A number of common pain conditions in people share deep muscle pain as a hallmark. At the APS 2018 Scientific Summit, a symposium titled “Muscle Pain Mechanisms: It’s Time to Dive Deep” focused on some of the latest research in this area. The symposium was dedicated to the memory of Edward R. Perl and William D. Willis Jr., both of whom made seminal contributions to the fields of pain and neuroscience.
Michael Jankowski, Cincinnati Children’s Hospital Medical Center, US, discussed his work on muscle pain, including recently published data (Ross et al., 2014; Ross et al., 2016; Ross et al., 2017; Queme et al., 2017; Ross et al., 2018) and some ongoing work. His lab uses an ischemic myalgia model, where transient occlusion of the brachial artery in the forelimb is followed by tissue reperfusion. This model recapitulates human phenotypes such as ongoing/spontaneous pain, mechanical hypersensitivity, muscle weakness, and decreased physical activity. Patients with ischemic myalgia often do not respond to standard analgesics, so novel treatment options are needed.
Previous studies have shown that muscle afferents are more efficient than cutaneous fibers at driving central sensitization, and that muscle injury sensitizes muscle nociceptors (Wall and Woolf, 1984; McMahon and Wall, 1989). The Jankowski laboratory hypothesized that ischemia and reperfusion (I/R) injury results in upregulation of specific genes within the dorsal root ganglia (DRG), which leads to response alterations in the muscle and distinct functional changes in afferent subtypes innervating muscle tissue. Ultimately, this drives increased pain behaviors. At the symposium, Jankowski showed that the pro-inflammatory cytokine interleukin 1 beta (IL1β) and glial cell-derived neurotrophic factor (GDNF) are both upregulated in muscle tissue after I/R, and their respective receptors (IL1r1 and GFRα1) are also upregulated in the DRG.
Considering the increase in muscle IL1β,Jankowski and colleagues tested whether blocking the IL1r1 receptor would affect the muscle afferent phenotype (determined by measuring changes in the afferent response to muscle metabolites/chemosensitivity) as well as pain-related behaviors. Indeed, inhibition of IL1r1 prevented I/R-induced changes in muscle afferent phenotypes. And, blocking IL1r1 also inhibited pain-related behaviors that follow I/R.
Next, the group investigated the downstream effect of IL1β and found that ASIC3, an ion channel that regulates mechanical sensitivity and chemosensitivity, was increased by 80-fold after I/R injury. Nerve-specific small interfering RNA (siRNA) knockdown of ASIC3 expression reversed spontaneous pain as well as mechanical hypersensitivity seen in the injured animals.
Since GFRα1 levels were also elevated in the DRG after I/R, the researchers used nerve-specific siRNA targeting here, too, to understand the role of this receptor in I/R pain. They found that siRNA targeting of GFRα1 also prevented mechanical hypersensitivity and partially prevented spontaneous pain following injury. Furthermore, GFRα1 activation was linked to increased expression of P2X5, which is a ligand-gated ATP receptor. Knockdown of GFRα1 resulted in decreased P2X5 expression, and P2X5-targeted siRNA knockdown also decreased I/R-related pain behaviors.
In sum, the research shows that I/R injury alters mechanical thresholds in muscle afferents, as well as the number of functional chemosensitive fibers, which likely drives spontaneous pain behavior, evoked mechanical hypersensitivity, reduced grip strength (a measure of muscle function), and alterations in activity levels. This all occurs through mechanisms relying on IL1β-ASIC3 and GDNF-P2X5 activity.
Clinical studies show that women often have greater pain sensitivity than men do, and various ischemic myalgias are more prevalent in women than in men. Therefore, Jankowski is also investigating sex-dependent mechanisms of ischemic myalgia pain in mice. This work shows that females also develop spontaneous pain after I/R injury, display evoked mechanical hypersensitivity, and exhibit a loss of muscle strength as do male mice with the same injury, but different, sex-dependent mechanisms of primary muscle afferent sensitization may underlie these behavioral changes. For instance, Jankowski and colleagues have reported sex differences in DRG mRNA expression after I/R injury, with significant elevations of ASIC3 in males but not females. Conversely, females had significant increases in the transient receptor potential vanilloid type 1 (TRPV1) ion channel—more than double those seen in males.
