipsilateral (acid injected, fatigued; n = 2 male, 2 female) or contralateral (untreated; n = 2 male, 2 female) muscle with 40 nm psalmotoxin-1 prior to fatigue. Muscle withdrawal thresholds were measured at baseline and 24 h after induction of the pain model Statistical Analysis Data are reported as means +/- S.E.M. Muscle force, withdrawal thresholds, and ph measurements were analyzed with repeated measures ANOVA followed by post-hoc testing with a Tukey test. For ph measurements, differences between groups at baseline and after treatment were assessed by independent t-

79 64 tests and differences within groups from baseline to post-treatment were assessed by paired t-tests. Significance for the repeated measures ANOVA and Tukey test was set at To control for multiple comparisons, Bonferroni correction was applied to all of the t-tests for differences in ph, adjusting the level of significance to Results Muscle fatigue is not changed by genetic deletion of the ASIC1a or ASIC3 or by antagonism of ASIC1a or ASIC3. The force of muscle contractions across fatiguing stimuli were recorded for ASIC1a-/-, ASIC3-/-, and wildtype mice. The force of contraction decreased significantly across time in all three groups. However, there were no significant differences between groups in initial force, rate of fatigue, or post-fatigue force (repeated measures ANOVA, F 2,17 = 2.452, p = 0.116). Similarly, pretreatment with either psalmotoxin-1 (repeated measures ANOVA, F 2,18 = 1.168, p = 0.333) or ApeTx2 (repeated measures ANOVA, F 2,17 = 2.461, p = 0.115) did not alter the initial force, rate of fatigue, or post-fatigue force (fig. 3-1) Fatiguing muscle contractions produce significant decrease in muscle ph. The effect of muscle fatigue on muscle ph was assessed by inserting a ph probe into the belly of the gastrocnemius muscle before and after either 6 m of

80 65 fatiguing muscle contractions or 6 m of inactivity. For both groups, the initial ph of the muscle was comparable: 7.14 (+/ SEM) and 7.09 (+/ SEM) for the fatigued and inactive groups, respectively. After fatiguing muscle contractions, muscle ph dropped to 6.86 (+/ SEM) while the inactive group remained relatively unchanged at 7.15 (+/ SEM). Muscle fatigue had a significant effect on muscle ph (repeated measures ANOVA, F 1,7 = , p < 0.05). At baseline, there were no significant differences between groups (Student s t-test, t 7 = 1.230, p = 0.258). The ph of muscles exposed to fatiguing muscle contractions decreased significantly from baseline (Paired t-test, t 4 = 5.778, p = 0.004) and was significantly lower than the inactive controls (Student s t-test, t 7 = 4.736, p = 0.002). The ph of inactive control muscles did not change from baseline (Paired t-test, t 3 = 1.452, p = 0.243) (fig.3-2) Genetic deletion of ASIC3, but not ASIC1a, prevents decreases in muscle withdrawal threshold after treatment with acidic saline and fatiguing muscle contractions. The roles of ASIC1 and ASIC3 in the development of exercise enhanced muscle pain were tested by comparing muscle withdrawal thresholds in ASIC1-/-, ASIC3-/- mice after exposure to acidic saline and fatiguing muscle contractions and compared to wild-type controls. As previously shown (148), wild type mice showed a significant decrease in the muscle withdrawal threshold. ASIC1a-/- mice also showed a significant decrease in muscle withdrawal threshold, but ASIC3-/-

81 66 mice showed no decrease and were significantly different from both wildtype and ASIC1a-/- mice (fig. 3-3, repeated measures ANOVA with post-hoc Tukey test, F 2,18 =20.614, p < 0.001) Blockade of ASIC1 and ASIC3 during the fatigue task prevents decreases in muscle withdrawal threshold. To confirm the results from ASIC1a-/- and ASIC3-/- mice, we blocked ASIC1 and ASIC3 with psalmotoxin-1 and ApeTx2, respectively, prior to the fatigue task. Mice treated with vehicle (saline) showed a significant decrease in muscle withdrawal threshold. Withdrawal thresholds in mice with the ASIC3 antagonist ApeTx2 were significantly higher than controls. The effect of ApeTx2 occurred in a dose-dependent manner with the 70 and 200 µm doses significantly higher than vehicle. (fig 3-4A, repeated measures ANOVA with post-hoc Tukey test, ApeTx2: F 4,3 9=10.643, p <0.001 ). Surprisingly, in contrast to the findings in ASIC1a -/- mice, blockade of ASIC1 with psalmotoxin-1 also prevented the decrease in withdrawal thresholds (fig 3-4B, repeated measures ANOVA with post-hoc Tukey test, F 4,38 =8.674, p < 0.001). Because of the discrepancy between behavior in ASIC1a -/- mice and mice treated with the ASIC1 antagonist, psalmotoxin-1 was tested in ASIC1a-/- mice to examine for non-specific effects. ASIC1a-/- mice pre-treated with either psalmotoxin-1 or saline 5 minutes before fatiguing muscle contractions showed similar decreases in muscle withdrawal threshold (fig 3-5A, repeated measures ANOVA, F 1,6 =0.628, p = 0.458).

