Enriched Oxygen Mixtures in Athletes

Currently, Hyperbaric Oxigen (HBO) is a widely used treatment for several conditions. There are 14 indications for HBO, officially recognized by the Undersea and Hyperbaric Medical Society (UHMS), but research is discovering other interesting applications.

HBO plays an important role in enhancing antioxidant defense mechanisms by increasing radical oxygen species (ROS) and nitric oxide species (NOS). This controlled oxidative stress has been shown to stop the vicious circle of inflammation - damage - hypoxia already seen in several diseases. Increased neoangiogenesis has been demonstrated at pressures of 2 atmospheres absolute (ATA), while effects helping ischemic tissues need pressures between 2.5 and 2.8 ATA to develop. Also, stem cell proliferation and mobilization have been demonstrated after HBO treatments.

During sports activities, metabolism generates waste products - mostly CO2, lactic acid, but also ROS. HBO could be useful in modulating antioxidant mechanisms and increasing stem cell mobilization, thus helping cells in the recovery after training and sportive competitions.

The authors hypothesize that:

1. HBO can reduce oxidative stress and induce stem cell mobilization in healthy professional athletes;
2. hyperoxic mixtures can reduce oxidative stress and induce stem cells mobilization in healthy professional athletes;
3. HBO at low pressures (L-HBO at 1.45 ATA) is at least comparable to conventional HBO (at 2.5 ATA) in reducing oxidative stress and increasing stem cell mobilization.

The Authors will include healthy athletes. These will be randomly assigned to a control group, a L-HBO group, a HBO group, a 30% O2 group, or a 50% O2 group.

The Authors will assess oxidative stress changes and stem cells proliferation before and after 20 L-HBO/HBO/30% O2 mix/50% O2 mix treatments, and after 2 months after the end of treatments.

Subjects will be recruited through public announcements in local gyms and gathered to explain the protocol. Those willing to participate will sign a written informed consent and recruited. To be included, all the subjects will undergo a general medical screening to allow hyperbaric treatments. This will include weight, height, non-invasive arterial blood pressure, and heart rate measurements.

After inclusion, subjects will be randomly assigned to three arms using an electronic number generator by personnel not directly involved in the experiment:

* Arm 1(control): no intervention.
* Arm 2 (L-HBO): treated with oxygen at 1.45 ATA for 60 min (inclusive of compression and decompression times, and an air break of 3 minutes breathing air);
* Arm 3 (HBO): treated with oxygen at 2.5 ATA for 60 min (inclusive of compression and decompression times, and an air break of 3 minutes breathing air).
* Arm 4 (30% O2): breathing an air mixture with 30% of oxygen at atmospheric pressure (1 ATA).
* Arm 5 (50% O2): breathing an air mixture with 50% of oxygen at atmospheric pressure (1 ATA).

Subjects included in Arm 2, 3, 4, 5 will undergo a total of 20 treatments. They will follow a personalized diet proportional to their energetic expenditure.

The Authors will identify 3 time-points in the protocol:

TIME 0 (T0): immediately after inclusion, before any treatment or experiment; TIME 1 (T1): at the end of HBO treatments; TIME 2 (T2): 2 months after the end of HBO treatments.

The following exams will be performed on the included subjects:

* a standardized panel including Complete Blood Count (CBC), creatinine, Blood Urea Nitrogen (BUN), C reactive protein, and VES will be performed at T0, T1, and T2.
* oxidative stress markers will be analyzed on blood, urine, and saliva samples. On blood samples (T0; T1; T2), the Authors will measure IL-1 beta, IL-6, TNF-alfa, reactive oxygen species and total antioxidant capacity (by paramagnetic resonance), total (tot) and reduced (red) aminothiols (by fluorescence spectroscopy), 3-nitrotyrosine (3-NT) (by competitive immunoassay).

On urine samples (T0; T1; T2), the Authors will assess lipid peroxidation by measuring 8-isoprostane concentration (by competitive immunoassay), nitrite and nitrate (NO2/NO3) concentration (by colorimetry based on the Griess reaction), inducible Nitric Oxide Synthase (by ELISA commercially available kit), creatinine, neopterine, and uric acid concentrations, 8-oh-2-deoxyguanosine (by competitive immunoassay).

On saliva samples (T0; T1; T2) the Authors will measure reactive oxygen species and total antioxidant capacity (by paramagnetic resonance), and cortisol (by competitive immunoassay).

* stem cells will be analyzed on blood samples (at T0, T1, T2) (by flow cytometry).

Blood samples (approximately 6-12 ml) will be drawn from the veins of the forearms (preferentially on the non-dominant limb); plasma and erythrocytes will be separated by centrifuge at 1000×g for 10 min at 4°C. Urine samples will be collected by voluntary voiding in sterile containers. 1 mL of saliva will be obtained by Salivette devices (Sarstedt, Nümbrecht, Germany). The subjects will be instructed to refrain from drinking, eating, smoking, brushing their teeth, and using mouthwash in the 30 min before salivary collection.

All samples will be stored in multiple aliquots at - 80 °C until assayed and thawed only once before analysis.

With this setting, blinding of patients and investigators will be impossible due to different structural characteristics. However, outcome assessors will be blinded to patients' allocation.

Eligibility Criteria:
Inclusion Criteria:

* professional athletes
* performing at least 3 training sessions/week

Exclusion Criteria:

* previous pneumothorax
* problems with compensation maneuvers
* known epilepsy
* active smoker