In addition to these acute changes, Jankowski also touched on an ongoing project investigating sex-dependent differences in the transition from acute to chronic ischemic myalgia pain. This work uses a two-hit I/R injury model in which a second I/R injury induces chronic pain, and has revealed different genetic changes in males versus females. For example, the second I/R hit leads to an extreme upregulation of P2X5 receptors in females that is completely absent in males.
Overall, Jankowski and colleagues show that I/R injury sensitizes muscle afferents, ultimately leading to pain. They have also uncovered two molecular pathways (IL1β-ASIC3 and GDNF-P2X5) that could potentially be targeted to better treat patients with deep muscle pain. This work highlights the importance of using both male and female rodents in preclinical research.
Kathleen Sluka, University of Iowa, Iowa City, US, and colleagues study peripheral and central mechanisms of chronic musculoskeletal pain in animal models and in humans. One area of interest is physical activity-modulated pain and fatigue, which is clinically relevant since physical inactivity is a major public health concern.
Sluka first turned to human studies, explaining that patients with chronic pain typically report physical fatigue and increased pain with physical activity, which in turn makes them less likely to participate in any physical activity, setting up a vicious cycle. To investigate this in a laboratory setting, she and her colleagues asked fibromyalgia patients to perform a physical activity task (Valpar peg test) lasting 15 minutes (Dailey et al., 2016). During this task, patients move pegs and shapes on a panel from shoulder height to four to six inches above the head and from overhead to waist height. After the task, patients with fibromyalgia reported significantly larger increases in pain and fatigue compared to healthy controls.
Next, Sluka described an animal model her lab has developed to study muscle pain in rodents. This model features acidic saline injections paired with a fatigue task. The injections alone do not cause hypersensitivity. But when they are combined with a fatigue task (such as electrical stimulation of the muscle for six minutes) or with activity-induced fatigue (forcing the animals to run on a running wheel for two hours), the animals show significant decreases in muscle withdrawal thresholds to pinch of the gastrocnemius muscle. Meanwhile, in a study of healthy human subjects (Frey Law et al., 2008), injection of acidic saline into the anterior tibialis muscle produced muscle pressure pain locally, which then evolved into referred pain in 60 percent of subjects. Here, significant sex differences were observed, with 80 percent of females developing referred pain compared to only 40 percent of males.
When muscles become fatigued, they release metabolites such as ATP, lactate, and protons, which can act on nociceptive neurons and cause pain. So Sluka has also asked whether such factors alone, when injected into muscle, could decrease muscle withdrawal thresholds, or if a combination was required. Results revealed that a combination of these factors was needed for behavioral sensitization in rats (Gregory et al., 2015).
Sluka and colleagues have also investigated whether their fatigue/acid-induced pain model requires the expression of acid-sensing ion channel 3 (ASIC3; Gregory et al., 2016). To do so, they used ASIC3 knockout mice and an ASIC3 inhibitor, APETx2, which is injected into the gastrocnemius muscle before a fatigue test. Knockouts and wild-type animals that received the ASIC3 inhibitor locally into the gastrocnemius muscle at higher doses did not develop chronic muscle pain. However, downregulating ASIC3 in muscle afferents using microRNA (miRNA) injection had no effect on mechanical withdrawal thresholds. This suggested that fatigue metabolites might not act directly on sensory nerve afferents, but rather via a different mechanism. This led the group to focus on muscle macrophages, whose numbers are increased in the fatigue/acid-induced pain model. They used liposomes containing clodronate to eliminate these cells and found that this prevented hyperalgesia from developing in their model. Other findings from Sluka’s studies further support a role for resident macrophages in muscle as contributors to muscle pain (Gong et al., 2016).
Run, run, run
The studies described above used sedentary animals, so the researchers next turned their attention to active animals, since physical activity is thought to decrease muscle pain. Here, they gave mice access to a running wheel in their home cages and compared them to animals without access to the wheel (Leung et al., 2016). The researchers found that running prevented mechanical hypersensitivity following acidic saline injections.
