Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2026 Jan 8.
Published in final edited form as: Brain Res. 2025 Sep 9;1866:149940. doi: 10.1016/j.brainres.2025.149940

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

Zhao Jiang 1, Tharun T Alamuri 1, Darren L Yang 1, Tyler Annarino 2, Eric R Muir 2, Tim Q Duong 3
PMCID: PMC12779082  NIHMSID: NIHMS2126663  PMID: 40935311

Abstract

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.

Keywords: stroke, ischemia, molecular hydrogen, minocycline, neuroprotection, rats

1. Introduction

Stroke is one of the leading causes of death in the US and worldwide (1). Much of the difficulty in identifying effective treatments for stroke has come from the complexity and multiplicity in pathophysiology. Many single agent treatments have been tested in preclinical and clinical experiments, including antioxidants like molecular hydrogen (H2) and minocycline (M). Free radical species are downstream mediators of inflammation, excitotoxicity, and other cytotoxic cascades that all contribute to ischemic brain injury (2). Furthermore, such species increase during reperfusion, potentially leading to further injury. While many candidate drugs to date have shown suboptimal results in clinical trials, H2 and minocycline have shown promising results in recent years. Numerous experimental studies (3–8) and a clinical study (9) have shown H2 to be capable of reducing lesion volume and neurological deficits (10), providing cytoprotection beyond ischemia and in organs other than the brain (11), and protecting against inflammation and apoptosis (12). H2 likely works by scavenging reactive species involved with pathological oxidative stress (3) and may also reduce inflammation and apoptosis (12). Minocycline has a similar profile of anti-inflammatory and anti-apoptotic effects in experimental models of stroke (13, 14) via activation of NLRP3 (NOS-, LRR-, and pyrin domain-containing protein 3) (15, 16) and inactivation of PARP (poly-ADP-ribose-polymerase) (17) and MMP-9 (matrix metalloproteinase-9) (18). NLRP3 3), PARP, and MMP-9 are each proteins involved in the inflammatory and apoptosis pathways. Minocycline has been found to be neuroprotective in stroke animal models (13) and have marginal efficacy in a clinical trial (19).

However, effective neuroprotection in clinical stroke has remained a major challenge. One possible reason may be due to the multiplicity of ischemic injury pathways in stroke. Previous research has shown the efficacy of combination therapy in the treatment of stroke within rat models (20), while most clinical drug trials and many experimental studies have tested single agents. For example, combination of neuroprotective YM872, an AMPA receptor antagonist, and thrombolytic t-PA has been found to give better outcomes than t-PA alone (21). Combination therapy with H2 or with minocycline has been previously reported, as we recently reported that the combination therapy of H2 and minocycline was effective in improving outcomes (reducing infarct volume, improving neurologic function, attenuating hyperperfusion, and reducing white matter injury) in a rodent stroke model (22). Despite these promising findings, the optimal parameters for using H2 in clinical or preclinical stroke therapy remain poorly defined. In particular, the dose-response relationship of H2 has not been systematically investigated, making it difficult to determine the most effective concentration or duration of treatment. This gap in knowledge hinders efforts to translate preclinical success into clinical application and may partly explain the variability in outcomes across studies. Optimizing the dose response is important as it could improve future outcomes.

A deeper understanding of the dose-response effects of H2 will not only enhance its standalone efficacy but may also inform more effective combination regimens with agents like minocycline. This study was therefore designed with two goals: (1) to establish the dose-response profile of H2 in a well-established rat model of transient middle cerebral artery occlusion (MCAO), and (2) to evaluate whether the optimized H2 dose in combination with minocycline provides superior neuroprotection compared to H2 monotherapy. Treatments were administered at clinically relevant timepoints, immediately after reperfusion and again on post-stroke days 1 and 2, to simulate realistic treatment windows. Primary outcomes included behavioral and neurological assessments, as well as histological measurement of infarct volume at 7 days post-stroke. We hypothesized that H2 would display dose-dependent neuroprotective effects, and that its combination with minocycline at an optimal dose would provide greater benefit than H2 alone.

