Tag Archives: stres oxidativ

dissolved hydrogen for peritoneal dialysis patients to suppress oxidative stress in the peritoneal cavity

Abstract

Background

Oxidative stress (OS) related to glucose degradation products such as methylglyoxal is reportedly associated with peritoneal deterioration in patients treated with peritoneal dialysis (PD). However, the use of general antioxidant agents is limited due to their harmful effects. This study aimed to clarify the influence of the novel antioxidant molecular hydrogen (H2) on peritoneal OS using albumin redox state as a marker.

Methods

Effluent and blood samples of 6 regular PD patients were obtained during the peritoneal equilibrium test using standard dialysate and hydrogen-enriched dialysate. The redox state of albumin in effluent and blood was determined using high-performance liquid chromatography.

Results

Mean proportion of reduced albumin (ƒ(HMA)) in effluent was significantly higher in H2-enriched dialysate (62.31 ± 11.10%) than in standard dialysate (54.70 ± 13.08%). Likewise, serum ƒ(HMA) after administration of hydrogen-enriched dialysate (65.75 ± 7.52%) was significantly higher than that after standard dialysate (62.44 ± 7.66%).

Conclusions

Trans-peritoneal administration of H2 reduces peritoneal and systemic OS.

Background

Peritoneal deterioration is one of the most serious complications of peritoneal dialysis (PD) therapy, leading to ultrafiltration failure and the more severe complication of encapsulating peritoneal sclerosis (EPS). As the duration of PD increases, so does the risk of peritoneal deterioration []. More than 40% of patients in Japan who were on PD treatment for longer than 8 years stopped it due to the progression of peritoneal damage []. The pathological mechanisms of peritoneal damage are multi-factorial, but accumulated data have revealed the critical role of glucose degradation end-products (GDPs), i.e., chemically reactive carbonyl compounds. Methylglyoxal (MG) is one of the representative toxic GDPs, causing detrimental effects due to its rapid and indiscriminate oxidative nature [], and its production of toxic reactive oxygen species (ROS) such as hydroxyl radical, methyl radical, and undetermined carbon-centered radicals []. These used to be present in conventional dialysate, and also enter into the dialysate from uremic plasma []. Bio-compatible low-GDP dialysate is currently available, but a Japanese multicenter nationwide study, the NEXT-PD study [], revealed the occurrence of EPS even with the use of low-GDP solutions [under submission]. This indicates the need for novel therapeutic approaches to suppress possible insults from enhanced oxidative stress (OS) due to uremic oxidants in the peritoneal cavity.

Recently, the novel role of molecular hydrogen (H2) as an antioxidant has been revealed. H2 eliminates the hydroxyl radical in cultured cells and living organisms []. Interestingly, H2 does not influence other ROS, including superoxide, peroxide, and nitric oxide; these ROS play important physiological roles in body []. In humans, the safety of H2 has been tested, particularly in the field of deep diving. In contrast to general drugs, which usually have some harmful effects, no toxicity was found even at high concentrations of H2[]. H2 thus has therapeutic potential for pathological states related to ROS [].

The present study tested the effects of peritoneal dialysate containing a high concentration of molecular hydrogen (H2-enriched dialysate) as a novel anti-oxidant among patients treated with PD. As a result, we demonstrated that the use of hydrogen-enriched dialysate could reduce not only peritoneal, but also systemic OS in clinical settings.

Methods

Preparation of hydrogen-enriched dialysate

Hydrogen-enriched dialysate was prepared using MiZ nondestructive hydrogen dissolver (MiZ, Kanagawa, Japan), as reported elsewhere []. When commercial peritoneal dialysate is immersed in H2-enriched water, hydrogen permeates through the container, resulting in the H2 concentration of dialysate gradually increasing in a time-dependent manner (Figure 1). We prepared H2-enriched dialysate using this apparatus by immersing commercial peritoneal dialysate bags for more than 2 hr. Hydrogen-enriched dialysate was then applied as a test solution for peritoneal equilibrium testing.

An external file that holds a picture, illustration, etc. Object name is 2045-9912-3-14-1.jpg

MiZ nondestructive hydrogen dissolver (A) and the hydrogen concentration of peritoneal dialysate in hydrogen-saturated water (B). Hydrogen concentration of dialysate and hydrogen-saturated water around dialysate was measured using a dissolved H2 measurement apparatus DH-35A (DKK-TOA, Tokyo, Japan).

Patients

Six male PD patients were studied (mean age, 55 years; range, 44–71 years; length of PD, 39 ± 17 months; weight, 68.1 ± 16.1 kg; height, 166.2 ± 5.6 cm). The pathology underlying end-stage renal disease was as follows: chronic glomerulonephritis, n = 3; diabetic nephropathy, n = 2; and hypertensive nephropathy, n = 1. Patients with active infection, bleeding, liver dysfunction, collagen disease, systemic vasculitis, cardiovascular accident within 6 months, or malignancy were excluded from this study. Performance status of all patients was class 1 according to American Heart Association criteria []. All patients had been receiving daily continuous ambulatory PD (3–4 bags/day) using neutral low-GDP dextrose solution. The ethics committee of Fukushima Medical University approved this study protocol (Acceptance No. 1362) and written informed consent was obtained from all patients prior to enrollment.

