Biomarkers in chronic and experimental human muscle pain

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

Biomarkers in chronic and experimental human muscle pain

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

Literacy as part of professional knowing in a Swedish dental education

BACKGROUND: Academic reading and writing are seen as self-evident literacy competences in most contemporary higher educations, however, whether students also are introduced to professional literacy of relevance for dentistry during their education is a question. The purpose of this study is to analyze one of the Swedish dental programmes, with respect to its design, in relation to possible content of relevance for academic and professional literacy. Secondarily, to identify and analyze Swedish dental students' writing in an academic setting, i.e. what these students are expected to read and write, and how they write. METHODS: Data, for this ethnographically inspired case-study, was produced by observations and audio-recordings of lectures, copies of teachers' handouts and of volunteering students' notes, and a multiple-choice-test. Data-analysis was made in five steps, starting with macro-level data, i.e. curriculum and syllabuses, followed by the syllabuses for the two observed modules, the teacher-provided material, analysis of the students' notes, while in the fifth and final step, the results from the previous steps were compared, to find patterns of what students were expected to read and write, and what in the teacher-provided multimodal material that was emphasized in teachers' talk. RESULTS: This study showed that students were engaged in several types of literacy events, such as reading, finding and watching videos on their learning platform, writing, and following instructions. The study also showed that there is a recurrent academic content comprised of anatomy, physiology and pathology, while the professional content comprised of patient communication and anamnesis. Further, an integrated content was found and was initiated in teacher-constructed PowerPoints and by student-questions. Note-taking patterns varied between individual students, but the general pattern for this group of students were the use of complementary notes. This type of note-taking was used to make available further descriptions of the teacher-constructed text in PowerPoints, but also an independent text describing pictures shown on teachers' PowerPoints or the blackboard. CONCLUSION: Findings from the present study reveal that students either copy text from teachers' PowerPoint-slides, re-formulate text from teachers' PowerPoint-slides, or write complementing text to teachers' PowerPoint-slides. Further, the students individually choses type of note-taking based on situation. The study also revealed that the academic literacy - in the two modules during the fifth and sixth semesters of a dental education analyzed - mainly has a professional basis for reading, writing, and communication purposes. The study also showed that academic and professional literacy are closely connected through recurrent integration

Early steps towards professional clinical note-taking in a Swedish study programme in dentistry

Background: Higher education tends to focus on academic writing only, instead of emphasizing that professional texts are also used as a basis for communication in contexts with a variety of participators. When it comes to clinical notes, research is scarce and focused on technology and informatics. Therefore, the aim was to explore dental students' clinical notes, and specifically which aspects of the clinical notes characterizes clinical notes that are not sufficient enough for professional purposes. Methods: The object of analysis was the student's written completion of a teacher constructed protocol regarding oral mucosa, the dental apparatus including pathology on tooth level, oral hygiene, and a validated international clinical examination protocol of the temporomandibular region. The study was framed within the New Literacy Studies approach, and the clinical notes were analyzed using thematic analysis. Results: Within the clinical notes three themes were identified; a) familiar content; b) familiar content in new context; and c) new content. The forms of notes could refer to either categorizational clinical notes or descriptive clinical notes. Most students were able to write acceptable clinical notes when the content was familiar, but as soon as the familiar content was in a new context the students had difficulties to write acceptable notes. When it comes to descriptive notes students suffered difficulties to write acceptable notes both when it came to familiar content, or familiar content in a new context. Conclusions: Taken together, the results indicate that students have difficulties writing acceptable notes when they are novices to the content or context, making their notes either insufficient, too short or even wrong for professional purposes. With this in mind, this study suggests that there is a need to strengthen the demands on sufficient professional quality in clinical notes and focus on clinical notes already in the early stages of the different medical educations

