2.1 Population of the study
Between September 2016 and January 2018, 69 consecutive MOH inpatients, referring to the Headache and Drug Abuse Centre at the University of Modena and Reggio Emilia, were enrolled in the study. All patients were diagnosed by trained physicians, according to the International criteria [8]. After signing written informed consent, the collection of demographic data, the measurement of blood pressure and experimental procedures were completed on admittance, before withdrawal treatment was initiated. Patients were tested in the morning, before taking any therapy. A trained investigator performed the CPT tests. The blood and urine routine laboratory tests were performed on the second day of hospitalization, fasting from midnight. For each patient, the subsequent parameters were analysed: (1) prevalent acute headache medication used, classified in triptans, NSAIDs and mixtures (2) daily drug intake (DDI), mean number of acute headache medications consumed every day; (3) years of chronification (CHR), time elapsed since the beginning of chronic symptoms. Prevalent acute headache medication used and DDI were referred to a 3-month period before testing.
Forty-two age- and sex-matched healthy volunteers were enrolled as controls. They were recruited from the hospital´s medical staff and their relatives. On the experimental day, they also performed blood tests for renal function. Only healthy subjects without migraine and any headache disorders were included in the study, except for tension-type headache for no more than three days per month. The daily intake of medicines and the assumption of acute pain medications more than five times in the last month or in the previous 48 hours of the experimental procedures were considered exclusion criteria for the study. For all study participants, exclusion criteria were: headache on admittance to the centre, altered results in routine laboratory tests, other non-cephalic chronic painful conditions, and autoimmune, hepatic, renal, oncological, cardiovascular, psychiatric or neurological relevant pathologies.
2.2 Serum samples collection and storage
Venous blood was collected in vacutainer tubes and allowed to clot at room temperature for 1 h. Serum was obtained by centrifugation at 2,000 x g for 10 min at 4 °C; a mixture of protease inhibitors (Sigma-Aldrich) was added to prevent protein degradation and alteration. Samples were divided into aliquots and stored at – 80 °C until analysis.
2.3 ELISA test
Globally, 111 serum samples, which were comprised of 69 MOH patients and 42 healthy individuals, were analysed by ELISA test. Especially, the expression level of the following proteins was evaluated: L-PGDS (human Prostaglandin D Synthase, Lipocalin-type, ELISA kit, BioVendor, Brno, CZ), VDBP (human Vitamin D-binding protein, ELISA kit, Cusabio Biotech, USA), APOE (human apolipoprotein E, Apo-E, ELISA kit, Cusabio Biotech, USA) and APOA1 (human apolipoprotein A1, Apo-A1, ELISA kit, Cusabio Biotech, USA). The assays were performed according to the manufacturer’s protocols and instructions.
L-PGDS and APOA1 assays employ the quantitative sandwich enzyme immunoassay technique, while the VDBP and APOE kits use the competitive inhibition enzyme immunoassay method. Briefly, in the first case, diluted serum samples, standards and blanks were dispensed in the microplate wells containing immobilized pre-coated specific antibody for L-PGDS and APOA1, respectively. After 1 h incubation, a horseradish peroxidase (HRP)-conjugated antibody specific for L-PGDS and APOA1 was added in each well of the plate, which was incubated further for 1h. Following a wash to remove any unbound reagent, a substrate solution was added and allowed to react with HRP-conjugate. A blue colour proportional to the amount of sample protein bound in the initial step developed. The reaction was interrupted by an acid solution, which lead to a yellow product. The absorbance was measured at λ 450 nm in a microplate reader (Multiscan FC, Thermo Scientific, USA). Protein concentrations were calculated from a standard curve generated by the protein stock solution furnished with the kit.
VDBP and APOE kits used a microtiter plate pre-coated with the respective proteins. Standards and samples (50 μL) were added to the plate wells, together with the same amount of HRP-conjugated antibody specific for the protein, and incubated at 37 °C. This step was followed by aspiration and washing repeatedly for five times before adding 90 μL of tetramethylbenzidine (TMB) substrate solution to each well (in this case, the colour developed in an opposite way to the amount of protein present in the serum sample). After 10 min incubation at 37 °C in the dark, the reaction was interrupted by adding 50 μL of stop solution and the colour intensity was measured within 5 min at λ 450 nm.
2.4 Cutaneous pain thresholds
All the CPTs assessments were performed in absence of a visible headache attack. Calibrated von Frey-like filaments (Touch-Test® Sensory Evaluators filaments, North-Coast Medical Inc., CA) were applied sequentially in scheduled areas, in increasing order for two seconds each to determine the basal CPTs, asking the patient when the touch became a painful or very uncomfortable sting. Assessments were repeated three times and the filaments were used in each location in the following sequence, representing the increased strengths measured in grams (e.g. 0.008; 0.02; 0.04; 0.07; 0.16; 0.4; 0.6; 1; 1.4; 2; 4; 6; 8; 10; 15; 26; 60; 100; 180 and 300 g). The test investigated three cutaneous areas: temple, cheekbone and chin areas (exploring first, second and third division of the trigeminal nerve, respectively), assessed bilaterally in random order. Landmarks definitions were: temple, the skin over the pars orbitalis of frontal and sphenoid bones; cheekbone, the skin over periorbital area of zygomatic bone; chin, the skin of the chin, including the innervation territories of the lower alveolar nerves.
2.5 Statistical analysis
Quantitative variables were expressed as a mean ± standard deviation. Qualitative variables were expressed as a percentage. Comparison of quantitative variables between MOH patients and healthy individuals was performed using independent t test, whereas comparison of qualitative variables was performed using χ2. All p-values are two-sided, with a level of significance of 1%. To correlate serum L-PGDS, VDBP, APOE and APOA1 with CPTs, data were analysed with the Pearson correlation coefficient or, if one of the two variables was not normally distributed, Spearman's rank correlation coefficient was used. R software (version 3.4.3) was used to perform statistical analysis. The level of significance for correlation tests was set at 5%.