2. Methods

2.1. Experimental design

Animal experiments were approved by Institutional Animal Care and Use Committee of Stony Brook University. Randomized, vehicle-controlled and double-blinded designs and guidelines of good laboratory practice were strictly followed to prevent bias (23, 24). Male and female Sprague-Dawley rats (250-350g, Charles River Laboratories, Wilmington, MA) were housed in a 12-hour dark and 12-hour light cycle. Rats had ad libitum access to irradiated rodent chow and autoclaved water.

Ischemic stroke was induced using a 60-min MCAO model for determining dose response curves for H2 and H2M. Focal brain ischemia was induced under 1.5-2.0% isoflurane using the intraluminal suture occlusion method via reversed access from the external carotid artery (25, 26). For reperfusion, rats were moved out of the scanner and the occluder withdrawn from the external carotid artery so blood flow from the common carotid artery to the brain was restored. The rectal temperature was maintained at 37.0±0.5º C. Respiration rate (via force transducer) was monitored while the animal was in the magnet.

2.2. Hydrogen and minocycline treatment:

We applied treatments directly after reperfusion, followed by daily treatments 1 and 2 days after MCAO. H2 (or vehicle control) was administered by gavage. Minocycline (or vehicle control) was administered via intraperitoneal injection.

H2:

Near-saturated hydrogen-enriched water (2.8–3.5 ppm) was prepared using magnesium-based hydrogen-producing tablets (Rejuvenation H2 tablets, Drink HRW, Oxnard, CA). Twenty-four hours prior to treatment, two tablets were dissolved in 500 mL of water within a sealed, fully-filled 500 mL plastic container at 4°Cto create pressurized hydrogen-enriched water. The dissolved hydrogen concentration was verified prior to each use with a hydrogen test kit (H2Blue, H2 Sciences Inc., Henderson, NV). The average concentration of H2 water was 3.18 ± 0.21 ppm (range from 2.7 to 3.9 ppm). To minimize hydrogen dissipation, the water was promptly administered via gastric gavage. Based on prior studies and preliminary data, doses of hydrogen-enriched water of 5, 10, 20, and 30 mL/kg were explored. The group receiving 30 mL/kg was added on based on results from the other groups, and were thus done unblinded. Vehicle control was produced similarly but using placebo tablets (Drink HRW). All rats were given the same total dose of fluid (20 mL/kg, except for the 30 mL/kg group) by giving a needed volume of vehicle control water in addition to the H2 water.

H2+Minocycline:

Previous studies demonstrated that neuroprotective doses of minocycline align with antibiotic dosing in both preclinical and clinical studies (27). Minocycline dose was not optimized since its dose-response curve has already been well established by previous studies. A 20 mg/kg dose corresponds to achieving neuroprotective plasma levels reported in rats with transient focal ischemia (28, 29). Therefore, to enhance the anti-inflammatory and antioxidant effects of hydrogen in combination therapy, a dose of minocycline of 20 mg/kg was selected for this study.

A total of n=66 rats were employed in this study and grouped as below, i) H2-water dose optimization (male rats only; n=6 for 5, 10, and 30 mL/kg groups; n=8 for vehicle-control and 20 mL/kg groups); ii) optimal H2-water dose and combined H2M (n=8 additional males with 20 mL/kg H2 + minocycline; n=8 for females for vehicle-control, 20 mL/kg H2, and 20 mL/kg H2 + minocycline groups).

2.3. Behavioral and histological outcomes:

The outcomes to determine efficacy of the different treatments were neurological Garcia scores and histological infarct volume at 7 days after stroke. The Garcia scores evaluate spontaneous activity, limb movements, trunk responses, and vibrissae sensitivity, with scores ranging from 6 (severe deficits) to 18 (normal). Fresh brain tissue was then collected and sliced for 2,3,5-Triphenyltetrazolium Chloride (TTC) staining. Infarct volume was manually measured from TTC-stained image as described previously (30).

2.4. Statistical analysis

Data in text and plots are given as mean ± standard deviation (SD). Statistical analysis was performed using R (version 4.3.3). Q-Q plots were used to assess notable deviation from normality, and non-parametric tests were used for data with potential non-normal distributions. To assess differences among H2 dose in male mice, a one-way ANOVA was used for Garcia scores and a Kruskal-Wallis rank sum test was used for lesion volumes. A two-way ANOVA was used to compare between males and females and between controls, optimal H2 dose, and H2M combination. Post hoc analysis was done using t-tests or Wilcoxon rank sum tests with Benjamini-Hochberg correction to compare between groups. Regression was performed to fit a quadratic curve to the Garcia scores and lesion volumes as a function of H2 dose. A p-value < 0.05 was considered significant.

