Effect of Systemic Ozone Therapy on Corneal Endothelium (OZON-CORNEA)

STUDY DESIGN AND PARTICIPANTS

This is a prospective controlled cohort study conducted at [Institution Name]. The study protocol was approved by the Local Ethics Committee and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent.

The ozone group comprised 48 consecutive patients scheduled to receive systemic ozone therapy for various indications including fibromyalgia (16.7%), chronic pain (22.9%), knee osteoarthritis (29.2%), disk herniation (10.4%), peripheral arterial disease (6.3%), and sports injuries (14.6%). The control group consisted of 48 healthy individuals matched for age (±2 years), sex, and body mass index (BMI, ±3 kg/m²) who did not receive ozone therapy.

Exclusion criteria for both groups included: (1) history of ocular surgery or trauma, (2) corneal dystrophy or degeneration, (3) glaucoma or ocular hypertension, (4) diabetes mellitus, (5) contact lens use within the preceding 3 months, (6) active ocular surface disease, and (7) systemic conditions known to affect corneal endothelium.

OZONE THERAPY PROTOCOL

Systemic ozone therapy was administered via major autohemotherapy (MAH) using a medical-grade ozone generator (Ozonosan Alpha Plus, Herrmann Apparatebau GmbH, Germany). Approximately 100 mL of the patient's blood was drawn into a sterile glass bottle containing 3.8% sodium citrate as an anticoagulant. The blood was then exposed to a precisely measured ozone-oxygen mixture at a concentration of 40 µg/mL. The ozonated blood was gently mixed and reinfused intravenously over 10-15 minutes. This procedure was performed once daily for 10 consecutive sessions.

OPHTHALMIC EXAMINATIONS

All participants underwent comprehensive ophthalmic examinations including: slit-lamp biomicroscopy, best-corrected visual acuity (BCVA) measurement using Snellen chart (converted to logMAR), intraocular pressure (IOP) measurement by non-contact tonometry (Topcon CT-80), and corneal endothelial evaluation by non-contact specular microscopy (Topcon SP-3000P, Japan).

Specular microscopy was performed by a single experienced technician blinded to group allocation. Three images were captured from the central cornea of each eye, and the image with the highest cell count and clarity was selected for analysis. The following parameters were recorded: endothelial cell density (ECD, cells/mm²), percentage of hexagonal cells (Hex%), coefficient of variation in cell area (CV, %), and central corneal thickness (CCT, µm). Measurements were obtained at four time points: baseline (T0, before ozone therapy), 1 month (T1, immediately after completing 10 sessions), 3 months (T2), and 6 months (T3) after baseline.

BIOCHEMICAL ANALYSIS

In the ozone group, venous blood samples were collected at each time point. Serum levels of malondialdehyde (MDA, µmol/L) were measured by the thiobarbituric acid reactive substances (TBARS) method. Superoxide dismutase (SOD, U/mL) and glutathione peroxidase (GSH-Px, U/mL) activities were determined by spectrophotometric methods.

STATISTICAL ANALYSIS

Statistical analysis was performed using SPSS version 26.0 and Python 3.11 with SciPy and StatsModels libraries. Normality was assessed using the Shapiro-Wilk test. Between-group comparisons at baseline were performed using independent t-test or Mann-Whitney U test. Within-group changes over time were analyzed using repeated measures ANOVA or Friedman test, followed by Wilcoxon signed-rank test. Between-group comparisons of changes from baseline (delta values) were performed using independent t-test or Mann-Whitney U test. Effect sizes were calculated as Cohen's d and r (Z/√N). Statistical significance was set at p < 0.05.

PRIMARY OUTCOME MEASURES

• Change in endothelial cell density (ECD) from baseline to 6 months
• Change in hexagonality (Hex%) from baseline to 6 months
• Change in coefficient of variation (CV) from baseline to 6 months
• Change in central corneal thickness (CCT) from baseline to 6 months
• Change in intraocular pressure (IOP) from baseline to 6 months

SECONDARY OUTCOME MEASURES

• Change in malondialdehyde (MDA) levels from baseline to 6 months (ozone group only)
• Change in superoxide dismutase (SOD) levels from baseline to 6 months (ozone group only)
• Change in glutathione peroxidase (GSH-Px) levels from baseline to 6 months (ozone group only)
• Correlation between changes in oxidative stress markers and corneal endothelial parameters
• Subgroup analyses by age, sex, BMI, and ozone therapy indications

FOLLOW-UP SCHEDULE

• T0: Baseline assessment (before ozone therapy)
• T1: 1 month after baseline (immediately after completing 10 ozone sessions)
• T2: 3 months after baseline
• T3: 6 months after baseline (primary endpoint)

SAFETY MONITORING

All adverse events were documented throughout the study. Slit-lamp biomicroscopy was performed at each visit to detect any ocular abnormalities. Best-corrected visual acuity was monitored to ensure no visual deterioration occurred.