**1. Introduction**

102 Biomarker

Tsuji, T.; Fukuwatari, T. Sasaki, S. & Shibata, K. (2010b). Urinary excretion of vitamin B1, B2,

Tsuji, T.; Fukuwatari, T. Sasaki, S. & Shibata, K. (2011). Twenty-four-hour urinary water-

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free-living elderly, female Japanese. *Nutr Res*, Vol.30, pp. 171-178.

children. *Public Health Nutr*, Vol.14, pp. 327-333.

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soluble vitamin levels correlate with their intakes in free-living Japanese school

intravenously administered riboflavin in healthy humans. *Am J Clin Nutr*, Vol.63,

GPA. (2003). Folate catabolite excretion is responsible to changes in dietary folate

#### **1.1 Epidemiology**

Approximately 20% of the European population report severe chronic pain (Breivik et al., 2006), with higher prevalences in women and in lower income groups (Gerdle et al., 2004; Larsson et al., 2007). Common chronic pain conditions are localized neck-shoulder pain including trapezius myalgia (prevalence in population 10-20%) (Lidgren, 2008), chronic whiplash associated disorders (WAD) (prevalence in the population 1.5%) (Guez et al., 2002), and chronic widespread pain (CWSP) (prevalence in population 5-10%) (Gerdle et al., 2008a). Chronic pain is associated with disability, low quality of life, and substantial socioeconomic costs (Breivik et al., 2006; Phillips, 2006; SBU, 2006).

### **1.2 Development of chronic myalgia – Chronic trapezius myalgia as an example**

There is a connection between physical demands, psychosocial demands, and the risk of persistent muscle pain (Bernard, 1997; Punnett & Wegman, 2004); however, the mechanisms behind chronic myalgia are poorly understood. Myalgia usually starts with a feeling of tiredness and stiffness. At the beginning, the initial intermittent stage, pain can be alleviated for short or long periods. Chronic regional myalgia (CRM) in the neck-shoulder area often gradually becomes more easily triggered and more diffuse and can be spread to include most of the body (CWSP). CWSP includes fibromyalgia, a subgroup characterized by widespread hyperalgesia. The risk factors for the transition from a local/regional pain condition to CWSP are poorly understood (Larsson et al., in press). The diagnoses CRM (e.g., chronic trapezius myalgia) and CWSP are settled by careful anamnesis and clinical examinations that reveal tender muscle at palpation corresponding to the reported painful areas.

#### **1.3 Neurobiological alterations in chronic pain**

Acute pain results from a complex integrated series of events at peripheral and central levels. In healthy subjects, mechanisms related to acute pain might not necessarily be valid in subjects with subchronic, intermittent, or chronic pain.

Potential Muscle Biomarkers of Chronic Myalgia in Humans –

**1.5 The bio-psycho-social model of chronic pain** 

**1.6 Central versus peripheral causes for chronic pain** 

2010; Staud, 2010; Staud et al., 2009; Woolf, 2011).

**1.7 The microdialysis technique** 

A Systematic Review of Microdialysis Studies 105

stimuli, temperature, and chemical substances such as serotonin H+, (5-HT), bradykinin (BKN), glutamate, prostaglandin E2 (PGE2), substance P, nerve growth factor (NGF), ATP, and potassium (Coutaux et al., 2005; Mense, 1993; 2009). Administration of any of these substances, alone or in combination, results in excitation of nociceptors (Mense, 1993). The relative effectiveness of these substances is unknown. In a pathophysiological situation due to trauma or inflammation, a combination of substances acts on the nociceptors (the inflammatory "soup" or "cocktail")(Mense, 2009). The nociceptor is not a static detector as plastic changes can occur such as peripheral sensitization (Woolf & Ma, 2007). A sensitized nociceptor has a lowered threshold for activation and can thus be activated by stimuli that are normally innocuous (Coutaux et al., 2005; Mense, 1993). Several substances – e.g., H+, NO, K+, ATP, bradykinin (BKN), PGE2, NGF, TNF-α, IL-6, and glutamate – are known to cause peripheral sensitization (Coutaux et al., 2005; Mense, 2009; Momin & McNaughton, 2009). The action of these substances is mediated by their specific receptors mainly found in three classes: 1) G protein coupled receptors; 2) receptor tyrosine kinases; and 3) ionotropic receptors/ion channels (Linley et al., 2010). Sensitization is often accompanied by an increase in the sensitive area (Mense, 1993). In addition, other alterations of the nociceptors, including activation of silent nociceptors, have been found as the result of injury or inflammation (Schaible et al., 2009). When persistent alterations in the nociceptors as the result of induced gene transcription and protein synthesis drive pain in the absence of noxious stimuli these alterations represent a pathological condition (Woolf & Ma, 2007).