3. Results

3.1. H2 Dose Response Curves

Averaged results for all groups are summarized in Table 1. The highest mean Garcia score was seen in the group receiving 20 mL/kg of H2-enriched water. As seen in Figure 1, Garcia score increased with H2 dose in the males, reaching a peak of 12.0 ± 0.8 in the 20 mL/kg group and slightly dipping to 11.7 ± 1.2 in the 30 mL/kg group. H2 treatment significantly improved Garcia scores (p=3.2E-8, ANOVA). The Garcia score in males was significantly higher in the 20 mL/kg group compared to the 0, 5, and 10 mL/kg groups (p<0.01, t-tests comparing optimal dose with all other H2 doses) but was not significantly different from the 30 mL/kg group (p=0.54, t-test). The dose response curve, including males and females, had an R2 coefficient of 0.751 with a quadratic fit (Figure 2), with a maximum at 23 mL/kg.

Table 1.

Group averaged Garcia scores and lesion volumes (mean±SD).

Sex Treatment Group n Garcia score Lesion volume (mm3)
Male Control 8 8.50 ± 0.53 138.52 ± 47.79
H2, 5mL/kg 6 10.00 ± 0.89 131.79 ± 69.11
H2, 10mL/kg 6 10.67 ± 0.82 101.94 ± 37.24
H2, 20mL/kg 8 12.00 ± 0.76 67.16 ± 29.01
H2, 30mL/kg 6 11.67 ± 1.21 92.39 ± 24.28
H2M 8 13.25 ± 1.28 42.67 ± 11.73
Female Control 8 8.63 ± 0.92 125.96 ± 30.32
H2, 20mL/kg 8 11.88 ± 0.99 82.04 ± 25.00
H2M 8 13.00 ± 0.93 58.26 ± 17.79
Open in a new tab

Figure 1.

Figure 1.

Open in a new tab

Group averaged neurological Garcia scores at seven days after stroke induction, showing the dose response of hydrogen-enriched water treatment. Mean±SD. *p<0.05 compared to 20 mL/kg group in males from post-hoc test.

Figure 2.

Figure 2.

Open in a new tab

Hydrogen-enriched water dose-response curve of Garcia scores. A quadratic fit of the data is shown (from combined males and females).

The smallest mean lesion volume was also seen in the group receiving 20 mL/kg. As shown in Figure 3 and Figure 4, lesion volume decreased with H2 dose in the males, reaching a low of 67.2 ± 29.0 mm3 in the 20 mL/kg group and slightly increasing to 92.4 ± 24.3 mm3 in the 30 mL/kg group. H2 treatment significantly reduced the lesion volume (p= 0.013, Kruskal-Wallis). Lesion volume of the 20 mL/kg group was significantly different than the 0 and 5 mL/kg groups (p<0.05, Wilcoxon rank sum tests comparing optimal dose with all other H2 doses). The dose response curve including males and females showed a R2 coefficient of 0.289 with a quadratic fit (Figure 5), with a minimum at 22 mL/kg.

Figure 3.

Figure 3.

Open in a new tab

Example TTC-stained images for males (M) and females (F) from each treatment group. H2 dose or H2M group are indicated.

Figure 4.

Figure 4.

Open in a new tab

Group averaged stroke lesion volume at seven days after stroke induction, showing the dose response of hydrogen-enriched water treatment. Mean±SD. *p<0.05 compared to 20 mL/kg group in males from post-hoc test.

Figure 5.

Figure 5.

Open in a new tab

Hydrogen-enriched water dose-response curve of stroke lesion volumes. A quadratic fit of the data is shown (from combined males and females).

3.2. H2M

H2M combination provided further improvement of Garcia score compared to optimal H2 treatment. Comparing controls, 20 mL/kg H2 treatment, and 20 mL/kg H2 + M treatment, there were significant differences in Garcia scores (p<2E-16, ANOVA) and lesion volumes (p=2.4E-9, ANOVA). There were no significant differences between males and females or interactions between treatment and sex for Garcia score (p>0.75, 2-way ANOVA) and lesion volume (p>0.30, 2-way ANOVA). As such, males and females were combined for post hoc comparisons between groups (Figure 6). There were significant differences of Garcia scores (p<0.01, t-tests) and lesion volumes (p<0.01, t-tests) between any two groups, with the H2M group showing most improved outcomes for both measures.