82 67 To rule out effects of psalmotoxin-1 outside the treated muscle, wild type mice were pre-treated with 40 nm psalmotoxin in either the ipsilateral (acid injected, fatigued) muscle or contralateral (untreated) muscle. Decreases in muscle withdrawal threshold were significantly greater in mice treated in the contralateral muscle compared to those treated in the ipsilateral muscle (fig 3-5B, repeated measures ANOVA, F1,6=53.54, p < 0.001). 3.5 Discussion These results indicate that six minutes of electrically stimulated muscle contractions not only results in substantial fatigue, but also physiologically relevant accumulation of protons, in the detectable range for ASICs. While a number of channels can detect protons, heteromeric channels expressing ASIC3 are necessary for the exercise enhanced pain effect since genetic deletion of ASIC3 prevents the development of hyperalgesia. ASIC1a is also a component of these heteromeric ASIC channels, but is not necessary since hyperalgesia still develops after genetic deletion of ASIC1. Together, these data suggest that exercise enhance muscle pain is driven by local accumulation of protons in exercising muscle, which activate heteromeric ASICs on nociceptors Muscle fatigue decreases ph The current study shows that muscle fatigue results in a significant decrease in the intramuscular ph to 6.8 (from 7.12 at rest) with 6 m of fatiguing contractions in the gastrocnemius muscle. This is in agreement with prior work

83 68 showing a in ph between (from 7.1 at rest) with fatigue in muscle, depending on the intensity of the muscle activity (251, 252, 256, 257). Venous effluent from muscle decreases to a similar degree with exercise (0.3 ph units to ph 7.1) but starts at a higher resting ph (7.4) (258, 259). Sustained exposure to ph in the range could lead to steady state desensitization of ASIC channels(61).since muscle nociceptors are located in the adventitia of arteries (63), they are in close proximity to both blood and intramuscular fluid. While the milieu surrounding these nerve terminals is not well understood, it may be the case that the terminals are typically surrounded by ph 7.4 serum and only transiently exposed to the more acidic intramuscular fluid, such as during muscle contractions. In this way, decrease in intramuscular ph may contribute to the exercise enhanced pain effect ASICs are required for development of muscle hyperalgesia Decreases in extracellular ph can be detected through a variety of receptors, including TRP(260) and ASICs(58). In particular, ASIC3 is necessary for mechanical hyperalgesia induced by repeated acid injections in muscle and inflammation; while ASIC1a contributes to hyperalgesia after muscle inflammation, but not repeated acid injections(66, 75, 139). The current study is consistent with these results and shows that ASIC3-/- mice do not develop mechanical hyperalgesia after exposure to the exercise-enhanced pain model,

84 69 while ASIC1-/- mice still develop mechanical hyperalgesia comparable to wild type mice. Muscle afferents express ASIC1a and ASIC3, possess heteromeric ASIC channels, and respond to ph within the ranges produced by fatiguing muscle contractions(63, 64, 68, 250). Muscle afferents from ASIC1a-/- mice demonstrate reduced ph sensitivity, desensitize slower, and recover faster after exposure to drops in ph. In contrast, ASIC3-/- muscle afferents show no change in ph sensitivity, but have a slower rate of desensitization and slower recovery from desensitization (64). ASIC3 may be required for the development of mechanical hyperalgesia for several reasons: the faster kinetics of recovery may be necessary to reset the receptor between fatiguing contractions; interaction with P2X receptors may result in synergistic combination of fatigue byproducts(76, 77)(see Chapter 4);or may enhance trafficking of the channel to the cell membrane(261). It is curious that ASIC1 is not necessary for exercise-enhanced muscle pain, given that loss of this subunit reduces ph sensitivity in the physiological range(64). These prior studies measured acid-evoke currents in vitro. It is possible that the in vivo ph sensitivity of peripheral nerve terminals is different than isolated DRGs due to other metabolites present in the surrounding milieu(76), interactions with other proteins(77), or other factors. The present study shows, however, that mechanical hyperalgesia is primarily mediated by ASIC3, and that ASIC1, while present in the wild type channel, is not necessary for the development of mechanical hyperalgesia.

