Optimized dose of hydrogen-enriched water with minocycline combination therapy in experimental ischemic stroke
Jiang Z, Alamuri TT, Yang DL, Annarino T, Muir ER, Duong TQ. · Brain Research. 2025;1866:149940.
Study at a glance
Preclinical
Sixty-six male and female Sprague-Dawley rats subjected to 60-minute middle cerebral artery occlusion followed by reperfusion.
Hydrogen-enriched water by gastric gavage immediately after reperfusion and again on days 1 and 2; doses were 5, 10, 20 or 30 mL/kg. Combination groups also received minocycline 20 mg/kg intraperitoneally. · Near-saturated water was made with two magnesium-based Rejuvenation H2 tablets in 500 mL water for 24 hours at 4°C. Measured H₂ averaged 3.18 ± 0.21 ppm (range 2.7-3.9 ppm). Placebo tablets produced vehicle water.
Three administrations: immediately after reperfusion and on post-stroke days 1 and 2; neurological score and infarct volume were assessed on day 7.
The 20 mL/kg group had the best mean Garcia score and smallest mean infarct volume. In males, 20 mL/kg differed from vehicle and lower doses but not from 30 mL/kg for Garcia score (p=0.54); infarct volume differed from vehicle and 5 mL/kg, not every dose. H₂ plus minocycline produced better outcomes than H₂ alone; no sex difference was detected in the tested optimal-dose arms.
Preclinical pilot study; only the selected arms included females, the estrous cycle was not monitored, the 30 mL/kg group was unblinded and added after other results, treatment began immediately after reperfusion, and minocycline was tested at one fixed dose. Commercial H₂ and placebo tablets and a commercial H₂ test kit were used. NIH grants funded the study; no explicit conflict declaration was identified in the article, so absence of a conflict is not inferred.
What kind of evidence is this?
Preclinical
Randomized, vehicle-controlled, double-blinded rat dose-response study; the 30 mL/kg group was added later and was unblinded
Other neurological conditions
H₂-rich water
Information not yet classified
Some editorial classification fields are still pending. The source-reported outcomes and result are shown below; Hydrogenology does not infer a positive or negative signal from prose automatically.
Methods
Sixty-six male and female Sprague-Dawley rats subjected to 60-minute middle cerebral artery occlusion followed by reperfusion.
66 rats. Male dose groups: vehicle n=8, H₂ water 5 mL/kg n=6, 10 mL/kg n=6, 20 mL/kg n=8 and 30 mL/kg n=6; additional male H₂ 20 mL/kg plus minocycline n=8. Female vehicle, H₂ 20 mL/kg and combination groups each had n=8.
Three administrations: immediately after reperfusion and on post-stroke days 1 and 2; neurological score and infarct volume were assessed on day 7.
Hydrogen-enriched water by gastric gavage immediately after reperfusion and again on days 1 and 2; doses were 5, 10, 20 or 30 mL/kg. Combination groups also received minocycline 20 mg/kg intraperitoneally.
H₂ dissolved in water — H₂ only, not Brown's gas.
Near-saturated water was made with two magnesium-based Rejuvenation H2 tablets in 500 mL water for 24 hours at 4°C. Measured H₂ averaged 3.18 ± 0.21 ppm (range 2.7-3.9 ppm). Placebo tablets produced vehicle water.
Placebo-tablet vehicle water at matched fluid volume; H₂ dose groups and H₂-plus-minocycline groups.
Outcomes and reported result
Garcia neurological score and TTC-stained infarct volume at seven days, with dose-response modelling and sex comparisons.
The 20 mL/kg group had the best mean Garcia score and smallest mean infarct volume. In males, 20 mL/kg differed from vehicle and lower doses but not from 30 mL/kg for Garcia score (p=0.54); infarct volume differed from vehicle and 5 mL/kg, not every dose. H₂ plus minocycline produced better outcomes than H₂ alone; no sex difference was detected in the tested optimal-dose arms.
The complete free PMC article, allocations, intervention preparation, measured H₂ concentration, all dose groups, outcome table, null comparisons, stated limitations and funding were checked.
A reported association, difference or mechanism is not automatically a clinical benefit.
Limitations and applicability
Preclinical pilot study; only the selected arms included females, the estrous cycle was not monitored, the 30 mL/kg group was unblinded and added after other results, treatment began immediately after reperfusion, and minocycline was tested at one fixed dose. Commercial H₂ and placebo tablets and a commercial H₂ test kit were used. NIH grants funded the study; no explicit conflict declaration was identified in the article, so absence of a conflict is not inferred.
Experimental ischemic stroke in rats; it does not establish a human stroke treatment or dosing schedule.
No single-study GRADE certainty rating is assigned. Read how records and evidence assessments are prepared.
Sources and record status
PMID: 40935311 · DOI: 10.1016/j.brainres.2025.149940
Free full article in PubMed Central.
Complete PMC article and PubMed metadata; full-text extraction checked 9 August 2026.
10 August 2026
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