Figure 6.

Figure 6.

Open in a new tab

Group averaged outcomes from control, optimal H2 dose, and H2M groups for A) Garcia scores and B) lesion volumes. Mean±SD. *p<0.05 compared to control group, #p<0.5 compared to H2 group, post-hoc tests of combined males and females.

4. Discussion

This double-blinded study applied behavioral measurements and histological infarct volumes to assess the progressive treatment effects of H2 water and H2 combination therapy with minocycline, and to additionally identify the optimal doses of H2. It was hypothesized that optimal H2M dose has superior efficacy compared to optimal M or H2 dose alone. The primary findings were: i) highest mean Garcia score and lowest mean lesion volume were observed in the 20 mL/kg H2 water group, and ii) 20 mL/kg H2+M treatment further improved mean Garcia score and lesion volume when compared to 20 mL/kg H2 alone. To our knowledge, this is the first study to analyze the dose response curve of H2 combined with another potential stroke therapeutic.

4.1. Dose Response

Consistent with previous studies (3, 12, 22), a decrease in lesion volume and an increase in Garcia score was observed with H2 alone and with H2M. Minocycline by itself has also been shown to have neuroprotective effects (28, 31, 32). It is likely that the increase in Garcia score was in part due to the decrease in lesion volume. Our previous study (22) also found beneficial effects on cerebral blood flow and white matter integrity with H2M compared to H2 alone. Although not directly analyzed in this study, prevention of hyperperfusion and reperfusion injury as well as reduction in white matter damage, observed by improved diffusion MRI parameters, is likely responsible. On the cellular level, this is likely due to the many cytoprotective and anti-inflammatory properties of both H2 and minocycline.

The greatest neuroprotective effects for both lesion volume reduction and Garcia score improvement were observed in the group receiving 20 mL/kg H2-enriched water. Li et al. previously studied the effect of hydrogen-rich saline (0.85 mM) on stroke model in a dose-dependent manner, finding that 10 mL/kg H2 had greater neuroprotective effects than 5 mL/kg H2 (33). To our knowledge, however, no other studies have elucidated the dose-response relationship for H2. Our findings suggest that a higher dose of up to 20 mL/kg H2 with a higher H2 concentration of 3.18 ppm may provide greater therapeutic benefits, while an even higher dose of 30 mL/kg showed no further benefit and displayed a small, non-significant trend of worse outcomes for both neurological score and lesion volume. The volume of water administered at the higher doses is large for rats, so it is useful to show that the efficacy peaks or plateaus prior to the maximum dose given. Minocycline has also been found to exert its effects in a dose-dependent manner. A meta-analysis of studies of various rodent neurological disease models found that, for rat stroke models, a moderate dose of around 45 mg/kg is most effective compared to higher and lower doses (34). However, a dose of 20 mg/kg has also been found to achieve significant results (29); thus a dose of 20 mg/kg was used in this study to explore the further benefit of combination therapy without allowing minocycline to overpower the effects of H2. While those receiving lower doses achieved suboptimal results due to less medication in their systems, those receiving higher doses may have achieved suboptimal results due to toxicity or other drug interactions. However, this would need to be further analyzed on a molecular level to test such hypotheses.

