Study Design
A double-blind, randomized, 1:1 ratio, placebo-controlled study of healthy middle-aged master athletes. The study was done between May 2018 and December 2020 and the protocol was approved by the Shamir Medical Center institutional review board. The study was registered in the National Institutes of Health (NIH) clinical trials registry, number NCT03524989 (30/04/2018). The study was performed in the Shamir Medical Center. All methods were performed in accordance with the relevant guidelines and regulations in accordance with the Declaration of Helsinki.
Subjects
Thirty-seven healthy master athletes, aged 40–50, who performed aerobic sports at least four times a week at moderate-high performance for their age group, with no significant musculoskeletal injury in the past 3 months, were enrolled. Exclusion criteria included: previous treatment with HBOT for any reason during the last 3 months, debilitating significant musculoskeletal injury, lung pathologies, middle or inner ear pathologies, claustrophobia, chronic illness, chronic medications or active smoking. Athletes were recruited via advertisements and social media. Informed consent was obtained from all subjects.
All the athletes were requested to continue their current training regimen, with no changes in volume or training intensity.
Randomization and Masking
After signing an informed consent, the athletes were randomly assigned (1:1) to either the HBOT or the SHAM-placebo groups. The randomization code was generated by a nurse coordinator who was masked to the study and was not involved in the execution of the study. Until study closure, the treatment codes were available only to this nurse and the HBOT technicians. Participant enrollment was done by physicians who were masked to the study randomization. Assessors were also blinded to the athletes’ intervention assignment.
To evaluate the blinding, following the first session, the athletes were asked to discreetly answer a two-question questionnaire about their perception of whether they were allocated to the treatment or SHAM group.
All data were stored in a dedicated database and were checked for accuracy and completeness. A masked data review was done before code-breaking and analysis, according to a standard procedure at our unit.
Interventions
Both the HBOT and SHAM-placebo protocols were conducted in a multiplace Starmed-2700 chamber (HAUX, Germany). Pressure gauges and informative screens within the chamber were disconnected for the athletes’ blinding. The protocol comprised of 40 daily sessions, five sessions per week, within a 2-month period. The athletes were instructed to maintain their usual training program throughout the study.
The HBOT protocol included breathing 100% oxygen by mask at 2ATA for 60 min with no air breaks. Compression/decompression rates were 1 m/min.
The SHAM-placebo protocol included breathing 21% oxygen by mask at 1.02 ATA for 60 min. In order to achieve blinding and have the athletes perform pressure equalization, compression to 1.2 ATA was performed for the first 5 min (0.4 m/min), followed by decompression to 1.02 ATA (0.4 m/min) in the following 5 min. The minimal added 0.02 ATA was mandatory for blinding and for avoiding chamber door opening during the session.
Outcomes
The athletes were evaluated at baseline, 1–2 weeks prior to their interventional protocol and 1–2 weeks after the last HBOT/SHAM session.
Cardiopulmonary Maximal Exercise Test (CPET)
Exercise tests were conducted on an E100 cycle ergometer (COSMED, Rome, Italy). Gas exchange was measured by a Quark CPET system (COSMED), with breath-by-breath sampling technology and integrated heartrate and exercise ECG monitoring and recording with a 12-lead ECG system (COSMED, Rome, Italy). Data were collected on a dedicated computer using the Omnia Metabolic Modules software (COSMED). Before each test, the gas analyzers and flow meter were calibrated. The start of the protocol included a one-minute rest without pedaling, followed by a 2-min warm up. The testing protocol included a ramp power increase of 30 watts every minute starting from 0 W, while pedaling cadence had to be maintained at 70 rpm. Exhaustion was reached when cadence could not be maintained above 70 rpm or when a participant terminated the test. Subsequently, a three-minute recovery with a pedaling workload of 0 W was initiated.