85 70 Surprisingly, the ASIC antagonist studies did not match the results of the ASIC knockout mice studies. While the ASIC3 antagonist ApeTx2 prevented the development of exercise-enhanced pain similar to ASIC3-/- mice, the ASIC1 antagonist psalmotoxin-1 also prevented exercise enhanced pain despite the fact that ASIC1-/- mice develop exercise enhanced pain similar to wild type mice. Given that wild type ASICs are most likely composed of ASIC1a/2a/3 heteromeric channels(64),this suggests psalmotoxin-1 is able to disrupt the function of wild type channels. Previous work indicates that psalmotoxin-1 is highly specific for ASIC1a and ASIC1b homomeric channels, and the current study shows that psyalmotoxin-1 has no effect in preventing the development of hyperalgesia in ASIC1-/- mice. Interestingly, prior work shows psalmotoxin-1 blocks current through ASIC1a homomeric channels, but not ASIC1a/3 or ASIC1a/ASIC2a channels(262). In contrast, opening of ASIC1b is facilitated by psalmotoxin- 1(263). Further, site directed mutagenesis (264, 265) and crystal structures of psalmotoxin-1 binding to ASIC1a ( ) indicate that hydrophobic interactions between psalmotoxin-1 and helix 5 are crucial for psalmotoxin-1 binding. In other ASIC subtypes, these residues differ substantially and likely prevent the hydrophobic residues of psalmotoxin-1 from binding (264, 266). Despite this specificity for the hydrophobic patch at helix 5, the polar residues of psalmotoxin- 1 extend into the acidic pocket of the adjacent subunit which is conserved across subtypes (266, 267). Thus, it is possible that in wild type ASICs, the hydrophobic

86 71 patch of psalmotoxin-1 binds to helix 5 of the ASIC1a subunit and the polar region extends into the adjacent acidic pocket. Alternatively, psalmatoxin-1 could produce analgesia through modulating endogenous opioid release. Intracerebral injection of psalmotoxin-1 produces analgesia by stimulating the release of enkephalin (255). It is possible that psalmatoxin-1 could have systemic effects after injection into the muscle. However, this is unlikely since injection into the contralateral gastrocnemius muscle had no effect on the development of the exercise-induced hyperalgesia. Taken together, these data indicate that psalmotoxin-1 prevents the development of mechanical hyperalgesia by altering the function of ASICs in an ASIC1adependent manner. Further, data from both ASIC antagonists indicate that ASIC activation of nociceptors is necessary for the development of exercise enhanced mechanical hyperalgesia ASICs and fatigue-enhanced hyperalgesia While we find no significant effect of ASIC1a or ASIC3 on muscle fatigue induced by electrical stimulation, other work has suggested ASIC1a and ASIC3 may contribute to muscle fatigue. Our laboratory previously showed that male ASIC3+/+ mice muscle showed less fatigue than male ASIC3-/- mice and female ASIC3+/+ mice (1h Rota-Rod task); this difference did not occur in female mice (269). Further, female ASIC1a-/- mice show decreased grip duration compared to wild types and males(270). The discrepancy between these studies may be

87 72 explained by several factors, including the longer duration of exercise, the taskspecific nature of muscle fatigue, and the role of voluntary effort in task failure. In the current study we use a short fatigue task (6m) resulting in fatigue lasting 10 m in a single muscle(148), as compared to a much longer (3 h) and widespread protocol that produced fatigue lasting (2 h) in our prior study(269). Notably, when the duration of fatigue task was reduced in our prior study (1.5h), there was no difference in fatigue between ASIC3-/- and WT mice in males or females (269). Further, different muscles were studied. The current study measured contraction of the gastrocnemius muscle, which is involved in maintaining posture and fatigues slowly (204). The paw flexors, however, are composed of fast twitch fibers and fatigue at a different rate (271, 272). Finally, in the present study muscle contractions were generated with electrical stimulation which are not affected by the voluntary effort of the animal. In contrast, exercise on the Rota- Rod and grip force both depend on the voluntary effort of the animal. While the muscles may be capable of generating similar forces, the perception of effort may differ, leading to earlier voluntary release in ASIC3-/- mice. Patients with chronic fatigue syndrome report worsening of their fatigue symptoms with exercise, which is correlated with significant upregulation of ASIC3 mrna measured in blood samples, suggesting ASIC3 may contribute to the subjective feeling of fatigue (246). Related to this, in female ASIC1a-/- mice ASIC1a was found to play a role presynaptically in motor neurons (270). This presynaptic inhibition of neuromuscular transmission would be overridden by the direct electrical

88 73 stimulation of the peripheral ending of motor neurons used in the current study. Therefore, despite the decreased voluntary performance, the muscles of both ASIC1a-/- and ASIC3-/- mice are capable of generating comparable force contractions as wild type muscle, which indicate that ASIC1a and ASIC3 are not necessary for normal muscle development or that compensatory changes allow equivalent performance on this particular task. Further, antagonism of either the ASIC1a or ASIC3 receptor does not effect the rate at which muscle force declines during fatiguing muscle contractions, suggesting that neither receptor contributes to the fatigue seen in this protocol Clinical Implications Worsening of pain after exercise remains a significant barrier to adherence to an exercise regimen that is crucial for treating diseases like myofascial pain syndrome, fibromyalgia, and low back pain(8, 12, 245). Many mechanisms likely contribute to the development of pain after exercise, including tissue damage and inflammation, but for fibromyalgia patients whose symptoms acutely worsen with even light or moderate exercise that does not damage muscle, targeting ASICs may provide symptomatic relief(10, 11). More than that, studies show consistent exercise improves many of the symptoms of in people with fibromyalgia(8)and prevents the development of chronic and exercise-induced pain in animals(273). By inhibiting ASICs prior to exercise in the early period of an exercise regimen, it