4.2. Limitations and future perspectives

The limitations of this study are: i) This pilot study employed only a single MCAO duration of 60 min with treatment administered immediately after reperfusion as well as 1 and 2 days after stroke onset. Future studies will need to investigate different occlusion durations, possible delayed treatment effects, and possible rebound edema after ending treatment after day 2. ii) It has been shown that changes in hormone levels over the estrous cycle in rats can affect stroke outcomes (35, 36). The estrous cycle in females was not monitored herein, which may have increased variability in the outcomes. Results in males and females were highly consistent, suggesting this had minimal effects herein. iii) As the dosing study requires many groups and thus large N, we decided to conduct initial dose-response studies on males only to reduce animal use. The results of the control, optimal H2-only, and H2+minocycline analysis strongly supported a lack of sex-based differences for H2/H2M treatment in this stroke model, suggesting this was a reasonable decision. iv) A fixed dose of minocycline was used in combination with varying H2 doses. Future studies will need to employ varying doses of minocycline to determine optimal dosing. v) The molecular mechanisms of H2 and minocycline alone have been previously examined individually, but the mechanisms of combined H2M neuroprotection have not been studied. Future studies will need to confirm if minocycline still provides anti-inflammation and anti-apoptotic effects when combined with H2. Multiparametric MRI evaluation to assess short- and long-term tissue outcomes are planned similar to those reported here (37–40). vi) Our study considers only minocycline as a potential target for combination therapy. Other studies have also reported neuroprotective effects with agents such as dimethyl sulfoxide and methylene blue in models of ischemic stroke (41, 42); therefore, combination therapy with H2 and other therapeutics may have even greater neuroprotective benefits.

5. Conclusion

This is the first study to analyze the dose response curve of H2 enriched water combined with another potential stroke therapeutic. The H2 dose response showed the greatest efficacy to improve neurological score and reduce lesion volume at a dose of 20 mL/kg of 3.2 ppm concentration H2 water, with the efficacy plateauing at higher dose. Additionally, minocycline combined with the optimal H2 dose showed further significant improvement of neurological score and lesion volume. Combination treatment with complementary neuroprotective drugs may thus provide improved outcomes compared to single-drug therapies. Both hydrogen and minocycline have proven safety profiles in humans and could be readily deployed in acute settings, especially if optimal dosing is determined.

Funding:

This work was supported by the National Institutes of Health (grant numbers R01-NS045879 and R01-NS129936).