Maximal Exercise Tests
A blinded physiologist performed analysis of each CPET test separately, masked from the athletes’ name, group allocation, date of performance and whether the test was a baseline or post-intervention measurement. The breath-by-breath dataset was averaged in epochs of seven breaths and both VO2Max and VO2AT (VO2 at the ventilatory anaerobic threshold) were determined according to classic criteria (based on the plateau in the VO2 plot with increasing workload for VO2Max [17] and minute ventilation (VE), respiratory exchange ratio (RER), end-tidal partial pressure of oxygen (PetO2), ratio of minute ventilation to oxygen consumption (VE /VO2) and the ratio of minute ventilation to carbon dioxide (VE /VCO2) for VO2AT [18]). Compared parameters were maximal power output, maximal oxygen consumption (VO2Max), anaerobic oxygen consumption (VO2AT), breathing reserve (BR), RER, heartrate, VE and volume of CO2 expired (VCO2).
Mitochondrial Respiration
Muscle samples were taken from the gluteus maximus by a fine-needle biopsy technique using a TruCut biopsy needle and a 14G puncture cannula after prepping, draping and local anesthesia. The gluteus maximus muscle was chosen to minimize any interference with the athletes’ daily training and increase participation rates. Athletes underwent muscle biopsies at baseline (1–2 weeks prior to intervention) and upon its completion (1–2 weeks post-intervention). Muscle tissue of about 5–10 mg was immediately put in ice-cold biopsy preservation solution (BIOPS). Samples were immediately transferred to saponin solution for membrane permeabilization for 25 min followed by two cycles of 10 min each in respiration medium (MiR05) prior to experimentation. Samples were analyzed by high-resolution respirometry.
Mitochondrial respiration was measured using the Oroboros® Oxygraph-2 K (Oroboros Instruments, Innsbruck, Austria). This device allows to simultaneously record the O2 concentration in two parallel chambers, calibrated for 2 ml of respiration medium (MiR05). Mitochondrial respiration was quantified in terms of oxygen flux (JO2) based on the rate of change of the O2 concentration in the chambers normalized for wet tissue volume. Two mg wet weight samples were added to each Oxygraph chamber and normalized to the amount of tissue per chamber.
The titration sequence used for the human muscle samples was as follows: 10 mM pyruvate, 5 mM malate, stepwise titration of 2.5 mM ADP, 10 μM cytochrome c, 10 mM glutamate, 10 mM succinate, stepwise titration of 0.5 μM carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), 0.5 μM rotenone and 5 μM antimycin A. A measure of the proton leak (leak) was obtained following the addition of pyruvate and malate. A stepwise titration of ADP to saturated concentrations allowed us to selectively quantify the activity of complex I (CI) (the oxygen consumption rate through the NADH pathway). Ten μM cytochrome c was added as an internal control to survey the integrity of the outer mitochondrial membrane. Glutamate was added to evaluate its additive effect on CI activity. The maximum oxidative capacity through complex I + II (maximum OxPhos) was determined after the addition of the FADH2 pathway substrate succinate. Subsequent injections of the uncoupler FCCP allowed obtaining the maximum respiratory activity in the uncoupled state (maximum uncoupled). Finally, the selective complex II activity (CII) was obtained at the end of the titration sequence by adding the complex I inhibitor rotenone in the maximum uncoupled state. In a final step, complex III was inhibited by the administration of antimycin A.
Mitochondrial Markers
Muscle samples were taken from the gluteus maximus by a fine-needle biopsy technique (see above) at baseline (1–2 weeks prior to intervention) and upon its completion (1–2 weeks post-intervention). Following membrane permeabilization by saponin, samples were immersed in 4% paraformaldehyde for 6 h and then transferred to 70% ethanol for preservation until paraffin embedding. Due to the small size of the sample following immersion, it was not organized in a specific orientation. Paraffin-embedded sections were de-paraffinized, rehydrated and washed in phosphate-buffered saline (PBS). Antigen retrieval was performed in 1 mM EDTA, pH 8.0 for 40 min. After blocking with 5% normal goat serum (Abcam, ab7481) supplemented by 2% BSA for one hour at RT, sections were incubated with primary antibodies for MNF1/2 (1:100), PGC-1 alpha (Abcam ab54481, 1:200) and OPA1 (Abcam ab157457, 1:300) overnight at 4 °C, followed by a secondary antibody for one hour at 4 °C, and followed by either goat anti-mouse or goat anti-rabbit secondary antibodies (Abcam 1:500) for one hour at RT. The sections were then mounted using Fluoroshield mounting medium with DAPI (ab104139). Control samples were exposed to only the secondary antibody to rule out unspecific staining.
