Salivary inflammatory markers in tension-type headache and migraine: a case-control study 1st Department of Neurology, Eginition Hospital, National and Kapodistrian University of Athens, Faculty of Medicine, Athens, Greece. Vassilisis Sophias Avenue 72-74, 115 ABSTRACT OBJECTIVES: We investigated whether headache attacks are associated with changes in CRP, IL-1ß and IL-6 in saliva. We also investigated whether these markers in tension-type headache (TTH) and migraine could be influenced by comorbidities such as depression and anxiety. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essay METHODS: This case-control study involved 13 patients with migraine, 9 patients with TTH who attended our headache clinic, and 15 age-matched healthy controls between January to March 2016. We had access to the their demographic characteristics, headache characteristics, anxiety, and depression as measured by the Hamilton Anxiety Rating Scale (HAM-A) and the Beck Depression Inventory (BDI). Salivary IL-6, IL-1ß and CRP were collected at distinct time points such as A- headache free period, B - during headache, C- one day after headache attack and measured by ELISA kit. RESULTS:IL-1β significantly decreased from time point A to point B, whereas it increased from time point B to C in the headache groups. Both headache types had higher IL-1ß levels at time point B compared to controls. No significant differences were found in the temporal variation of CRP, IL-1ß and IL-6 levels between migraine and TTH (p>0.05). CRP was negatively correlated with HAM-A and BDI scores. IL-6 measured at time point A was negatively correlated with BDI scores. CONCLUSION: For the first time, the existence of a similar variation in IL-1ß was demonstrated in both migraine and TTH patients. Migraineurs had elevated levels of IL-1ß during the attack compared to controls. CRP and IL-6 were correlated with lower anxiety and depression symptom scores before or immediately after the headache period in the patient groups. Keywords: Tension-type headache (TTH), migraine, inflammation, Interleukin (IL)-1ß, Interleukin IL-6, salivary markers. Introduction Tension-type headache (TTH) and migraine are the most common types of primary headache, with a high lifetime prevalence of 78% and 18%, respectively, and a high overall lifetime cost of life [1,2]. However, their pathophysiology is still being studied. Both peripheral and central mechanisms have been implicated. Increased nociception from tense muscles may be the primary cause of both TTH and migraine, possibly aided by a temporary central change in pain control [3]. Inflammation not only generates but also maintains pain in peripheral structures, while promoting sensitization of central nervous system structures involved in nociception [4]. An elevated level of C-reactive protein (CRP) may be an indicator of proinflammatory state in migraine patients. Activation of brain tissue triggers the release of peptides from the perivascular regions of the trigeminal, causing inflammation and dilation of extraparenchymal vessels. Repeated migraine attacks are associated with inflammatory arteriopathy of the cranial vessels [5]. Interleukin (IL)-1ß is also an important mediator of the inflammatory response. The induction of cyclooxygenase-2 by this cytokine in the central nervous system could contribute to inflammatory pain hypersensitivity [6].IL-1ß may play a causal role in migraine by activating neuronal and glial cells to release cyclooxygenase-2, which in turn could induce neuroinflammation. Serum IL-1ß levels were significantly elevated in patients with chronic TTH, which contributes to central sensitization and increased general hyperalgesia [7]. Interleukin 6 (IL-6) acts as a proinflammatory cytokine secreted by T cells and macrophages to stimulate the immune response. Both serum and CSF levels of IL-6 were elevated in subjects with episodic/chronic forms of TTHed migraine [8]. Serum IL-6 levels were significantly higher in migraine patients during attacks compared to controls [9]. Both TTH and migraine have been associated with psychiatric comorbidities, such as depression and anxiety [10,11]. These disorders have previously been associated with increased expression of IL-6, IL-1ß and CRP [12-14]. No studies to date have assessed whether the increase in these salivary proinflammatory cytokines in TTH and migraine may be secondary to psychiatric comorbidities. All the above studies mainly focused on blood and cerebrospinal fluid samples [15]. Saliva collection is a simple, non-invasive and economical method with clear advantages in the field of psychoneuroendocrinological research [16]. However, no research has yet been conducted with the aim of comparing salivary levels of CRP, IL-1ß and IL-6 in patients with TTH and migraine compared to age-matched controls. The main aim of this study was to investigate whether migraine attacks and TTH are associated with changes in the concentration of inflammatory markers. The secondary objective was to investigate whether cytokine levels in TTH and migraine could be influenced by psychiatric comorbidities such as depression and anxiety. Materials and methods. Study design and population This case-control study enrolled 37 subjects (22 migraine, TTH patients and 15 healthy volunteers) who attended an outpatient headache clinic at the Department of Neurology of Aeginition Hospital in Athens between January and March 2016. Subjects of both sexes. Patients aged between 18 and 60 years suffering from primary headache (TTH) and migraine, according to the International Classification of Headache Disorders, 3rd edition (beta version) were enrolled [ 17]. Patients underwent a complete physical and neurological examination. During the recruitment day, all patients who visited the outpatient clinic were asked to participate in the study after meeting the eligibility criteria and providing informed consent. The main exclusion criteria were (1) abnormal plasma levels of hs-CRP, IL-1ß and IL-6 (>10 mg/L) (2) smoking cigarettes >1 pack/day; (3) current pregnancy, breastfeeding, or use of hormonal contraceptives (4) alcohol or substance abuse (5) use of medications such as antiplatelet agents, anticoagulants, statins, or hormonal medications (6) Patients with headache with a recent history of a disease with levels known high levels of inflammatory markers (7) Patients on anti-inflammatory therapy (8) other primary or secondary headaches (9) serious psychiatric diseases (10) oral health problems. Healthy control subjects aged 18 to 50 years were consecutively recruited from hospital staff, laboratory personnel, and patients' relatives. Inclusion criteria for control subjects were (1) absence of primary headaches such as migraine, TTH; (2) absence of other neurological or systemic diseases; and (3) presence of a match with migraine patients on age (± 2 years), gender. Exclusion criteria for control subjects were the same asgroups with headaches. Initially, participants were fully informed about the purpose of the study and their responsibilities and subsequently completed the Hamilton Anxiety [18] (HAM-A), the Beck Depression Inventory Scale [19] (BDI) and provided demographic characteristics . All patients were required to keep a headache diary during the four-week run-in period. Each week for up to four weeks to complete the intervention, participants were contacted to ensure compliance and appropriate use of the technique. One month after the end of the study, a follow-up visit was carried out to ensure the completion of the questionnaires, both in the headache group and in the control group. Informed consent was obtained from all patients. The study protocol was approved by the Institutional Review Board (IRB) of Aeginition Hospital in Athens (IRB approval number: 638/5.11.2015) and complies with the Helsinki Declaration [20]. Psychometrics The Hamilton Anxiety Rating Scale (HAM-A) is a psychological questionnaire used by clinicians to assess the severity of a patient's anxiety [18]. Each of the 14 items contains six items of anxious symptoms (mood, mental tension, fears, insomnia, intellectual difficulties and depressed mood) and eight items of somatic symptoms (muscular, sensory, cardiovascular, respiratory, gastrointestinal, genitourinary, other autonomic) . and behavior during the interview). Each symptom group is rated on a scale from zero (not present) to four (severe) with a total score range of 0 to 56, where <17 indicates mild severity, 18–24 mild to moderate severity, and 25– 30 moderate to moderate. serious [18]. The Beck Depression Inventory (BDI) is a 21-item self-report assessment inventory that measures attitudes and symptoms characteristic of depression [19]. It consists of items relating to symptoms of depression such as hopelessness and irritability, cognitions such as guilt or feelings of punishment, as well as physical symptoms such as tiredness, weight loss and lack of interest in sex. Each of these items is scored on a scale of 0 to 3. Higher total scores indicate more severe depressive symptoms. The validated Greek version of HADS showed high internal consistency and stability (Cronbach's α = 0.82) [21]. Saliva sample collection All participants underwent saliva sample collection (Sarstedt, Nuembrecht, Germany) according to the instructions for correct sample collection. Whole unstimulated saliva accumulating on the floor of the mouth was collected from patients and healthy volunteers into high-quality polypropylene vials using the passive drooling technique. All participants were instructed not to eat a large meal or brush their teeth within 60 minutes prior to sample collection, and to avoid foods high in sugar or acidity or with a high caffeine content immediately prior to collection of the sample and also to rinse the mouth with water to remove food residues and wait at least 10 minutes after rinsing to avoid dilution of the sample before collecting saliva. Finally, all samples were stored in a plastic container at 2-4°C until analysis. Saliva was collected from participants at 8.00 am to rule out any confounding factors caused by circadian rhythm. All headache subjects were asked to collect headache-free baseline salivary samples at study screening when they were headache-free for at least 48 hours (time point A). They collected additional samples during moderate/severe headache (time point B) and phaseof self-defined resolution 24 hours after the headache attack (time point C). Healthy control subjects were asked to collect samples at the time of study screening (time point D). Sample analysis The morning samples were stored in the refrigerator at 4°C and at the end of the day they were taken to the laboratory where they were centrifuged at 3000 rpm at 4°C and the supernatant was aliquoted into polypropylene cryovials. The vials were frozen at −80°C until analysis. Salivary transferrin levels were measured by competitive immunoassay kits, and IL-6, IL-1ß, and CRP levels were measured by sandwich ELISA kits (Salimetrics Europe, Ltd. UK). Transferrin levels were used as a screening tool for the presence of blood in saliva samples, and samples with transferrin values ​​greater than 1 mg/dl were considered candidates for exclusion in other saliva tests. The cortisol assay has a sensitivity < 0.007 µg/mL and an inter-assay coefficient of variation < 11% while these characteristics are 0.07 pg/mL and 8 for IL-6, 0.37 pg/mL and 7 for IL -1β, 10 pg/mL and 11.2 for CRP as well as 0.08 mg/dL and 7.2 for transferrin respectively. Statistical analysis Quantitative variables are presented with mean and standard deviation (SD). Qualitative variables are presented with absolute and relative frequencies. Fisher's exact test was used for comparisons of proportions. Student's t tests were calculated for comparisons of mean values ​​when the distribution was normal. Mann-Whitney test for comparing study variables between two groups when the distribution was not normal. Wilcoxon signed-rank tests were used for time differences and results were considered significant at p0.999). The mean age at migraine onset was 26.5 years (SD=15.3 years), and 9 patients (40.9%) had TTH. All saliva samples had transferrin levels below 1 mg/dl and were therefore all used in the remainder of the saliva tests. The levels of CRP, IL-1ß and IL-6 measured at different time points for the two study groups are shown in Table 2. In both TTH and migraine subjects, only IL-1ß was found to significantly decrease from time point A to time point B, while from time point B to time point C a significant increase was recorded. Between-group comparisons showed that the headache groups had higher IL-1ß levels at time point B compared to controls at time point D. Table 3 shows CRP, IL-1ß, and IL-6 levels for those with migraine and those with TTH. No significant differences were found at any time point. Repeated measures analysis of variance showed no significant differences in the temporal variation of CRP, IL-b and IL-6 levels between subjects with migraine and those with TTH (p>0.05). Significantly higher values ​​for the BDI were found for those with TTH compared to those with migraine (Table 4). Correlation coefficients of CRP, IL-1ß, and IL-6 levels with scores on the HAM-A and BDI are presented in Table 5. CRP measured at time point A was negatively correlated with HAM-A and BDI scores. Furthermore, IL-6 measured at time A was negatively correlated with BDI scores. Discussion The main findings of this study were as follows: 1) IL-1ß was found to significantly decrease from time point A to time point B, while a significant increase was recorded from time point B to time point C. 2) All headache subjects had higher IL-1ß levels at time point B than controls at time point D. 3) No differences were foundsignificant in the temporal variation of CRP, IL-1ß and IL-6 levels between migraine and TTH 4) CRP was negatively correlated with HAM-A and BDI scores. 5) IL-6 measured at time point A was negatively correlated with BDI scores. The ability to measure these markers in saliva as a stress-free method highlights the novelty of this study in the psychobiological basis of headache research. Most previous studies have investigated circadian immune variations using blood samples but not saliva samples. In the few previous studies that have investigated salivary inflammatory markers and their circadian patterns, the results are contradictory. Sjögren et al. (2006) reported that salivary IL-6 levels in the evening were higher than those in the morning [22]. Fuori et al. (2012) demonstrated that salivary CRP levels upon awakening were higher than those before bedtime and that salivary CRP levels were moderately stable over a two-year period [23]. Izawa et al (2012) demonstrated that salivary IL-6 levels peaked upon awakening, gradually decreased from morning to midday, and peaked again at midnight, before participants went to sleep. Salivary CRP levels peaked upon awakening and were lowest throughout the day [24]. To date, however, no studies have investigated the variability of inflammatory cytokines, including IL-6 and CRP, over multiple days in headache sufferers. For the first time, the present study shows a similar change in salivary IL-1ß in both migraine and TTH. This change in IL-1ß concentration may reflect feedback inhibition of cytokine concentrations by endogenous cortisol, which is subject to circadian variation. This variation may have important implications for future studies in terms of the timing of salivary sampling during measurement for the assessment of primary headaches. The novelty of the present study is that it demonstrates an increase in salivary levels of IL-1ß in both migraineurs and TTH subjects during the headache period. One possible hypothesis for the ictal increase in proinflammatory molecules in TTH is that myofascial pain triggers the release of inflammatory mediators leading to excitation of peripheral afferent nerves. However, normal levels of inflammatory mediators have been found in the painful trapezius muscle in patients with chronic TTH [25]. Another interpretation is that the spinoreticulothalamic tract may be activated by peripheral stimuli, stimulating more circulating molecules such as corticotropin-releasing hormone, vasopressin, and beta-endorphin, which trigger the production of cortisol which may activate the peripheral release of cytokines [26]. This theory is in line with a previous study suggesting that cytokines during headache attacks are involved in the pain process [15]. Although the findings summarized in this section are from blood or cerebrospinal fluid samples, our new findings appear to be similar, but further research is needed into the biological implications of salivary inflammatory mediators in headache sufferers. In the present study, interesting and novel evidence demonstrated that higher levels of CRP and IL-6 were correlated with lower anxiety and depression symptom scores before or immediately after the day of the headache attack. The question of whether there is a relationship between elevated inflammatory markers and depression is a more complex question and would require longitudinal studies, preferably with a large cohort. A particular difficulty with longitudinal studies is that of confounding variables, such as biological, psychological, and social factors that can..