Increased levels of intramuscular cytokines in patients with jaw muscle pain

Background: The aim of this study was to investigate cytokine levels in the masseter muscle, their response to experimental tooth-clenching and their relation to pain, fatigue and psychological distress in patients with temporomandibular disorders (TMD) myalgia. Methods: Forty women, 20 with TMD myalgia (Diagnostic Criteria for TMD) and 20 age-matched healthy controls participated. Intramuscular microdialysis was performed to sample masseter muscle cytokines. After 140 min (baseline), a 20-minute tooth-clenching task was performed (50% of maximal voluntary contraction force). Pain (Numeric rating scale 0-10) and fatigue (Borg's Ratings of Perceived Exertion 6-20) were assessed throughout microdialysis, while pressure-pain thresholds (PPT) were assessed before and after microdialysis. Perceived stress (PSS-10) and Trait Anxiety (STAI) were assessed before microdialysis. Results: The levels of IL-6, IL-7, IL-8 and IL-13 were higher in patients than controls (Mann Whitney U-test; P's < 0. 05) during the entire microdialysis. IL-6, IL-8 and IL-13 changed during microdialysis in both groups (Friedman; P's < 0.05), while IL-1 beta, IL-7 and GM-CSF changed only in patients (P's < 0.01). IL-6 and IL-8 increased in response to tooth-clenching in both groups (Wilcoxon test; P's < 0.05), while IL-7, IL-13 and TNF increased only in patients (P's < 0.05). Patients had higher pain and fatigue than controls before and after tooth-clenching (P < 0.001), and lower PPTs before and after microdialysis (P < 0.05). There were no correlations between cytokine levels, pain or fatigue. Also, there were no differences in stress or anxiety levels between groups. Conclusions: In conclusion, the masseter levels of IL-6, IL-7, IL-8 and IL-13 were elevated in patients with TMD myalgia and increased in response to tooth-clenching. Tooth-clenching increased jaw muscle pain and fatigue, but without correlations to cytokine levels. This implies that subclinical muscle inflammation may be involved in TMD myalgia pathophysiology, but that there is no direct cause-relation between inflammation and pain

Influence of Polymorphisms in the HTR3A and HTR3B Genes on Experimental Pain and the Effect of the 5-HT3 Antagonist Granisetron

The aim of this study was to investigate experimentally if 5-HT3 single nucleotide polymorphisms (SNP) contribute to pain perception and efficacy of the 5-HT3-antagonist granisetron and sex differences. Sixty healthy participants were genotyped regarding HTR3A (rs1062613) and HTR3B (rs1176744). First, pain was induced by bilateral hypertonic saline injections (HS, 5.5%, 0.2 mL) into the masseter muscles. Thirty min later the masseter muscle on one side was pretreated with 0.5 mL granisetron (1 mg/mL) and on the other side with 0.5 mL placebo (isotonic saline) followed by another HS injection (0.2 mL). Pain intensity, pain duration, pain area and pressure pain thresholds (PPTs) were assessed after each injection. HS evoked moderate pain, with higher intensity in the women (P = 0.023), but had no effect on PPTs. None of the SNPs influenced any pain variable in general, but compared to men, the pain area was larger in women carrying the C/C (HTR3A) (P = 0.015) and pain intensity higher in women with the A/C alleles (HTR3B) (P = 0.019). Pre-treatment with granisetron reduced pain intensity, duration and area to a lesser degree in women (P < 0.05), but the SNPs did not in general influence the efficacy of granisetron. Women carrying the C/T & T/T (HTR3A) genotype had less reduction of pain intensity (P = 0.041) and area (P = 0.005), and women with the C/C genotype (HTR3B) had less reduction of pain intensity (P = 0.030), duration (P = 0.030) and area compared to men (P = 0.017). In conclusion, SNPs did not influence experimental muscle pain or the effect of granisetron on pain variables in general, but there were some sex differences in pain variables that seem to be influenced by genotypes. However, due to the small sample size further research is needed before any firm conclusions can be drawn

Polymorphisms in the HTR2A and HTR3A Genes Contribute to Pain in TMD Myalgia

Background: The aim of this study was to investigate if single nucleotide polymorphisms (SNPs) related to monoaminergic neurotransmission, in particular the serotonergic pathway, contribute to pain perception in patients with temporomandibular disorder (TMD) myalgia and if there is a correlation to jaw function as well as psychosocial factors such as stress, anxiety and depression. Materials and Methods: One hundred and seventeen individuals with TMD myalgia were included. A venous blood or saliva sample was taken for genetic analyses and genotyped regarding HTR2A (rs9316233) HTR3A (rs1062613), HTR3B (rs1176744), SERT (5-HTTLPR) and COMT (rs4680). A clinical examination according to Diagnostic Criteria for TMD (DC/TMD) was performed and axis II data (psychosocial factors) were compared between participants with different genotypes for each gene using Kruskall–Wallis test. The characteristic pain intensity (CPI) was tested for correlations to scores for the Perceived Stress Scale, Generalized Anxiety Disorder, and Patient Health Questionnaires using Spearman's rank correlation test with Bonferroni correction for multiple testing. To further explore data factor analysis was performed to identify latent factors associated to the outcome variables. Results: Participants carrying at least one copy of the rare allele of the HTR2A (rs9316233) and HTR3A (rs1062613) had higher CPI compared with the participants with the homozygous common genotype (P = 0.042 and P = 0.024, respectively). Correlation analyses showed several significant positive correlations between CPI on one hand, and self-reported psychosocial distress and jaw function on the other hand for several genotypes that mostly were weak to moderate. The factor analysis identified two latent variables. One was positively correlated to the HTR3B gene, jaw function and self-reported parafunctions, and the other was positively correlated to psychological distress and negatively correlated to SERT. Conclusion: Taken together, the polymorphism rs1062613 in the HTR3A gene contributes to pain intensity in TMD myalgia. This together with positive interactions between pain variables and psychological factors in genotypes strengthens that pain and psychological distress are related. Further research is needed to explore this as well as the influence of gene-to-gene interactions on pain and psychological distress

Chocolate intake and muscle pain sensation: A randomized experimental study.