The net result of the above mentioned and other alterations are clinically registered as pain hypersensitivity – an increased responsiveness to nociception and sometimes to innocuous stimuli. In clinical management of chronic pain, a bio-psycho-social model (Gatchel et al., 2007) is preferred since the above mentioned complex blend of factors – neurobiological, psychological (e.g., depression, catastrophizing, and anxiety), coping styles, and contextual factors – contribute to the development and maintenance of chronic pain (Alonso et al., 2004; Asmundson & Katz, 2009; Börsbo et al., 2008; Dersh et al., 2001; Ericsson et al., 2002; Means-

One of the consequences of the discovery of central sensitization is that CNS can change pain –e.g., amplification, duration, degree, and spatial extent – so that pain no longer directly reflects the peripheral noxious situation (Woolf, 2011). It is unknown whether a chronic pain condition can be driven by established central alterations such as central hyperexcitability, alterations in pain matrix, and alterations in descending mechanisms (facilitation) with very little or no peripheral stimuli or nociception. However, there are several indications that central alterations in nociceptive processing are driven by peripheral tissue alterations (Gerdle et al., 2008c) and peripheral nociceptive input (Schneider et al.,

Concerns have been expressed at the lack of success in translating basic science data using animals into clinical analgesics (Lascelles & Flecknell, 2010). Microdialysis may be able to

Christensen et al., 2008; Ocañez et al., 2010; Sofat et al., 2011; Sullivan et al., 2001).

Pace et al. suggested two types of persistent chronic pain: 1) nociceptive/inflammatory pain and 2) neuropathic pain (Pace et al., 2006). The present study mainly discusses nociceptive/inflammatory pain. Chronic pain is more complex than acute pain as extensive short-term and long-term plastic and sometimes permanent changes (including peripheral and/or central hyperexcitability/sensitization) of the pain transmission system can occur at different levels (Kuner, 2010; Reichling & Levine, 2009) and by the modification of psychological (e.g., attentional, emotional, and anticipation status) and context factors (Grachev et al., 2000; Hunt & Mantyh, 2002; Petersen-Felix & Curatolo, 2002; Schmidt-Wilcke, 2008; Wilder-Smith et al., 2002; Woolf & Salter, 2000). Different structures in the brain – vaguely labelled as the pain matrix (Iannetti & Mouraux, 2010; Lee & Tracey, 2010; Legrain et al., 2011) – are dynamically involved in processing of nociception and pain (including emotions, cognitions, and motivation) (Ossipov et al., 2010). In patients with chronic pain conditions, a pain matrix shows different types of alterations including morphological changes (Apkarian, 2008; Schweinhardt & Bushnell, 2010), indicating that different chronic pain conditions exhibit unique anatomical "brain signatures" (Baliki et al., 2011).

Descending supraspinal control of spinal nociception originates from many brain regions (Heinricher et al., 2009; Ossipov et al., 2010). The descending supraspinal control includes a dynamic balance between inhibiting and facilitating mechanisms that can be altered due to behavioural, emotional, and pathological states (Heinricher et al., 2009; Ossipov et al., 2010). When the system shifts towards inhibition, hyposensibility or lack of pain in spite of inputs from peripheral tissue may result (Heinricher et al., 2009; Kuner, 2010; Porreca et al., 2002; Ren & Dubner, 2002; Robinson & Zhuo, 2002; Wilder-Smith et al., 2002). The evolutionary value of this is that the organism can ignore pain during critical situations, e.g., flight or fight scenarios (Kuner, 2010). A facilitating shift of the descending system has been reported for different groups of patients with persistent pain (Heinricher et al., 2009; Julien et al., 2005; Kuner, 2010; Porreca et al., 2002; Ren & Dubner, 2002; Robinson & Zhuo, 2002; Wilder-Smith et al., 2002).

Decreased production of substances such as endorphins or gamma-aminobutyric acid (GABA), the most common inhibitory neurotransmitter in the central nervous system, may contribute to disturbances in pain inhibition. Animal and human studies have confirmed that the endogenous opioid system and GABA play a role in the modulation of pain. However, the understanding of the mechanisms, including the peripheral balances between nociceptive and anitinociceptive processes, behind chronic myalgia is incomplete. The analgesic properties of *exogenous* cannabinoids have been recognized for many years. Data clearly implicate endocannabinoids as endogenous tonic pain controlling molecules (Agarwal et al., 2007; Richardson et al., 1998; Walker & Huang, 2002), but little is known as to whether *peripheral* endocannabinoid signalling is disturbed in human pain.