References

  • 1.Martin SS, Aday AW, Almarzooq ZI, Anderson CAM, Arora P, Avery CL, et al. 2024 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation. 2024;149(8):e347–e913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Chan PH. Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab. 2001;21(1):2–14. [DOI] [PubMed] [Google Scholar]
  • 3.Ohsawa I, Ishikawa M, Takahashi K, Watanabe M, Nishimaki K, Yamagata K, et al. Hydrogen acts as a therapeutic antioxidant by selectively reducing cytotoxic oxygen radicals. Nat Med. 2007;13(6):688–94. [DOI] [PubMed] [Google Scholar]
  • 4.Cui Y, Zhang H, Ji M, Jia M, Chen H, Yang J, et al. Hydrogen-rich saline attenuates neuronal ischemia--reperfusion injury by protecting mitochondrial function in rats. J Surg Res. 2014;192(2):564–72. [DOI] [PubMed] [Google Scholar]
  • 5.Hugyecz M, Mracsko E, Hertelendy P, Farkas E, Domoki F, Bari F. Hydrogen supplemented air inhalation reduces changes of prooxidant enzyme and gap junction protein levels after transient global cerebral ischemia in the rat hippocampus. Brain Res. 2011;1404:31–8. [DOI] [PubMed] [Google Scholar]
  • 6.Li F, Gong Q, Wang L, Shi J. Osthole attenuates focal inflammatory reaction following permanent middle cerebral artery occlusion in rats. Biol Pharm Bull. 2012;35(10):1686–90. [DOI] [PubMed] [Google Scholar]
  • 7.Liu Y, Liu W, Sun X, Li R, Sun Q, Cai J, et al. Hydrogen saline offers neuroprotection by reducing oxidative stress in a focal cerebral ischemia-reperfusion rat model. Med Gas Res. 2011;1(1):15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Nemeth J, Toth-Szuki V, Varga V, Kovacs V, Remzso G, Domoki F. Molecular hydrogen affords neuroprotection in a translational piglet model of hypoxic-ischemic encephalopathy. J Physiol Pharmacol. 2016;67(5):677–89. [PubMed] [Google Scholar]
  • 9.Ono H, Nishijima Y, Ohta S, Sakamoto M, Kinone K, Horikosi T, et al. Hydrogen Gas Inhalation Treatment in Acute Cerebral Infarction: A Randomized Controlled Clinical Study on Safety and Neuroprotection. J Stroke Cerebrovasc Dis. 2017;26(11):2587–94. [DOI] [PubMed] [Google Scholar]
  • 10.Ohta S Molecular hydrogen as a preventive and therapeutic medical gas: initiation, development and potential of hydrogen medicine. Pharmacol Ther. 2014;144(1):1–11. [DOI] [PubMed] [Google Scholar]
  • 11.Zhang G, Li Z, Meng C, Kang J, Zhang M, Ma L, et al. The Anti-inflammatory Effect of Hydrogen on Lung Transplantation Model of Pulmonary Microvascular Endothelial Cells During Cold Storage Period. Transplantation. 2018;102(8):1253–61. [DOI] [PubMed] [Google Scholar]
  • 12.Li H, Luo Y, Yang P, Liu J. Hydrogen as a complementary therapy against ischemic stroke: A review of the evidence. J Neurol Sci. 2019;396:240–6. [DOI] [PubMed] [Google Scholar]
  • 13.Yrjanheikki J, Tikka T, Keinanen R, Goldsteins G, Chan PH, Koistinaho J. A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window. Proc Natl Acad Sci U S A. 1999;96(23):13496–500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hewlett KA, Corbett D. Delayed minocycline treatment reduces long-term functional deficits and histological injury in a rodent model of focal ischemia. Neuroscience. 2006;141(1):27–33. [DOI] [PubMed] [Google Scholar]
  • 15.Lu Y, Xiao G, Luo W. Minocycline Suppresses NLRP3 Inflammasome Activation in Experimental Ischemic Stroke. Neuroimmunomodulation. 2016;23(4):230–8. [DOI] [PubMed] [Google Scholar]
  • 16.Xu L, Fagan SC, Waller JL, Edwards D, Borlongan CV, Zheng J, et al. Low dose intravenous minocycline is neuroprotective after middle cerebral artery occlusion-reperfusion in rats. BMC Neurol. 2004;4:7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dawson TM, Dawson VL. Nitric Oxide Signaling in Neurodegeneration and Cell Death. Adv Pharmacol. 2018;82:57–83. [DOI] [PubMed] [Google Scholar]
  • 18.Chaturvedi M, Kaczmarek L. Mmp-9 inhibition: a therapeutic strategy in ischemic stroke. Mol Neurobiol. 2014;49(1):563–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Malhotra K, Chang JJ, Khunger A, Blacker D, Switzer JA, Goyal N, et al. Minocycline for acute stroke treatment: a systematic review and meta-analysis of randomized clinical trials. J Neurol. 2018;265(8):1871–9. [DOI] [PubMed] [Google Scholar]
  • 20.O’Collins VE, Macleod MR, Donnan GA, Howells DW. Evaluation of combination therapy in animal models of cerebral ischemia. J Cereb Blood Flow Metab. 2012;32(4):585–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Suzuki M, Sasamata M, Miyata K. Neuroprotective effects of YM872 coadministered with t-PA in a rat embolic stroke model. Brain Res. 2003;959(1):169–72. [DOI] [PubMed] [Google Scholar]
  • 22.Jiang Z, Alamuri TT, Muir ER, Choi DW, Duong TQ. Longitudinal multiparametric MRI study of hydrogen-enriched water with minocycline combination therapy in experimental ischemic stroke in rats. Brain Res. 2020;1748:147122. [DOI] [PubMed] [Google Scholar]