Mitochondrial mass was evaluated with the MitoTracker Green FM dye (Molecular Probes) as previously described [19]. Briefly, following deparaffinization and rehydration, the sections were incubated for 30 min at room temperature with 100 nM MitoTracker Green FM followed by rinses with PBS.
Stained slides were imaged for fluorescence using a Lionheart™ FX Automated Fluorescent Microscope. Total intensity divided by total area in three random fields (for each experimental sample) was measured with Lionheart™ FX Gene 5 image analysis software. Fluorescence intensity values for each experimental group were averaged and presented as mean fluorescent intensity (MFI).
Pulmonary Function
Measurements of pulmonary functions were performed using the KoKo Sx1000 spirometer (Nspire Health, USA), 1–2 weeks prior to and after the last HBOT/SHAM session. The equipment was calibrated using a 3–l syringe before performing measurements according to the manufacturer’s instructions. Measurements were performed by a trained technician. The forced expiratory maneuvers were performed as recommended by the guidelines [19].
The forced vital capacity (FVC) forced expiratory volume in 1 s (FEV1), the Tiffeneau–Pinelli index (FEV1/FVC) and peak expiratory flow rate (PEF) were taken as the highest readings obtained from at least three satisfactory forced expiratory maneuvers. Mean forced mid-expiratory flow rate (FEF25–75%) and forced expiratory flow rates at 25, 50 and 75% of FVC expired (FEF25%, FEF50% and FEF75%) were taken as the best values from flow–volume loops not differing by > 5% from the highest FVC.
Body Composition
An Inbody720 body composition analyzer (Biospace Co., South Korea) was used to detect the human body composition based on recommendations provided in the user manual. The athletes’ bare feet stood on the pedal plate electrode, hands naturally hanging down and holding the hand electrode gently, and the angle between the trunk and upper limbs was maintained at ~ 15°. Indexes included basal metabolic rate, lean body weight, intracellular fluid, extracellular fluid, body water content, skeletal muscle, body fat, abdominal obesity, etc. Weight was measured in kilograms with the athletes barefoot in minimal clothing by a digital scale (Beurer, Germany), and height was measured in centimeters. BMI was calculated as the weight in kilograms divided by the square of the height in meters (kg/m2). Athletes underwent body composition analysis at baseline, 1–2 weeks prior to intervention and upon its completion, and 1–2 weeks post-intervention.
Physical Measurements
A trained physical therapist, who was masked from group allocation, performed physical measurements including range of motion, vertical jump, maximal quadriceps power, a step test and an agility test.
Safety
Athletes were monitored for adverse events including: barotraumas (either ear or sinuses) and oxygen toxicity (pulmonary and central nervous system).
Statistical Analysis
Continuous data are expressed as means ± standard-deviation. Normal distributions for all variables were tested using the Kolmogorov–Smirnov test. Unpaired and paired t tests were performed to compare variables between and within the two groups. Net effect sizes were evaluated using Cohen's d method.
Categorical data are expressed in numbers and percentages and were compared by chi-square tests. Univariate analyses were performed using chi-square/Fisher’s exact test to identify significant variables (P < 0.05).
To evaluate HBOT’s effects on physical performance, a within-subject repeated measures ANOVA model was used to test the main interaction effect between time and group. To evaluate HBOT’s effects on mitochondrial respiration, a within-subject analysis of covariance (ANCOVA) model was used to test the main interaction effect between time and group. The false discovery rate (FDR) method was used for multiple comparisons correction.
Due to non-normal distribution, mitochondrial mass markers were analyzed using the Mann–Whitney U test. Relative changes (in percentages) were calculated as post-intervention to baseline difference divided by the baseline and multiplied by 100.
Sample Size
Sample size was calculated based on Burgos et al. pilot study [16] on 12 young soccer players which showed a moderate effect size on VO2Max. A moderate effect size of 0.3 in VO2Max in a repeated measures ANOVA design, with a power of 85% and an alpha of 5%, requires 14 athletes in each arm. Adding a 15% dropout rate would require 32 subjects in total.