#### **1.4 Muscle nociception and peripheral sensitization**

Neurophysiological studies have indicated that small-diameter, slowly conducting afferent nerve fibres from skeletal muscle –free nerve endings of group III (Aδ) and IV afferent (C) fibres – have to be excited to elicit pain (Mense, 2003). The nociceptor is specialized to respond to noxious stimuli and communicate this information to the CNS. Nociceptors or noxious stimulus detectors (Woolf & Ma, 2007) are sensitive to chemical substances released from damaged or overloaded cells and excessive tissue deformation (Coutaux et al., 2005; Mense, 1993). Nociceptors respond to single or combinations of stimuli: noxious mechanical

Pace et al. suggested two types of persistent chronic pain: 1) nociceptive/inflammatory pain and 2) neuropathic pain (Pace et al., 2006). The present study mainly discusses nociceptive/inflammatory pain. Chronic pain is more complex than acute pain as extensive short-term and long-term plastic and sometimes permanent changes (including peripheral and/or central hyperexcitability/sensitization) of the pain transmission system can occur at different levels (Kuner, 2010; Reichling & Levine, 2009) and by the modification of psychological (e.g., attentional, emotional, and anticipation status) and context factors (Grachev et al., 2000; Hunt & Mantyh, 2002; Petersen-Felix & Curatolo, 2002; Schmidt-Wilcke, 2008; Wilder-Smith et al., 2002; Woolf & Salter, 2000). Different structures in the brain – vaguely labelled as the pain matrix (Iannetti & Mouraux, 2010; Lee & Tracey, 2010; Legrain et al., 2011) – are dynamically involved in processing of nociception and pain (including emotions, cognitions, and motivation) (Ossipov et al., 2010). In patients with chronic pain conditions, a pain matrix shows different types of alterations including morphological changes (Apkarian, 2008; Schweinhardt & Bushnell, 2010), indicating that different chronic pain

Descending supraspinal control of spinal nociception originates from many brain regions (Heinricher et al., 2009; Ossipov et al., 2010). The descending supraspinal control includes a dynamic balance between inhibiting and facilitating mechanisms that can be altered due to behavioural, emotional, and pathological states (Heinricher et al., 2009; Ossipov et al., 2010). When the system shifts towards inhibition, hyposensibility or lack of pain in spite of inputs from peripheral tissue may result (Heinricher et al., 2009; Kuner, 2010; Porreca et al., 2002; Ren & Dubner, 2002; Robinson & Zhuo, 2002; Wilder-Smith et al., 2002). The evolutionary value of this is that the organism can ignore pain during critical situations, e.g., flight or fight scenarios (Kuner, 2010). A facilitating shift of the descending system has been reported for different groups of patients with persistent pain (Heinricher et al., 2009; Julien et al., 2005; Kuner, 2010; Porreca et al., 2002; Ren & Dubner, 2002; Robinson & Zhuo, 2002; Wilder-

Decreased production of substances such as endorphins or gamma-aminobutyric acid (GABA), the most common inhibitory neurotransmitter in the central nervous system, may contribute to disturbances in pain inhibition. Animal and human studies have confirmed that the endogenous opioid system and GABA play a role in the modulation of pain. However, the understanding of the mechanisms, including the peripheral balances between nociceptive and anitinociceptive processes, behind chronic myalgia is incomplete. The analgesic properties of *exogenous* cannabinoids have been recognized for many years. Data clearly implicate endocannabinoids as endogenous tonic pain controlling molecules (Agarwal et al., 2007; Richardson et al., 1998; Walker & Huang, 2002), but little is known as

Neurophysiological studies have indicated that small-diameter, slowly conducting afferent nerve fibres from skeletal muscle –free nerve endings of group III (Aδ) and IV afferent (C) fibres – have to be excited to elicit pain (Mense, 2003). The nociceptor is specialized to respond to noxious stimuli and communicate this information to the CNS. Nociceptors or noxious stimulus detectors (Woolf & Ma, 2007) are sensitive to chemical substances released from damaged or overloaded cells and excessive tissue deformation (Coutaux et al., 2005; Mense, 1993). Nociceptors respond to single or combinations of stimuli: noxious mechanical

to whether *peripheral* endocannabinoid signalling is disturbed in human pain.

**1.4 Muscle nociception and peripheral sensitization** 

conditions exhibit unique anatomical "brain signatures" (Baliki et al., 2011).