  • 23.Macleod MR, Fisher M, O’Collins V, Sena ES, Dirnagl U, Bath PM, et al. Good laboratory practice: preventing introduction of bias at the bench. Stroke. 2009;40(3):e50–2. [DOI] [PubMed] [Google Scholar]
  • 24.Duong TQ, Fisher M. Applications of diffusion/perfusion magnetic resonance imaging in experimental and clinical aspects of stroke. Curr Atheroscler Rep. 2004;6(4):267–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Meng X, Fisher M, Shen Q, Sotak CH, Duong TQ. Characterizing the diffusion/perfusion mismatch in experimental focal cerebral ischemia. Ann Neurol. 2004;55:207–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Shen Q, Meng X, Fisher M, Sotak CH, Duong TQ. Pixel-by-pixel spatiotemporal progression of focal ischemia derived using quantitative perfusion and diffusion imaging. J Cereb Blood Flow and Metab. 2003;23:1479–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Fagan SC, Waller JL, Nichols FT, Edwards DJ, Pettigrew LC, Clark WM, et al. Minocycline to improve neurologic outcome in stroke (MINOS): a dose-finding study. Stroke. 2010;41(10):2283–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Naderi Y, Sabetkasaei M, Parvardeh S, Zanjani TM. Neuroprotective effect of minocycline on cognitive impairments induced by transient cerebral ischemia/reperfusion through its anti-inflammatory and anti-oxidant properties in male rat. Brain Res Bull. 2017;131:207–13. [DOI] [PubMed] [Google Scholar]
  • 29.Parvardeh S, Sheikholeslami MA, Ghafghazi S, Pouriran R, Mortazavi SE. Minocycline Improves Memory by Enhancing Hippocampal Synaptic Plasticity and Restoring Antioxidant Enzyme Activity in a Rat Model of Cerebral Ischemia-Reperfusion. Basic Clin Neurosci. 2022;13(2):225–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Jiang Z, Chen C-H, Chen Y-Y, Han J-Y, Riley J, Zhou C-M. Autophagic effect of programmed cell death 5 (PDCD5) after focal cerebral ischemic reperfusion injury in rats. Neuroscience letters. 2014;566:298–303. [DOI] [PubMed] [Google Scholar]
  • 31.Tanaka M, Ishihara Y, Mizuno S, Ishida A, Vogel CF, Tsuji M, et al. Progression of vasogenic edema induced by activated microglia under permanent middle cerebral artery occlusion. Biochem Biophys Res Commun. 2018;496(2):582–7. [DOI] [PubMed] [Google Scholar]
  • 32.Jin Z, Liang J, Wang J, Kolattukudy PE. MCP-induced protein 1 mediates the minocycline-induced neuroprotection against cerebral ischemia/reperfusion injury in vitro and in vivo. J Neuroinflammation. 2015;12:39. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Li J, Dong Y, Chen H, Han H, Yu Y, Wang G, et al. Protective effects of hydrogen-rich saline in a rat model of permanent focal cerebral ischemia via reducing oxidative stress and inflammatory cytokines. Brain Res. 2012;1486:103–11. [DOI] [PubMed] [Google Scholar]
  • 34.Li C, Yuan K, Schluesener H. Impact of minocycline on neurodegenerative diseases in rodents: a meta-analysis. Rev Neurosci. 2013;24(5):553–62. [DOI] [PubMed] [Google Scholar]
  • 35.Branyan TE, Aleksa J, Lepe E, Kosel K, Sohrabji F. The aging ovary impairs acute stroke outcomes. J Neuroinflammation. 2023;20(1):159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Carswell HV, Dominiczak AF, Macrae IM. Estrogen status affects sensitivity to focal cerebral ischemia in stroke-prone spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol. 2000;278(1):H290–4. [DOI] [PubMed] [Google Scholar]
  • 37.Shen Q, Ren H, Fisher M, Bouley J, Duong TQ. Dynamic tracking of acute ischemic tissue fates using improved unsupervised ISODATA analysis of high-resolution quantitative perfusion and diffusion data. J Cereb Blood Flow Metab. 2004;24(8):887–97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Huang S, Shen Q, Duong TQ. Artificial neural network prediction of ischemic tissue fate in acute stroke imaging. J Cereb Blood Flow Metab. 2010;30(9):1661–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Tanaka Y, Nagaoka T, Nair G, Ohno K, Duong TQ. Arterial spin labeling and dynamic susceptibility contrast CBF MRI in postischemic hyperperfusion, hypercapnia, and after mannitol injection. J Cereb Blood Flow Metab. 2011;31(6):1403–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Shih YY, Wey HY, De La Garza BH, Duong TQ. Striatal and cortical BOLD, blood flow, blood volume, oxygen consumption, and glucose consumption changes in noxious forepaw electrical stimulation. J Cereb Blood Flow Metab. 2011;31(3):832–41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Shen Q, Du F, Huang S, Rodriguez P, Watts LT, Duong TQ. Neuroprotective efficacy of methylene blue in ischemic stroke: an MRI study. PLoS One. 2013;8(11):e79833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Bardutzky J, Meng X, Bouley J, Duong TQ, Ratan R, Fisher M. Effects of intravenous dimethyl sulfoxide on ischemia evolution in a rat permanent occlusion model. J Cereb Blood Flow Metab. 2005;25(8):968–77. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES