Optimized dose of hydrogen-enriched water with minocycline combination therapy in experimental ischemic stroke

Background: Ischemic stroke remains a leading cause of death and disability worldwide, with limited effective treatments due to the complexity of its pathophysiology. Molecular hydrogen (H₂) and minocycline (M), both possessing anti-inflammatory and antioxidant properties, have shown individual neuroprotective potential in preclinical models. However, the optimal therapeutic dosing of H₂, particularly in combination with other agents, remains undefined. Objective: This study aimed to (1) determine the dose–response relationship of hydrogen-enriched water in a rat model of transient middle cerebral artery occlusion (MCAO), and (2) evaluate whether optimized H₂ dosing combined with minocycline provides superior neuroprotection compared to H₂ monotherapy. Methods: Sixty-six male and female Sprague-Dawley rats underwent 60-minute MCAO followed by treatment with varying doses (5–30 mL/kg) of hydrogen-enriched water (3.2 ppm), alone or in combination with minocycline (20 mg/kg). Treatments were administered post-reperfusion as well as on days 1 and 2. Behavioral outcomes (Garcia score) and infarct volumes (TTC staining) were assessed at 7 days post-stroke. Results: The optimal H₂ dose was 20 mL/kg, which produced the highest Garcia scores and lowest infarct volumes. A dose-dependent effect was observed with a quadratic fit (R² = 0.751 for Garcia scores; R² = 0.289 for lesion volume). Combination therapy with H₂ and minocycline significantly outperformed H₂ monotherapy in both neurological recovery and infarct reduction, with no sex differences observed. Conclusion: Hydrogen-enriched water shows a dose-dependent neuroprotective effect in experimental ischemic stroke, with 20 mL/kg identified as the optimal dose. Combined therapy with minocycline further enhances outcomes, supporting the potential of dual-agent strategies for improved stroke treatment. These findings provide a foundation for translational development of H₂-based combination therapies in clinical settings.