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.