Animals

It is well known that incidence rate of FM is high in women1,2,3. Hence, we used 36 female Wistar rats (aged 12 weeks old and 170 ± 10 g body weight) in the current study. The rats were housed in controlled-temperature (21 ± 2 °C) and humidity (65%) rooms, under a 12:12 h light-dark cycle, with ad libitum access to water and food until use. Hot plate and Von Frey tests were performed between 9:00 and 10.00 a.m. at baseline, 5th and 19th days of the experiments.

Availability of data and materials

Data and approve of rats were taken from Experimental Animal Research Center of Suleyman Demirel University (SDU) according to protocol number (HADYEK-21438139-320). All methods in the manuscript were performed in accordance with the relevant guidelines and regulations of SDU by including a statement in the methods section to this effect. The dataset and analyses were generated in Neuroscience Research Center of SDU and they are available from the corresponding author on reasonable request. Graphics and tables in the manuscript were prepared by the corresponding author.

Study groups

The rats were divided into four groups as follows: The control group (n = 8) had no FM and was not administrated. They received intraperitoneal (i.p.) 0.9% w/v saline solution for 2 weeks. In the Se group (n = 8), they received i.p. Se 1.5 mg/kg/over day for two weeks (total seven doses)15. In FM group (n = 10), the rats were exposed FM induction procedure4. Then, the rats received i.p. saline solution for 2 weeks. In FM + Se group (n = 10), the rats received Se (same as the Se group) after FM induction (same as the FM group).

Twelve hours after the last saline solution and Se dose administration, all rats were decapitated under propofol inhalation anesthesia in accordance with SDU experimental animal legislation. Selenium (Sodium selenite, Sigma Chemical Co., MO, USA) was diluted to appropriate concentration in sterile 0.9% w/v saline solution. In patch-clamp experiment and [Ca2+] i concentration assays, the DRGN and SciN were further treated with CumHPx (1 mM) or ADPR (1 mM) and capsaicin (10 μM) for activation of TRPM2 and TRPV1 channels, respectively and they were also blocked the TRPM2 channels antagonist, N-(p-amylcinnamoyl) anthranilic acid (ACA and 0.025 mM) and TRPV1 antagonist, capsazepine (CPZ and 0.1 mM). Doses of capsaicin and CPZ on TRPV1 in cells are changing between 100 nM and 0.1 mM. In a recent study31, we tested different doses of capsaicin and CPZ in the DRGN and TRPV1 against and antagonist doses of capsaicin and CPZ in the study was found as 10 μM and 0.1 mM, respectively. Therefore, the doses were used in the current study.

Induction of hyperalgesia

For produce a bilateral long-lasting hyperalgesia similar to fibromyalgia in humans, acidic saline solution (adjusted to pH 4.0) was injected into the gastrocnemius muscle in the rats4. Before the first injection of acidic or normal saline into the gastrocnemius muscle, paw withdrawal threshold values of the rats were recorded in order to record the baseline value. Each animal received two repeated injections of the acidic saline solution (100 μl) in the same unilateral gastrocnemius muscle under propofol inhaled anesthesia, a procedure which is repeated five days later4. After the second injection of the acidic saline, hyperalgesia in the animal is detected increase the paw withdrawal threshold by the von Frey filament and hot plate tests in the muscle.

Hyperalgesia tests

The assessment of hyperalgesia was measured by the using calibrated von Frey filaments (20PC Aesthe Model, NO. 160615, Muromachi Kikai Co., Ltd. Tokyo, Japan), to the plantar aspect of the hind paw of mice that were kept in suspended wire mesh cages. A response was indicated by lifting of the hind paw38.

Heat-controlled plate (Varioma, Thermo Fischer Inc., Langenselbold, Germany) for Hot Plate test was used to assess paw withdrawal latency to thermal nociceptive stimuli. The hot plate test apparatus consisted of an electrically heated surface kept at a constant temperature of 55.0 ± 0.6 °C. Each mouse was placed on hot plate, and the reaction time was measured until the mouse either demonstrated hind paw licking or jumping (‘up and down’ method). A cutoff time of 60 s was used to prevent tissue damage for hot plate test38. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength.

