Biomarkers in chronic and experimental human muscle pain

Abstract: The main aim of this thesis was to improve the knowledge ofthe peripheral mechanisms that may participate in the underlying pathophysiology of chronic temporomandibular disorders (TMD) myalgia i.e. pain that is experienced locally in the jaw muscles and has myofascial trigger points. The project examined the effect of the 5-hydroxytryptamine type 3 (5-HT3)- antagonist granisetron on experimentally induced muscle pain and whether specific genetic variants i.e. polymorphisms (SNPs) in the serotonergic system influences pain perception and the pain reducing effect of granisetron. The project also investigated the relationship between certain biomarkers; serotonin (5-HT), glutamate, metabolites and pro- and anti-inflammatory cytokines in jaw muscle pain. One part examined the 5-HT3- receptor antagonist granisetron, and its effect on experimentally induced masseter muscle pain in healthy participants. Also, whether certain polymorphisms in the serotonergic system are involved and may influence the pain response and the efficacy of granisetron. The SNPs (rs1062613, rs1176744) in the HTR3A/B genes were therefore investigated. 0.5 mL granisetron (Kytril® 1 mg/ml) or placebo (isotonic saline, 9 mg/mL) was injected in the masseter muscles in a randomized, placebo-controlled and double-blinded order, followed by a bilateral painful injection of either: a) acidic saline (0.5 mL, 9 mg/mL, pH 3.3) or b) hypertonic saline (HS, 0.2 mL, 58.5 mg/mL). The pain variables; pain intensity, pain duration, pain area and pain pressure threshold (PPT) were assessed. Another part in this project, used a microdialysis technique in order to investigate the intramuscular levels of several biomarkers in the masseter muscle, at rest and after experimentally induced muscle pain. HS injections and static tooth-clenching were used as experimental pain models in healthy, pain-free participants and in patients with TMD myalgia. The biomarkers and metabolites analyzed were; 5-HT, glutamate, lactate, pyruvate, glucose, glycerol as well as the pro- and anti-inflammatory cytokines; IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7 IL-8, IL-10, IL-12, IL-13, TNF, IFN- γ and GM-CSF. The results showed that granisetron had a pain reducing effect on experimentally induced masseter muscle pain, both by acidic saline and HS injections. The pain intensity, pain duration and pain area were significantly lower on the side pre-treated with granisetron, but did not have any effect on the PPT. Also, there were sex differences in pain variables and in response to granisetron. The 5-HT3 polymorphisms did not influence any pain variables in general or the pain reducing effect of granisetron. However, there were sex differences in regards to pain variables and the efficacy of granisetron. Women had higher pain intensity and larger pain area after experimentally induced masseter muscle pain, and less pain reduction (pain intensity, duration and area) of granisetron in specific genotypes of the 5-HT3 polymorphisms. Intramuscular microdialysis in the masseter muscles of healthy participants showed increased levels of 5-HT, glutamate and glycerol after evoked muscle pain with a HS injection, and 5- HT correlated positively to pain. In TMD myalgia patients, there were higher levels of the pro- and anti-inflammatory cytokines IL-6, IL-7, IL-8 and IL-13, throughout the microdialysis compared to healthy controls. The cytokines IL-6, IL-7, IL-8, IL-13 and TNF increased in response to experimental tooth-clenching in patients, and IL-6 and IL-8 increased in healthy controls. TMD myalgia patients reported higher pain and fatigue after tooth-clenching compared to controls. However, no correlation between the cytokine levels and pain and fatigue were found. In conclusion, the results of this thesis showed that granisetron had a pain reducing effect on experimentally evoked masseter muscle pain, with a generally better effect in men. None of the 5-HT3 polymorphisms investigated in this thesis, seemed to influence the experimentally induced muscle pain or the positive effect of granisetron. Nevertheless, there were some indications of gene-to-sex interactions in pain variables and granisetron effects. Therefore, one cannot completely exclude the possibility that polymorphisms in the serotonergic system may influence, predict or be a risk factor in developing chronic muscle pain. Further research is needed to systematically investigate multiple 5-HT polymorphisms in order to draw any further conclusions. Further, the levels of the biomarkers 5-HT, glutamate and glycerol, increased after experimentally induced muscle pain in the masseter muscles, but without any sex differences. In addition, patients with TMD myalgia constantly had elevated levels of the pro- and antiinflammatory cytokines IL-6, IL-7, IL-8 and IL-13 compared to healthy controls. The cytokine levels of IL-6, IL-7, IL-8, IL-13 and TNF increased in patients after tooth-clenching, and IL-6 and IL-8 increased in controls. This indicates that muscle inflammation could be involved in the multifactorial pathophysiology of chronic muscle pain. However, no correlations between the cytokine levels and pain and fatigue were found, indicating that there is no direct cause-relation effect between increased pain and cytokine release. Other peripheral mediators and mechanisms, such as central sensitization can therefore not be ruled out in the pathophysiology of chronic TMD myalgia.