89 74 may be possible to address the acute exacerbation of pain and fatigue with exercise, leading to greater adherence to a regular exercise program Summary In conclusion, these data implicate ASICs in the development of exercise enhanced pain through a decrease in ph from the fatigued muscle. Muscle fatigue reduces intramuscular ph from approximately 7.1 to 6.8, which is in the range for activating ASICs. Studies in knockout mice indicate ASIC3, but not ASIC1a, is the essential subunit within the wild type channel on muscle DRG; however, ASIC1a is present in the wild type channel and can serve as a binding site for psalmotoxin-1, an ASIC1a antagonist. In this way, pre-treatment with psalmotoxin-1 is able to prevent muscle pain similar to the ASIC3 antagonist ApeTx2. Therefore, ASICs are necessary for the development of exercise enhanced muscle pain, likely for their ability to detect the decreases in intramuscular ph over the course of fatiguing muscle contractions.

90 Figure 3-1 Measurement of force during fatiguing muscle contractions. No significant differences were observed between ASIC1-/- (n=7), ASIC3- /- (n=7), and wild type mice (n=7) (p = 0.116) (A). No significant differences were observed between mice pre-treated with the ASIC1 antagonist psalmotoxin-1(12 nm n = 4, 120 nm = 8, vehicle n = 9), ASIC3 antagonist ApeTx2 (20 µm n = 7, 200 µm n = 5, vehicle n = 9), or saline (psalmotoxin-1, p = and ApeTx2, p =0.115) (B & C). Mean + SEM. 75

91 Figure 3-2 ph of Muscle Before and After Fatiguing Muscle Contractions. Individual replicates for each ph measurement in before and after fatigue (n=5), and in inactive control (n=4) are shown in (A). The ph of fatigued muscle was significantly lower than baseline and the inactive group (B). *, p < , difference from baseline and inactive post treatment. Mean + SEM. 76

92 77 Figure 3-3 Comparison of muscle withdrawal threshold in ASIC1-/-, ASIC3-/-, and wild type mice in the exercise enhanced pain model. At baseline there were no significant differences in muscle withdrawal threshold between ASIC1a (n=7), ASIC3-/- (n=8), and wild type animals (n=5). The muscle withdrawal threshold of ASIC3-/- mice was significantly higher than both ASIC1-/- and wild type after treatment with the exercise enhanced pain model. No sex differences were observed within ASIC1a-/- and ASIC3-/- mice. There were no significant differences between ASIC1-/- and wild type mice. *, p < 0.05, difference from ASIC1-/- and wild type. Mean + SEM. Figure 3-4 Comparison of muscle withdrawal thresholds in mice pre-treated with ASIC3 (A) and ASIC1 (B) antagonists in the exercise enhanced pain model. All groups showed similar muscle withdrawal thresholds at baseline. Muscle withdrawal thresholds of animal pre-treated with moderate and high doses of the ASIC3 antagonist ApeTx2 (20 µm n = 14, 70 µm n = 8, 200 µm n = 7, vehicle n = 16, naïve n = 4) were significantly greater than vehicle or low dose ApeTx2 (A). *, p < 0.05, difference from saline controls. Muscle withdrawal thresholds of animals pre-treated with the ASIC1 antagonist Psalmotoxin-1 (12 nm n = 8, 40 nm n = 7, 120 nm n = 12, vehicle n = 16, naïve n =4) were significantly higher than the vehicle control(b). *, p < 0.05, difference from naive. Mean + SEM.

93 Figure 3-5 Specificity of psalmotoxin effects to muscle ASIC1a. (A)Muscle withdrawal threshold in ASIC1a knockout mice pre-treated with psalmotoxin-1 in the exercise enhanced pain model. ASIC1a knockout mice were pre-treated with either psalmotoxin (n = 4) or saline (n = 4) prior to fatiguing muscle contractions. There were no significant differences between groups (p = 0.458). (B)Muscle withdrawal threshold after injecting psalmotoxin-1 into the treated versus untreated muscle in the exercise enhanced pain model. Wild type mice were pretreated with psalmotoxin in either the ispsilateral (fatigued, acid injected; n = 4) or contralateral (untreated; n = 4) muscle prior to fatiguing muscle contractions. There were no significant differences between groups at baseline. Muscle withdrawal thresholds after exposure to the exercise enhanced pain model were significantly higher in mice treated with ipsilateral injection of psalmotoxin (*, p < 0.001). 78

