Salivary Inflammatory Markers in Tension Type Headache and Migraine
Salivary Inflammatory Markers in Tension Type Headache and Migraine
Data role of salivary inflammatory markers in migraine and Tension Type headache (TTH) are
 lacking. Τhe investigators studied whether headache attacks are associated with changes in C
 reactive protein (CRP), Interleukin -1β and Interleukin -6 in saliva in patients with Tension
 Type Headache and Migraine and age matched healthy controls . Τhe investigators, also
 investigated whether these markers could be influenced by comorbidities such as depression
 and anxiety.
Sixty subjects of both genders, aged from 18 to 60 years old, with primary headaches TTH and
 migraine, according to the International Classification of Headache Disorders, 3rd edition
 (beta version), were enrolled from the outpatient headache clinic of University Hospital of
 Athens between January to March 2016. 30 healthy control subjects aged matched were recruited
 mainly from hospital staff and patients' relatives.

 Initially, the participants completed the Hamilton Anxiety (HAM-A), Scale Beck Depression
 Inventory (BDI). All patients had to keep a headache diary during the four-week run-in
 period. All headache suffers were instructed to collected salivary headache-free baseline
 samples at the time of study screening when they had been free of headache for at least 48
 hours (time point A). All headache suffers collected additional samples during
 moderate/severe headache (time point B), and at self-defined resolution phase 24 hours of
 their headache attack (time point C). Healthy control subjects were instructed to collected
 samples at the time of study screening (time point D). Every week, until four weeks and one
 month after the end of the study, participants were contacted, in order to ensure the
 compliance and the appropriate use of the technique. One year later longitudinal data would
 also be obtain.

 Saliva Sample Collection Detailed instructions for the correct collection of saliva samples
 were given to all participants. Such instructions include avoiding eating a major meal and
 teeth brushing 60 minutes prior to sample collection. Also consumption of high sugar and
 caffeine content foods as well as high acidity foods have to be excluded before saliva sample
 collection. Mouth rinse with water in order to remove any food residue and saliva sample
 collection at least 10 minutes after mouth rinse was recommended. Unstimulated whole saliva
 that pooled on the mouth floor were collected from patients and healthy volunteers in high
 quality polypropylene vials by the passive drool technique. Finally, all samples were stored
 in a plastic container at 2-4 ° C until analysis. Saliva was collected from the participants,
 at 8.00 a.m. in the morning in order to rule out any confounding factor caused by circadian
 rhythm.

 Sample Analysis Morning samples were kept in the refrigerator at 4°C and at the end of the
 day were brought to the laboratory where they were centrifuged 3000 rpm at 4°C and the
 supernatant was aliquoted in to polypropylene Cryogenic vials. Vials were frozen in -80°C
 until analyzed. Saliva transferrin levels were measured by competitive immunoassay kits and
 Interleukin-6, Interleukin-1β and CRP levels were measured by sandwich ELISA kits.
 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 as candidates for
 exclusion in other salivary assays. Cortisol assay has a sensitivity of < 0.007 μg/ml and an
 inter-assay coefficient of variation of < 11% while these characteristics are 0.07 pg/ml and
 8 for Interleukin-6, 0.37 pg/ml and 7 for Interleukin-1β, 10 pg/ml and 11.2 for CRP as well
 as 0.08 mg/dl and 7.2 for transferrin respectively.
Inclusion Criteria:

 -  aged from 18 to 60 years old

 -  Tension type Headache (TTH) and migraine, fulfilling the criteria International
 Classification of Headache Disorders, 3rd edition (beta version)

 Exclusion Criteria:

 -  abnormal plasma CRP, Interleukin-1β and Interleukin-6 levels (>10 mg/L)

 -  smoking cigarettes > 1 pack/day;

 -  current pregnancy, lactation, or hormonal contraceptive use

 -  alcohol or substance abuse

 -  drug use such as anticoagulants, statins, or hormonal drugs

 -  anti-inflammatory therapy

 -  other primary or secondary headaches

 -  major psychiatric disease

 -  oral health problems.