BackgroundChocolate, as a cocoa-derived product rich in flavanols, has been used for medical and anti-inflammatory purposes. Therefore, the aim of this study was to investigate if the ingestion of different percentages of cocoa products affects the experimentally induced pain caused by intramuscular hypertonic saline injections in the masseter muscle of healthy men and women.MethodsThis experimental randomized, double-blind, and controlled study included 15 young, healthy, and pain-free men and 15 age-matched women and involved three visits with at least a 1-week washout. Pain was induced twice at each visit with intramuscular injections of 0.2 mL hypertonic saline (5%), before and after intake of one of the different chocolate types: white (30% cocoa content), milk (34% cocoa content), and dark (70% cocoa content). Pain duration, pain area, peak pain, and pressure pain threshold (PPT) were assessed every fifth minute after each injection, up until 30 min after the initial injection. Descriptive and inferential statistics were performed using IBM® SPSS (Version 27); significance level was set to pResultsThis study showed that intake of chocolate, no matter the type, reduced the induced pain intensity significantly more than no intake of chocolate (pConclusionIntake of chocolate before a painful stimulus had a pain-reducing effect no matter the cocoa concentration. The results indicate that perhaps it is not the cocoa concentration (e.g., flavanols) alone that explains the positive effect on pain, but likely a combination of preference and taste-experience. Another possible explanation could be the composition of the chocolate, i.e. the concentration of the other ingredients such as sugar, soy, and vanilla. ClinicalTrials.gov Identifier: NCT05378984

Influence of Polymorphisms in the <i>HTR3A</i> and <i>HTR3B</i> Genes on Experimental Pain and the Effect of the 5-HT₃ Antagonist Granisetron

<div><p>The aim of this study was to investigate experimentally if 5-HT₃ single nucleotide polymorphisms (SNP) contribute to pain perception and efficacy of the 5-HT₃-antagonist granisetron and sex differences. Sixty healthy participants were genotyped regarding <i>HTR3A (rs1062613)</i> and <i>HTR3B (rs1176744)</i>. First, pain was induced by bilateral hypertonic saline injections (HS, 5.5%, 0.2 mL) into the masseter muscles. Thirty min later the masseter muscle on one side was pretreated with 0.5 mL granisetron (1 mg/mL) and on the other side with 0.5 mL placebo (isotonic saline) followed by another HS injection (0.2 mL). Pain intensity, pain duration, pain area and pressure pain thresholds (PPTs) were assessed after each injection. HS evoked moderate pain, with higher intensity in the women (P = 0.023), but had no effect on PPTs. None of the SNPs influenced any pain variable in general, but compared to men, the pain area was larger in women carrying the C/C (<i>HTR3A</i>) (P = 0.015) and pain intensity higher in women with the A/C alleles (<i>HTR3B</i>) (P = 0.019). Pre-treatment with granisetron reduced pain intensity, duration and area to a lesser degree in women (P < 0.05), but the SNPs did not in general influence the efficacy of granisetron. Women carrying the C/T & T/T (<i>HTR3A</i>) genotype had less reduction of pain intensity (P = 0.041) and area (P = 0.005), and women with the C/C genotype (<i>HTR3B</i>) had less reduction of pain intensity (P = 0.030), duration (P = 0.030) and area compared to men (P = 0.017). In conclusion, SNPs did not influence experimental muscle pain or the effect of granisetron on pain variables in general, but there were some sex differences in pain variables that seem to be influenced by genotypes. However, due to the small sample size further research is needed before any firm conclusions can be drawn.</p></div

Pain distribution in the masseter muscle after HS injection alone and after pre-treatment with granisetron.

<p>The figures show the pain distribution in the masseter muscle after injections of hypertonic saline (HS) alone (first injection) and after pre-treatment with granisetron (0.5 mL) and placebo (0.5 mL isotonic saline) followed by a second injection of hypertonic saline (second injection) in 60 healthy participants (30 women and 30 age-matched men). The pain area after HS injection did not differ significantly between sexes. The pain area was significantly smaller after pre-treatment with granisetron (P < 0.001) with greater reduction in men than women (P = 0.007).</p