Smith et al., 2002).

stimuli, temperature, and chemical substances such as serotonin H+, (5-HT), bradykinin (BKN), glutamate, prostaglandin E2 (PGE2), substance P, nerve growth factor (NGF), ATP, and potassium (Coutaux et al., 2005; Mense, 1993; 2009). Administration of any of these substances, alone or in combination, results in excitation of nociceptors (Mense, 1993). The relative effectiveness of these substances is unknown. In a pathophysiological situation due to trauma or inflammation, a combination of substances acts on the nociceptors (the inflammatory "soup" or "cocktail")(Mense, 2009). The nociceptor is not a static detector as plastic changes can occur such as peripheral sensitization (Woolf & Ma, 2007). A sensitized nociceptor has a lowered threshold for activation and can thus be activated by stimuli that are normally innocuous (Coutaux et al., 2005; Mense, 1993). Several substances – e.g., H+, NO, K+, ATP, bradykinin (BKN), PGE2, NGF, TNF-α, IL-6, and glutamate – are known to cause peripheral sensitization (Coutaux et al., 2005; Mense, 2009; Momin & McNaughton, 2009). The action of these substances is mediated by their specific receptors mainly found in three classes: 1) G protein coupled receptors; 2) receptor tyrosine kinases; and 3) ionotropic receptors/ion channels (Linley et al., 2010). Sensitization is often accompanied by an increase in the sensitive area (Mense, 1993). In addition, other alterations of the nociceptors, including activation of silent nociceptors, have been found as the result of injury or inflammation (Schaible et al., 2009). When persistent alterations in the nociceptors as the result of induced gene transcription and protein synthesis drive pain in the absence of noxious stimuli these alterations represent a pathological condition (Woolf & Ma, 2007).

#### **1.5 The bio-psycho-social model of chronic pain**

The net result of the above mentioned and other alterations are clinically registered as pain hypersensitivity – an increased responsiveness to nociception and sometimes to innocuous stimuli. In clinical management of chronic pain, a bio-psycho-social model (Gatchel et al., 2007) is preferred since the above mentioned complex blend of factors – neurobiological, psychological (e.g., depression, catastrophizing, and anxiety), coping styles, and contextual factors – contribute to the development and maintenance of chronic pain (Alonso et al., 2004; Asmundson & Katz, 2009; Börsbo et al., 2008; Dersh et al., 2001; Ericsson et al., 2002; Means-Christensen et al., 2008; Ocañez et al., 2010; Sofat et al., 2011; Sullivan et al., 2001).

#### **1.6 Central versus peripheral causes for chronic pain**

One of the consequences of the discovery of central sensitization is that CNS can change pain –e.g., amplification, duration, degree, and spatial extent – so that pain no longer directly reflects the peripheral noxious situation (Woolf, 2011). It is unknown whether a chronic pain condition can be driven by established central alterations such as central hyperexcitability, alterations in pain matrix, and alterations in descending mechanisms (facilitation) with very little or no peripheral stimuli or nociception. However, there are several indications that central alterations in nociceptive processing are driven by peripheral tissue alterations (Gerdle et al., 2008c) and peripheral nociceptive input (Schneider et al., 2010; Staud, 2010; Staud et al., 2009; Woolf, 2011).

#### **1.7 The microdialysis technique**

Concerns have been expressed at the lack of success in translating basic science data using animals into clinical analgesics (Lascelles & Flecknell, 2010). Microdialysis may be able to

Potential Muscle Biomarkers of Chronic Myalgia in Humans –

**2. Methods** 

**2.2 Search strategy** 

**3. Results** 

**3.1 Chronic trapezius myalgia** 

**2.1 Inclusion and exclusion criteria** 

group, and if and how the authors handled RR.

**2.3 Positive outcome with respect to potential biomarker** 

concentrations in the patient group compared to the controls.

identified three articles concerning chronic tendinosis (**Table 8**).

A Systematic Review of Microdialysis Studies 107

in metabolic substances, pain-related substances (algesics), and anti-analgesics in different chronic muscle pain conditions (mainly myalgia) in humans using microdialysis. This systematic review was done to identify potential biomarkers – an objectively measured and

Studies that focused on chronic pain conditions affecting human muscles (myalgias) were included. To be included, the studies had to use microdialysis and had to use a patient group and a healthy control group, so articles concerning only healthy subjects have been excluded.