Preparation of brain, blood, primary DRGN and SciN samples

Details of isolations of DRGN and SciN were given in previous studies15,34. Briefly, the DRGN and SciN were minced with iridectomy scissors and incubated with enzymes including trysin (type III, Sigma) and 0.5 mg/ml collagenase (type XI, Sigma) in 5 ml DMEM at 37 °C in a shaking bath for 40 min after removing the attached nerves and surrounding connective tissues. To stop the enzymatic digestion 1.25 mg/ml soybean trypsin inhibitor (type II-S1, Sigma) was added. After dissociation with a sterile syringe, the DRGN and SciN suspensions of mediums were centrifuged at 1,500 g and the medium and high size neurons were removed for the analysis. The isolated neurons were transferred into a 35-mm culture dish and kept still for at least 30 min.

The brain was also taken as follows; the cortex was dissected out after the brain was split in the mid-sagittal plane. The gastrocnemius muscle samples from right legs were also taken. After preparing brain and muscle homogenates in ice-cold Tris-HCl buffer (50 mM, pH 7.4), they were stored at −85 °C for analyses of lipid peroxidation and antioxidants. Half of the muscle sample with DRGN and SciN was frozen at −85 °C for using Western blot analyses. Plasma and erythrocyte samples from anticoagulated blood (sodium EDTA) were obtained as described in a previous studies17,18 and they were stored at −85 °C.

Electrophysiology

Whole-cell voltage clamp recording was taken from the DRGN at 22–24 °C (EPC10 patch-clamp set, HEKA, Lamprecht, Germany). Resistances of the recording electrodes were adjusted to about 3–6 MΩ by a puller (PC-10 Narishige International Limited, London, UK). We used standard extracellular bath and pipette solutions as described in previous studies16,30. Holding potential of the patch-clamp analyses in the DRGNs was −60 mV. Voltage clamp technique was used in the analyses and current-voltage (I–V) relationships were obtained from voltage ramps from −90 to +60 mV applied over 200 milliseconds. All experiments were performed at room temperature (22 ± 1 °C).

In the experiments, TRPM2 is intracellularly (via patch pipette) gated by ADPR (1 mM), although they were extracellularly (via patch chamber) blocked by ACA (0.025 mM). TRPV1 was extracellularly gated by CAPS (10 μM), and the channels were extracellularly blocked by CPZ (0.1 mM). For the analysis, the maximal current amplitudes (pA) in a DRGN were divided by the cell capacitance (pF), a measure of the cell surface. The results in the patch clamp experiments are the current density (pA/pF).

Measurement of [Ca2+] i concentration in DRGN and sciatic nerve

The DGRN and SciN samples were separately seeded in sterile cell culture flasks. Then they were loaded with 4 μM Fura-2/AM in loading buffer for 45 min at 37 °C in the dark, washed twice with the phosphate buffer, incubated for an additional 30 min at 37 °C to complete probe de-esterification. The groups were exposed to the stimulations in a water-jacketed cuvette (37 °C) with continuous magnetic stirring. Fluorescence was detected by using a Carry Eclipse Spectrofluorometer (Varian Inc, Sydney, Australia). The fluorescence at 505 nm was measured at 1 second intervals after excitation at 340 nm and 380 nm, respectively.

For calibration of [Ca2+] i , maximum and minimum fluorescence values were obtained by adding the detergent Triton X-100 (0.1%) and the Ca2+ chelator EGTA (10 mM) sequentially at the end of each experiment. Calculation of the [Ca2+] i concentrations were described in previous studies39,40, assuming a Kd of 155 nM. The [Ca2+] i concentrations in TRPM2 and TRPV1 experiments were recorded by using the integral of the rise in [Ca2+] i for 120 seconds after addition of CumHPx (1 mM) or capsaicin (10 μM), respectively. The [Ca2+] i concentration is expressed as nanomolar (nM) taking a sample every second as previously described39,41.

Measurement of intracellular ROS production and mitochondrial membrane depolarization

Dihydrorhodamine (DHR)123 is an uncharged and nonfluorescent intracellular ROS production indicator and the level of intracellular ROS was assessed by fluorescence microplate reader (Infinite pro200, Tecan Life, Männedorf, Switzerland) with DHR123, which oxidizes to cationic rhodamine 123 in the presence of ROS, as described previously39. The resulting data were normalized using the control values. 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) dye was used for measurement of the mitochondrial membrane potential and it was determined in he a plate reader (Infinite pro200) by using JC-1 as described in previous studies40,42. The ROS and JC-1 values were expressed as fold-increase over the pretreatment level after calculating fluorescence units/mg protein.