94 79 CHAPTER IV EFFECT OF INTRAMUSCULAR PROTONS, LACTATE, AND ATP ON MECHANICAL HYPERALGESIA IN RATS 4.1 Abstract While common experience indicates that exercise is painful, the basis of pain after exercise is poorly understood. Strenuous exercise can lead to tissue damage, inflammation, and subsequent delayed onset muscle soreness; however, the stimuli that produce pain during muscle contractions are not clear. Classically, lactic acid has been thought to cause muscle pain during contractions and previous studies of cardiac afferents suggests lactic acid does in fact activate nociceptors more effectively than other tips of acid of the same ph. By-products of muscle activity like protons, lactate, and ATP have been shown to activate DRGs and can be painful when injected into the muscle of humans. Whether protons, lactate, and ATP have a dose-dependent effect on their own or produce a synergistic effect when combined has not been studied. Using a rat model of mechanical hyperalgesia, we tested each of these compounds individually over a range of physiologic and supra-physiologic concentrations to establish their effects on muscle withdrawal threshold 30 minutes after injection. Further, we combined all three compounds in a series of dilutions and tested their effect on muscle withdrawal threshold. Surprisingly, we found no dose-dependent effect on

95 80 mechanical hyperalgesia for protons, lactate, or ATP. Only ph 4 saline produce mechanical hyperalgesia alone. When combined, we found that only the most dilute combination of protons, lactate, and ATP produced significant mechanical hyperalgesia. These data indicate that while protons, lactate, and ATP may contribute to mechanical hyperalgesia after exercise, they are not sufficient in themselves to reproduce the effect 30 minutes after intramuscular injection. 4.2 Introduction Chronic muscle pain is a common condition contributing to disability in millions of people worldwide. The initiation of chronic muscle pain is poorly understood and likely multifactorial. Recent work indicates that fatiguing muscle contractions can trigger long lasting mechanical hypersensitivity that affects female mice more than males(148), a finding consistent with the 8:1 female predominance of fibromyalgia in humans(274). Further, local antagonism of acid sensing ion channels (ASICs) in the muscle prior to fatiguing muscle contractions prevents the development of mechanical hypersensitivity (see Chapter 3), indicating that detection of fatigue by-products may contribute to the development of muscle pain. However, decreases in ph alone are unlikely to be the cause of muscle pain, as conditions that lower systemic ph do not trigger widespread muscle pain(251, ). Pain is only produced acutely when ph is decreased by buffered solution and recovers within minutes after a return to normal ph(67).

96 81 ph is rapidly buffered in the muscle(66, 259), and thus long-term decreases in ph alone are not likely. Studies of rat DRG show that physiologic concentrations of protons, lactate, and ATP trigger calcium influx in a dose-dependent manner(104). Further, in humans infusion of these three compounds in combination produces subjective feelings of fatigue at low concentrations and pain at higher concentrations (278). While other molecules are also nociceptive in the muscle, these 3 substances are particularly interesting because properties of their receptors suggest they interact and enhance afferent activity. Previous studies show ASICs are normally bound by Ca2+ that prevents flux of ions through the channel. By chelating Ca2+, lactate can enhance the response to even modest decreases in ph(76, 279). ASICS can also physically interact with one or more members of purinergic receptor (P2X) family to enhance sensitivity to protons in the presence of ATP(77). Despite this molecular and cellular evidence, an interaction between protons, lactate, and ATP has not been confirmed behaviorally, nor has the nature of the interaction been studied. In this study, we test the effects of ph, lactate, ATP, and the non-hydrolyzable α,β-methyl-atp alone and in combination on the withdrawal threshold of the muscle. We hypothesize that combining ph, lactate and ATP will produce a synergistic effect and mimic the effects of fatigueenhanced muscle pain.

97 Materials and Methods These experiments were approved by the Animal Care and Use Committee at the University of Iowa. Male Sprague-Dawley rats ( g, Harlan, n=226) were used for these studies Muscle Withdrawal Threshold Muscle withdrawal thresholds (MWT) were measured by applying force sensitive tweezers to the belly of the gastrocnemius muscle as previously described, where lower thresholds indicate greater sensitivity. Rats were acclimated to a gardner s glove in two five minute sessions per day over two days prior to behavioral testing. On the day of testing, rats were placed in a gardener s glove, the hindlimb was held in extension, and the muscle was squeezed with force sensitive tweezers until the animal withdrew its hindlimb. An average of 3 trials per animal was taken at each time period. A decrease in withdrawal thresholds was interpreted as muscle hyperalgesia Drugs ATP (Sigma-Aldrich, 24 mm to 760 nm), α,β-methylene ATP (10 µm to100 µm, Sigma-Aldrich), lactate (Sigma-Aldrich, 1.5 M to 474 µm), acidic saline (ph 4 to 7), andα,β-methylene ATP, in 3 doses (10 µm to100 µm, Sigma- Aldrich). Solutions were prepared in 0.9% saline. A single 100µL intramuscular injection was given to the gastrocnemius muscle while the rat was anesthetized with 4% isoflurane.