Looking more closely at the differences between the “runner animals” and the sedentary ones, Sluka and colleagues saw changes in the ratio of M1 (pro-inflammatory) macrophages and M2 (anti-inflammatory) macrophages in muscle. Runner animals had significantly more M2 macrophages compared to inactive animals, which had more M1 macrophages. Further experiments showed that the anti-inflammatory cytokine IL-10 mediated the effects of physical activity on pain.
Results consistent with the above findings were also reported earlier this year using a nerve injury model (Bobinski et al., 2018). Also, human studies have revealed that fibromyalgia patients show improvements in the balance of pro- and anti-inflammatory cytokines in response to exercise (Ortega et al., 2012; Bote et al., 2014).
Looking to the central nervous system (CNS)
Sluka also discussed the CNS contribution to musculoskeletal pain, with a focus on the NR1 subunit of the NMDA glutamate receptor in the rostral ventromedial medulla (RVM). She and her colleagues decreased the expression of the NR1 subunit in the RVM by injecting NR1 miRNA and found that this strategy prevented hyperalgesia in response to repeated acidic saline injections into muscle (Da Silva et al., 2010). Conversely, NR1 overexpression in the RVM caused significant hyperalgesia, confirming that this subunit facilitates pain.
Furthermore, phosphorylation of NR1 leads to increased expression of NMDA receptors at the synapse, along with increased current flow. Sluka reported that physical activity (use of a running wheel) prevented muscle pain by reducing NR1 phosphorylation (Sluka et al., 2013). More recent findings from Sluka’s group have further elucidated the importance of the RVM in muscle pain (Lima et al., 2017).
Cheryl Stucky, Medical College of Wisconsin, Milwaukee, US, has studied sickle cell disease (SCD) for many years and has now started to investigate deep muscle pain in a mouse model of SCD (Disclosure: the current writer is a member of Stucky’s lab but is not working on SCD and was not involved in the data collection). SCD is an inherited blood disorder that affects nearly 100,000 Americans and roughly three million people worldwide. Patients with severe SCD have a homozygous gene mutation that causes hemoglobin to polymerize during oxygen deprivation, dehydration, and physiological stress. Patients with SCD and mice carrying the mutated human hemoglobin gene experience severe acute pain brought about by low oxygen content. Up to 40 percent of SCD patients and mice also develop chronic pain.
With the help of Bonnie Freudinger, from the Medical College of Wisconsin’s engineering laboratory, Stucky and colleagues have adapted the strain gauge forceps originally developed by Kathleen Sluka for use in the SCD mouse model. These forceps can be used to measure gastrocnemius muscle compression withdrawal thresholds in mice. Unpublished results showed that both male and female SCD animals had significantly lower withdrawal thresholds compared to healthy controls. The researchers also showed that SCD and control females were more sensitive than males to gastrocnemius muscle compression.
Researchers have used hypoxia (low oxygen environment) treatments to induce acute sickling crises in animals, with resultant acute pain. Stucky reported that muscle compression withdrawal thresholds in SCD animals subjected to hypoxia were even lower than their already low baseline values, whereas hypoxia treatment had no effect on control non-SCD animals.
Stucky and colleagues are also now investigating the molecular mechanisms underlying acute pain in SCD animals. Hypoxia treatment has been shown to change extracellular pH levels, so the group turned its attention to ion channels that sense changes in extracellular pH. One of these ion channels, acid-sensing ion channel 3 (ASIC3), has been linked to various pain states, notably to muscle pain. Unpublished data showed that intramuscular injection of ASIC3 inhibitor APETx2 reversed the muscle compression sensitivity seen after hypoxia treatment. Thus, inhibition of ASIC3 may reduce acute SCD pain caused by hypoxia-induced crises.
Together, the data indicate that SCD animals, similar to patients with SCD, experience not only cutaneous but also deep muscle pain. The findings raise the questions of why female mice in general are significantly more sensitive than males to gastrocnemius compression (as both SCD females and non-SCD females were more sensitive to muscle pinch), and whether ASIC3 inhibitors could be used to treat patients with acute SCD pain.
Francie Moehring is a postdoctoral research fellow at the Medical College of Wisconsin.