Assay for apoptosis, cell viability, and caspase 3 and 9 values

The apoptosis levels were determined in a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) by using a commercial kit of Biocolor Ltd. (Northern Ireland) as described in a previous study41. The method is based on loss of asymmetry in membranes of apoptotic neurons. We used to cell viability analyses as 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in the neurons as described elsewhere41 and absorbance values of MTT were recorded by the spectrophotometer (UV-1800) at 490 nm.

The determinations of caspase 3 and caspase 9 activities in the sciatic nerve and DRGN neurons were performed in the microplate reader (Infinite pro200) by using caspase 3 (N-acetyl-Leu-Glu-His-Asp-7-amino-4-methylcoumarin (AC-LEHD-AMC), Bachem, Bubendorf, Switzerland) and caspase 9 (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (ACDEVD-AMC), Sigma) substrates. Details of the assays were indicated in recent studies16,34. The apoptosis, MTT, caspase and 9 values were expressed as fold-increase after calculating fluorescence units/mg protein.

Lipid peroxidation, GSH-Px, reduced glutathione (GSH) and antioxidant vitamin analyses in plasma, erythrocytes, muscle (gastrocnemius) and brain

The GSH concentration and GSH-Px activity in the DRGN, SciN, muscle, brain homogenate and hemolyzed erythrocyte were spectrophotometrically (UV-1800) assayed at 412 nm by using the method of Sedlak and Lindsay43 and Lawrence and Burk22, respectively. Lipid peroxidation levels as malondialdehyde (MDA) in the DRGN, SciN, muscle, brain homogenate, plasma and hemolyzed erythrocyte were measured with the thiobarbituric-acid reaction by using method of Placer et al.44. The GSH-Px activity, GSH and MDA concentrations were expressed as international unit (IU) of GSH oxidized/min/μg protein (IU/μg pr) and μmol/g protein (μmol/μg pr), respectively. Method of Bradford was used in the muscle and brain homogenate, and hemolyzed erythrocytes for the determination of protein contents.

Results of recent studies indicated low vitamin A (retinol), vitamin E (α-tocopherol) and β-carotene concentrations in plasma of patients with FM. For further clarifying role of antioxidant vitamins in FM-induced oxidative stress, retinol, β-carotene and α-tocopherol were spectrophotometrically determined in the brain, gastrocnemius muscle and plasma samples by a modification of the method described by Suzuki and Katoh45 and Desai46. Calibration was performed using standard solutions of all-trans retinol, β-carotene and α-tocopherol in hexane. All antioxidant vitamin values in plasma, muscle and brain were expressed as μmol/l for plasma and μmol/g tissue for brain and muscle, respectively.

Western Blot analyses

Standard procedures are used in the Western Blot analyses of gastrocnemius muscle, SciN and DRGN16,34. Samples were homogenized in ice-cold RIPA buffer. The protein concentration in the supernatant was determined using the Bradford’s method. Membrane blots were incubated overnight at 4 °C with the following primary antibodies: caspase 3 (p17-specific Polyclonal Antibody), caspase 9 (p35/p10 Polyclonal Antibody), β actin (polyclonal antibody), poly-ADPR polymerase 1 (PARP1) (polyclonal antibody). The primary antibodies were purchased from Proteintech (Istanbul, Turkey) although secondary antibodies (Rabbit IgG, HRP-linked from donkey) were purchased from GE Healthcare (Amersham, UK). Relative levels of immunoreactivity in ECL Western HRP Substrate (Millipore Luminate Forte, USA) were quantified using Syngene G:Box Gel Imagination System (UK). Rabbit anti-β-actin (1:2000) was used as an internal control for the concentration of proteins loaded. Obtained values were expressed as relative density over the control level.

Statistical analyses

All data were represented as means ± standard deviation (SD). The data were analyzed by using 17.0 version of SPSS statistical program (Chicago, Illinois, USA). P value as ≤ 0.05 was considered to indicate a statistically significant difference. Presence of significance in the four groups was once detected by LSD test. Withdrawal thresholds of Hot Plate and Von Frey were analyzed by using a Dixon non-parametric test. Remaining p value levels of significances in the data were analyzed by using Mann-Whitney U test.

Compliance with Ethical Standards

The study was approved by the Local Experimental Animal Ethical Committee of Suleyman Demirel University (SDU) (Protocol number: HADYEK-21438139-320).