98 Protocol Baseline muscle withdrawal thresholds (MWTs) were measured on the morning of testing. The rats were anesthetized with 4% isoflurane and then injected intramuscularly with 100 µl of an algesic compound. 30 minutes after injection, MWT was measured again. Only a single dose of a single drug was injected into each rat. In the first phase of the study, rats were injected with a single compound dissolved in 0.9% normal saline. Proton concentration was adjusted with NaOH and HCl in normal saline and tested at ph 4, 4.5, 5, 6, and 7. ph. ATP was tested using half-log and log dilutions between 76 mm and 760 nm. Lactate was tested using half-log and log dilutions between 1.5 M and 474 µm. The ATP and lactate solutions were adjusted to ph 7.4. An additional series of experiments tested the effects of the non-hydrolyzable form of ATP, α,β-methylene ATP, in 3 doses (10 µm, 30 µm, 100 µm, Sigma-Aldrich). The second phase of the study used concentrations derived from the first phase to test for synergy between ph, ATP, and lactate. ATP, lactate, and protons were combined using highest ineffective concentrations found in the first phase: ph 6, 474 µm lactate, 2.6 nm ATP. This ratio was held constant across a series of 4 half-log dilutions. Saline (vehicle) and 3% carrageenan were used as a negative and positive control, respectively.

99 Results ph 4 saline produces significant decrease in muscle withdrawal thresholds. The effect of intramuscular injection of protons (ph) on muscle withdrawal thresholds was tested using acidic and normal saline (ph ). ph had a significant effect on muscle withdrawal threshold (fig. 4-1, repeated measures ANOVA, F 5,36 = , p <0.001). Of the ph values injected intramuscularly, only ph 4 saline produced a significant decrease in the muscle withdrawal threshold 30 minutes after injection relative to saline injected controls (Tukey test, p < 0.001) Lactate alone does not decrease muscle withdrawal threshold. The effect of intramuscular injection of lactate on muscle withdrawal threshold was tested using a number of concentrations, both in the physiologic and supra-physiologic ranges (450µM to 1.5 M). Lactate injected intramuscularly had no significant effect on muscle withdrawal thresholds over this range of doses (fig. 4-2,repeated measures ANOVA, F 6,59 = 1.988, p = 0.082) ATP alone does not decrease muscle withdrawal threshold. The effect of intramuscular injection of ATP on muscle withdrawal threshold was tested using a number of concentrations (24 mm to 760 nm). ATP

100 85 had a significant effect on muscle withdrawal threshold (fig. 4-3,repeated measures ANOVA, F 7,46 = 2.315, p = 0.042). Post-hoc testing showed no doses were significantly different from the saline control (Tukey test, p > 0.05) Lowest combination of protons, ATP, and lactate reduces muscle withdrawal threshold. Synergism between ph, ATP, and lactate was tested by combining ineffective concentrations of each compound and injecting half-log dilutions of this fixed ratio into the gastrocnemius muscle of rats. Muscle withdrawal thresholds at 30 minutes post-injection were compared to single-dose concentrations and saline controls. The concentration of this combination had a significant effect on muscle withdrawal threshold (fig. 4-4, repeated measures ANOVA, F 5,27 = , p < 0.001). Surprisingly, only the most dilute combination significantly decreased with muscle withdrawal threshold (post-hoc Tukey test, p = 0.001). This decrease was significantly less than the closest single dose of each of the compounds (repeated measures ANOVA, F 4,25 = 6.126, p = 0.001, Tukey test, p < 0.05 for each compound alone), and significantly less than saline controls (Tukey test, p <0.001).

101 α,β-methylene ATP reduces muscle withdrawal threshold. Because of the unexpected lack of effect of ATP on muscle withdrawal threshold, we tested the effect of the non-hydrolyzable α,β-methylene ATP on muscle withdrawal threshold. α,β-methylene ATP had a significant effect on muscle withdrawal threshold (fig. 4-5, repeated measures ANOVA, F 3,14 =14.028, p < 0.001). Each dose of α,β-methylene ATP decreased muscle withdrawal threshold similarly and was significantly less than saline controls (Tukey test, p < 0.05 for each dose). 4.5 Discussion The current study shows that intramuscular injection of acidic saline and an ATP analogue are sufficient to produce mechanical hyperalgesia in the muscle of rats; however, ATP and lactate had no effect on withdrawal thresholds. Further the combination of acid, lactate, and ATP produces mechanical hyperalgesia at concentrations that have no effect on muscle withdrawal thresholds when each is given alone. These data suggest that acid, lactate and ATP produce a synergistic response on muscle nociceptors to induce hyperalgesia Acidic ph and Pain Previous studies have shown that decreasing ph can produce pain in humans (67, 280) and animals (66, 169, 253, 281). In humans, intramuscular infusion of buffered ph 5.2 saline for 15 minutes produce pain and hyperalgesia

102 87 both local to the muscle and distantly at the ankle (67, 280). In mice, injection of a range of acid concentrations in the skin produces a short duration mechanical hyperalgesia (281). Similarly, an acute injection of ph 4.0 saline into muscle also produces a short duration decrease in withdrawal threshold of the paw in mice (66, 169, 253). The current study extends this finding and shows that there is also a reduction in withdrawal thresholds of the muscle after ph 4.0 saline, and this effect is eliminated by increasing ph 0.5 units (ph 4.5). Intramuscular ph likely does not decrease to the value of the injected solutions because of the buffering capacity of the physiologic fluid and clearance of fluid from the tissue. In fact, intramuscular injection of ph 4.0 reduces the ph in muscle to an average of ph 6.5 for less than 10 minutes(66).the hyperalgesia of the paw induced by ph 4.0 depends on ASIC3 as ASIC3-/- mice do not show this hyperalgesia(253). Decreases in muscle ph are also found in incisional models of muscle pain(282). Not only does ph decrease as low as 6.76 in the gastrocnemius(282), but incision also enhances the response of DRGs to exogenous application of mildly acidic saline solutions(283). Exercise also decreases muscle ph to similar levels (251, 284) and muscle fatigue decreases ph to 6.8 (Chapter 3). Yet, ph 6.6 solutions applied to DRG innervating muscle do not trigger calcium influx(104) nor is infusion of ph 6.6 saline reported as painful in human subjects(278).these data suggest that ph has a narrow range over which it can produce hyperalgesia, that the decrease requires ASIC3 for induction of hyperalgesia, and that other factors likely work in concert with ph to induce hyperalgesia and pain.

103 Lactate and Pain Previous studies in humans indicate that lactate alone does not produce hyperalgesia(278). In humans, lactate is normally present in plasma and interstitial fluid at approximately 1 mm, and can increase to 10 mm after exhausting exercise(44). In rats, incision of muscle also results in sustained increases in tissue lactate concentration (from approximately 1 mm at baseline to after incision)(285) and is associated with muscle pain(286). While no studies in animals have tested the effect of exogenously applied neutral lactate on muscle hyperalgesia, cardiac efferents show a greater response to lactic acid than acidic phosphate buffer of the same ph(5.42)(287). Lactate alone at a normal ph has minimal effects in increasing intracellular calcium concentration in isolated DRG neurons(104), but when ph is decreased the effects of lactate are potentiated(104). A similar trend is seen in humans: injection 50 mm neutral ph lactate into the hypothenar muscle results in no pain sensation, but lower concentrations of lactate produce pain when combined with protons and ATP (10 mm lactate, ph 7.2, 500 nm ATP) (278). In cell culture, lactate acts as a Ca2+ chelator and potentiates the response of ASICs to protons by facilitating the displacement of Ca2+ from the acidic pocket of ASICs where it prevents channel opening(76, 279). In the present study we show lactate by itself is not sufficient for the development of mechanical hyperalgesia. This suggests that even at high concentrations, where presumably a

104 89 substantial amount of free Ca2+ is chelated, protons are still needed to displace the Ca2+ bound to the acidic pocket and open ASIC channels ATP and Pain Previous studies have shown that injection of ATP into the muscle activates muscle nociceptors in animals(96) and is painful in humans(100). In humans, injection of low dose ATP (5micro molar) into the thumb is not painful(278), but (9-36 mm) in the trapezius muscle produced pain at rest and mechanical hyperalgesia (100). ATP binds to a range of both metabotropic (P2Y) and ionotropic (P2X) receptors (288). In addition to generating an inward current, ATP binding to P2X receptors can potentiate the response of ASIC3 to decreases in ph through direct physical interaction between ASIC3 and P2X receptors (77). Studies of the masseter show that P2X receptors are preferentially expressed on nociceptive neurons(98). Further, mechanical stress and muscle contraction leads to the upregulation of P2X3 and subsequent mechanical hyperalgesia in the muscle (42) In the present study, however, we show no muscle hyperalgesia despite using much higher concentration of ATP (24 mm) than the effective doses in humans(100). The basis for this difference is unclear but may represent differences in metabolism, volume of injection, or the muscle injected. In contrast, we show injection of a non-hydrolyzable form of ATP, α,β-methylene ATP, does produce mechanical hyperalgesia. This is consistent with studies showing α,β-methylene ATP activates muscle nociceptors(97) and produces

105 90 mechanical hyperalgesia(99) at similar concentrations used in the present study. In addition to being more stable, α,β-methylene ATP has lower affinity to P2X2, P2X4, and P2X5(289). It is possible that the ATP was degraded more rapidly than expected in our preparation or that the different affinities for P2X receptors contribute to the differences between the human and rat studies. In summary, ATP can activate DRG through purinergic receptors, P2X receptors are upregulated after inflammation and exercise, and P2X receptors can produce pain and hyperalgesia Synergism between ph, lactate and ATP Physiologic combinations of ph, lactate, and ATP have previously been shown to activate rat DRGs and produce pain in humans(104, 278). In the present study, however, we were only able to induce mechanical hyperalgesia in rats at the lowest dose combination (ph 7.5, 15 µm lactate, 76 nm ATP). Compared to the studies by Light and Pollak, the dose of lactate used in the present study was lower, but proton and ATP concentrations were higher. The lower lactate concentration may have been too low relative to the 1mM resting lactate in the muscle (44, 286). Without sufficient lactate to chelate Ca2+, ASICs may be less sensitive relative to conditions found during fatiguing muscle contractions. However, higher doses of lactate did not result in greater potentiation of the response in the current study. It is curious that the lowest concentration combination produced the greatest mechanical hyperalgesia. This may mean that

106 91 simply increasing concentration of these compounds is not sufficient to produce hyperalgesia; rather, the concentrations must be within a specific range for which the receptors are attuned. Despite these limitations, the fact that concentrations of protons, lactate, and ATP, which were unable to produce an effect alone, resulted in mechanical hyperalgesia when combined suggests these compounds are synergistic Clinical Implications Fatigue and muscle pain are two major contributing factors to disability in patients with diseases like fibromyalgia. The interaction between pain and fatigue may arise from common substances produced by the muscle by-products of muscle activity like protons, lactate, and ATP. Abnormal response to the presence of these compounds may contribute to the subjective feelings of fatigue and muscle pain (278). Further, previous work has shown that fatiguing muscle contractions can enhance the response to sub-threshold muscle insult, producing long lasting pain ((148),Chapter 2). The release of protons, lactate, and ATP during these fatiguing muscle contractions may contribute to the sensitization of muscle nociceptors peripherally or stimulate nociceptor activity directly, leading to enhanced neuron firing and subsequent central sensitization. While supraphysiologic concentrations of these compounds alone are not sufficient to produce mechanical hyperalgesia, this study indicates that in the right combination, even

107 92 relatively small concentrations are sufficient to produce hyperalgesia of the muscle Summary In summary, the current study shows that acidic saline or α,β-methylene ATP, but not lactate or ATP alone, is sufficient to produce mechanical hyperalgesia in rats. When combined, protons, lactate, and ATP produce mechanical hyperalgesia at low concentrations, but have no effect at higher concentrations. This indicates that these compounds have a synergistic interaction, but that development of mechanical hyperalgesia develops within a physiologically relevant range for which receptors like ASICs and P2Xs are sensitive.

108 93 Figure 4-1 Effect of acidic saline intramuscular injection on muscle withdrawal thresholds. Rats were injected with ph 4 (n = 12), ph 4.5 (n = 6), ph 5.0 (n = 6), ph 6.0 (n = 6), or neutral (n = 6) saline. There were no significant differences between groups at baseline. Muscle withdrawal thresholds were significantly lower in rats treated with ph 4 saline compared to higher ph solutions, which showed no change in muscle withdrawal threshold. Data are mean + SEM. (*, p < 0.001).

109 Figure 2 Effect of intramuscular lactate injection on muscle withdrawal thresholds. Rats were injected with lactate 470 µm (n = 8), 1.5 mm (n = 8), 4.7 mm (n = 8), 15 mm (n = 9), 47 mm (n = 8), 150 mm (n = 6), 470 mm (n = 6), 1.5 M (n = 6), or ph 7.0 saline (n = 6). No significant differences were observed between rats treated with saline and those treated with lactate (p > 0.05). Data are mean + SEM. 94

110 Figure 4-3 Effect of ATP intramuscular injection on muscle withdrawal threshold. Rats were injected with ATP 760 nm (n = 6), 7.6 µm (n = 6), 76 µm (n = 6), 760 µm (n = 12), 2.4 mm (n = 6), 7.6 mm (n = 6), 24 mm (n = 6), or ph 7.0 saline (n = 6). No significant differences were seen between animals treated with saline and those treated with ATP (p>0.05). Data are mean + SEM. 95

111 Figure 4-4 Effect of combinations of ph, lactate, and ATP on muscle withdrawal threshold. Rats were injected with either a combination of ATP, lactate, and protons, neutral saline (n = 6), or 3% carrageenan (n = 4). Iso1 consisted of ph 6.0, 474 µm lactate, and 2.4µM ATP (n = 6). Iso2 consisted of ph 6.5, 150µM lactate, 760 nm ATP (n = 6). Iso3 consisted of ph 7.0, 47 µm lactate, 240 nm ATP (n = 5). Iso4 consisted of ph 7.5, 15 µm lactate, 76 nm ATP (n = 6). The group with the lowest combination of ph, lactate, and ATP (Iso4) demonstrated significantly lower muscle withdrawal thresholds than the saline control (*, p < 0.05).Data are mean + SEM. 96

112 Figure 4-5 Effect of α,β-methylene ATP on muscle withdrawal threshold. Rats were injected with α,β-methylene ATP 10 nm (n = 4), 30 nm (n = 5), 100 nm (n = 5), or neutral saline (n = 7). Muscle withdrawal threshold decreased significantly in rats injected with either 10, 30, or 100 nm α,β-methylene ATP as compared to vehicle controls (*, p < 0.05) Data are mean + SEM. 97