This review identified the studies fulfilling the above criteria in the systematic review of Larsson et al. (Larsson et al., 2007). Furthermore, we modified their search strategy in the following ways: ((muscle OR pain) AND microdialysis) OR (muscle AND pain AND induced) AND (Humans[Mesh] AND (Clinical Trial[ptyp] OR Meta-Analysis[ptyp] OR

Using this strategy, PubMed was searched. From this search, the titles and abstracts were scrutinized. If the articles were relevant and necessary, they were read for further evaluation. We also checked reference lists of these articles. If the article was relevant according to our aim and inclusion criteria, we listed the results in tables. The tables of the different conditions reported the statistics concerning comparisons between the patient group and control group with respect to baseline data or corresponding data for all investigated substances. Moreover, we listed gender, flow rate, number of subjects in each

A specific substance was classified as a potential biomarker if the majority of studies (including the majority of subjects) showed significantly lower or significantly higher

Using the modified search strategy, we had 441 hits after searching PubMed. After screening, we identified 17 articles that fulfilled our inclusion criteria. Thirteen of these were not mentioned by Larsson et al. (Larsson et al., 2007). After scrutinizing these 13 articles and the articles selected by Larsson et al., we found 22 articles concerning different chronic pain conditions involving muscle. These articles are summarized in **Tables 1-7**. Moreover, we

For chronic trapezius myalgia, we identified seven groups of patients reported in ten studies (**Table 1**). The majority of the studies reported increases in the interstitial concentrations of

Review[ptyp]) AND English[lang] AND adult[MeSH] AND "last 10 years"[PDat]).

evaluated indicator of, e.g., normal pathogenic processes (Ptolemy & Rifai, 2010).

replace animal experiments (Langley et al., 2008). The microdialysis technique offers a wellestablished *in vivo* method for studying the local biochemistry of individual tissues in the body (Ungerstedt, 1991), e.g., nociceptive and metabolic mechanisms. This technique has been used in neuroscience to monitor neurotransmitter release, but has also found application in monitoring the biochemistry of peripheral tissues in both animals and humans (Ungerstedt, 1991).

Microdialysis mimics the function of a capillary blood vessel by perfusing a thin dialysis tube (catheter) implanted into the tissue with a physiological saline solution. Through simple diffusion, substances can move across the dialysis membrane along the concentration gradient. The chemical analysis of the dialysate reveals the composition of the extracellular fluid. Thus microdialysis allows for continuous sampling of compounds in the interstitial space of the muscle, where nociceptor free nerve endings terminate close to the muscle fibres, providing accurate information on regional biochemical changes before such compounds are diluted and cleared by the circulatory system. The trapezius muscle has been used as a *human model muscle* for chronic myalgia both due to its clinical importance and to its accessibility for invasive investigations. Some studies use the masseter, vastus lateralis, and gastrocnemius muscles to examine myalgia.

To determine the concentrations of small molecules such as lactate, pyruvate, glutamate, and glucose, a catheter with a 20 kDa cut-off is usually used (Waelgaard et al., 2006). To determine the concentrations of larger molecules such as cytokines, a catheter with a 100 kDa cut-off is usually used (Waelgaard et al., 2006).

A crucial parameter in microdialysis is relative recovery (RR): the ratio between substance concentrations in the dialysate to that in the perfusate (Afinowi et al., 2009; Dahlin et al., 2010 ; Ungerstedt, 1991). RR is used to determine the true concentration of extracellular fluid. Because the perfusate constantly flows across the membrane, a state of equilibrium will never be achieved and as a result the dialysate will only represent a certain percentage of the actual concentration of the extracellular fluid (Afinowi et al., 2009; Hamrin et al., 2002). Therefore, the final concentration in the dialysate partially depends on the flow rate of the perfusate. Low flow rate results in higher RR; high flow rates, result in lower RR. At a very low flow (i.e., < 0.3µl/min), the recovery is near 100%, but factors such as alterations in the osmotic pressure, temperature, weight cut-off, area of the membrane, concentration gradient, and composition of the perfusate can influence RR (Dahlin et al., 2010 ; Hamrin et al., 2002 ; Plock & Kloft, 2005 ). Between cytokines have been reported marked variation in RR; molecular weight correlated negatively with RR (Helmy et al., 2009).

#### **1.8 Aim**

Microdialysis has several important advantages, but studies with patients are expensive and time consuming. Hence a systematic review of the literature is needed. Systematic knowledge of the results of such studies might help provide new assessment approaches of patients with chronic myalgia, new treatments, and new rehabilitation techniques for patients with chronic myalgia. Most research on muscle pain has been conducted on animals; however, this review will primarily focus on human studies of neck and shoulder myalgia, for which the frequently affected trapezius muscle often serves as a model muscle. This study systematically reviews studies in the literature that have investigated alterations in metabolic substances, pain-related substances (algesics), and anti-analgesics in different chronic muscle pain conditions (mainly myalgia) in humans using microdialysis. This systematic review was done to identify potential biomarkers – an objectively measured and evaluated indicator of, e.g., normal pathogenic processes (Ptolemy & Rifai, 2010).
