Subjects
Nine male NPSPs (age: 20 ± 2 years) were enrolled from the same soccer team by the Sports Medicine Service of Modena and they performed evening-based soccer training sessions. All the enrolled athletes have obtained a certificate of competitive soccer activity from the Sports Medicine Service. That certificate is issued after physical examination, medical history evaluation, an ECG at rest and after a stress exercise test. The only exclusion criteria were the presence of inflammatory diseases or injuries occurred just before or during the study.
This study was performed in agreement with ethical recommendations of the Declaration of Helsinki, and the Ethics Committee of Area Vasta Emilia Nord approved all experiments (protocol number 88/2018/SPER/AUSLMO). Moreover, all the participants read and signed an informed consent.
Design
This observational study provides five consecutive WBC-t sessions, administered at the mornings. WBC-t was performed in a Cryomed chamber (Cryomed Italy, Milan, Italy) and consisted of short exposure (up to 3 min) to extremely cold air (-190° C) inside a chamber in which the subject’s hands and head remain outside, therefore not in contact with the cold stimulus. Subjects were dressed in underwear avoiding the presence of metal pieces. A sample of 40 mL of venous blood was collected from each subject before the first session of WBC-t (day 1) and promptly following the fifth and final WBC-t (day 5; Fig. 1).
Clinical chemistry, hematology and hormonal analyses
NPSPs blood and urine samples were collected before and after WBC-t and subjected to several laboratory analyses. Clinical chemistry, hematology, hormonal parameters, and urine were evaluated at the BLU Laboratory (NOCSAE, Baggiovara, Modena, certification #ISO90012015) according to hospital protocol. Regarding blood samples, the BLU Laboratory of Baggiovara evaluated a full set of fifty analytes, involving glucose, urea, creatinine, uric acid, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, bilirubin T, bilirubin D, T proteins, glutamic oxaloacetic transaminase (GOT), pyruvic glutamic transaminase (GPT), gamma-glutamil transferase (GGT), CK, amylase, sodium, potassium, iron, transferrin, % saturation, ferritin, phosphorus, lactic acid, C protein, S protein, white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelets, mean platelet volume (MPV), % and count of lymphocytes, monocytes, eosinophils, basophils, neutrophils and reticulocytes, thyroid stimulating hormone (TSH), T, C, growth hormone (GH), insulin growth factor (IGF)-1, peptide C, insulin, luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and progesterone. Clinical chemistry analytes were measured in the CoreLab on full-automated clinical chemistry platforms, based on state-of-the-art enzyme kinetic techniques, immunoturbidimetric techniques, colorimetric methods (Chemistry analyzerS Olympus 680 and LX20, Beckman Coulter, Brea, CA, USA). Complete blood count with formula was performed with Accucount technology for red, white cells and platelets and VCS technology, triple impedance counting, for leukocyte formula and spectrophotometric determination of hemoglobin, and reticulocytes (after staining with methylene blue) (Hematology Analyzers DXH 750 and DXH 800, Beckman Coulter, Brea, CA, USA). Blood circulating hormones were detected on full automated platforms based on CMIA methods (Chemiluminescent Immunoassay Analyzer: Architect, Abbott Laboratories, Chicago, Illinois; USA; DXI, Beckman Coulter, Brea, CA, USA; LiaisonXL, DiaSorin, Saluggia, Italy).”
Urine analysis evaluated specific gravity, pH, glucose, proteins, hemoglobin, ketones, bilirubin, urobilinogen, leukocyte esterase and nitrite. Urine analysis was made using dipstick and image capture (Urine Microscopy System IQ200 sprint connected to the Urine Chemistry Analyzer iChem Velocity, Beckman Coulter, Brea, CA, USA).
Blood processing and plasma analysis
Peripheral blood mononuclear cells (PBMCs) and plasma were isolated in our laboratory at the University of Modena and Reggio Emilia from venous blood using a density-gradient centrifugation standard method. Viable PBMCs were stored in liquid nitrogen and plasma was stored at -80 °C until use. Plasma analysis included four soluble factors (CCL-2, IL-2RA, IL-1RN and IL-18), DNA extraction and subsequent quantification of circulating mitochondrial (mt)DNA through droplet digital PCR (ddPCR). DNA was isolated from plasma samples with the QIAmp DNA Minikit, (Qiagen, Alameda, CA, USA), in accordance with the manufacturer’s instructions. MtDNA was quantified on a Bio-Rad QX200 ddPCR droplet system, by using the ddPCR Supermix for Probes, and ddPCR assay for the mtDNA gene ND2 (1 uL; UniqueAssayID: dHsaCPE5043508) and for the nuclear gene EIF2C1 (1 uL; UniqueAssayID: dHsaCP2500349). Reagents were from Bio-Rad, Hercules, CA, USA.
The four soluble factors CCL2, IL-18, IL-2RA and IL-1RA were analyzed from plasma samples by using Ella assays (Bio-Techne, MN, USA) following the manufacturer’s instructions [23].