Protocol

Patients underwent a simplified peritoneal equilibration test (fast PET) using standard dialysate, then underwent fast PET using hydrogen-enriched dialysate 2 weeks later. Fast PET was conducted in accordance with the method of Twardowski []. In brief, peritoneal dialysate (2 L of 2.5% dextrose-dialysate) was intraperitoneally infused with a Tenckhoff catheter, and the entire volume of dialysate was drained from the body after 240 min. The drained effluent was mixed well and 2 mL was collected as an effluent sample. Blood samples were obtained before and after fast PET, then 2 mL of serum was drawn after centrifugation and stored at −80°C for 1–4 weeks until analysis. Samples of serum and effluent collected to measure albumin redox were stored at −80°C for 1–4 weeks until analysis. During fast PET, blood pressure, cardiac pulse, and hydrogen concentration in the breath were measured repeatedly every 60 min. Breath hydrogen concentration was also measured in three cases just after, 15 min after, and 30 min after infusion of H2-enriched dialysate. Breath hydrogen concentration was measured using a biological gas (gas in the oral cavity) H2 measurement apparatus BGA-1000D (Aptec, Kyoto, Japan).

Measurement of albumin redox state

Human serum albumin (HSA) is a protein composed of 585 amino acids. The amino residue at position 34 from the N-terminus is a cysteine, containing a mercapto group (SH group). This mercapto group deoxidizes other substances according to the degree of surrounding OS and is itself oxidized. From the perspective of cysteine residues, HSA is a mixture of human mercaptoalbumin (HMA) in which the mercapto group is not oxidized, human non-mercaptoalbumin-1 in which disulfide bond formation is reversibly oxidized mainly by cysteine (HNA-1), and human non-mercaptoalbumin-2 which is strongly oxidized and forms a sulfinic (−SO2H) or sulfonic (−SO3H) group.

The redox state of HSA was determined using high-performance liquid chromatography (HPLC), as previously reported []. The HPLC system consisted of an autosampler (AS-8010; Tosoh, Tokyo, Japan; injection volume, 2 μL) and double-plunger pump (CCPM; Tosoh) in conjunction with a system controller (CO-8011; Tosoh). Chromatographs were obtained using a UV6000LP photodiode alley detector (detection area, 200–600 nm with 1-nm step; Thermo Electron, Waltham, MA, USA). A Shodex-Asahipak ES-502N 7C column (10 × 0.76 cm I.D., DEAE-form for ion-exchange HPLC; Showa Denko, Tokyo, Japan; column temperature, 35 ± 0.5°C) was used in this study. Elusion was performed as linear gradient elusion with graded ethanol concentrations (0 to 1 min, 0%; 1 to 50 min, 0 → 10%; 50 to 55 min, 10 → 0%; 55 to 60 min, 0%) for serum in 0.05 M sodium acetate and 0.40 M sodium sulfate mixture (pH 4.85) at a flow rate of 1.0 mL/min. De-aeration of the buffer solution was performed by bubbling helium.

HPLC profiles obtained from these procedures were subjected to numerical curve fitting with PeakFit version 4.05 simulation software (SPSS Science, Chicago, IL, USA), and each peak shape was approximated by a Gaussian function. Values for fractions of HMA, HNA-1, and HNA-2 to total HSA were then calculated (ƒ(HMA), ƒ(HNA-1), and ƒ(HNA-2), respectively).

Statistical analysis

Values are expressed as mean ± standard deviation unless otherwise stated. StatView version 5.0 statistical software (SAS Institute, Cary, NC, USA) was used for statistical analysis. The significance of collected data was evaluated using a paired t-test or 1-factor repeated-measures analysis of variance (ANOVA) followed by Scheffe’s test as a post-hoc test, as appropriate. For magnitude of correlation, Pearson’s correlation coefficient (R) was used. Differences or correlations were considered significant for values of P < 0.05.

Results

Table 1 shows changes in blood pressure, heart rate, and breath hydrogen concentration during fast PET. Regarding blood pressure and heart rate, no significant difference was seen between standard and H2-enriched dialysate (paired t-test). No significant changes were observed during fast PET in either standard or H2-enriched dialysate (1-factor repeated-measures ANOVA).

Table 1

The changes of blood pressure, cardiac pulse, and breath H2 concentration during fast PET

Standard dialysate H2-enriched dialysate
Blood pressure mmHg




   0 min


130 ± 12 / 79 ± 10


135 ± 13 / 81 ± 10


   60 min


130 ± 11 / 79 ± 5


131 ± 14 / 82 ± 12


   120 min


125 ± 9 / 79 ± 7


134 ± 8 / 80 ± 14


   180 min


123 ± 12 / 75 ± 12


136 ± 5 / 78 ± 12


   240 min


128 ± 9 / 78 ± 7


132 ± 9 / 81 ± 13


Pulse /min




   0 min


81 ± 7


82 ± 12


   60 min


76 ± 6


79 ± 12


   120 min


74 ± 6


78 ± 14


   180 min


77 ± 4


78 ± 17


   240 min


78 ± 7


81 ± 15


Breath H2 ppm




   0 min


4.7 ± 6.6


3.2 ± 2.0


   60 min


1.8 ± 1.3


8.3 ± 7.5*


   120 min


3.0 ± 1.7


8.5 ± 11.0


   180 min


4.2 ± 2.8


5.8 ± 4.8


   240 min 5.5 ± 6.7 7.2 ± 4.6

*; p < 0.05 vs. standard dialysate.

Changes in breath hydrogen concentration in all cases are shown in Table 1 and Figure 2 (A, B). Although no significant changes were observed during fast PET in both standard and H2-enriched dialysate, the hydrogen concentration at 60 min was significantly higher in H2-enriched dialysate than in standard dialysate.

An external file that holds a picture, illustration, etc. Object name is 2045-9912-3-14-2.jpg

Change in breath hydrogen concentration during fast PET. A) Hourly change in PET using standard dialysate. No significant changes were observed. B) Hourly change during PET using H2-enriched dialysate. The hydrogen concentration at 60 min was significantly higher in H2-enriched dialysate than in standard dialysate. C) Breath hydrogen concentrations before, just after, 15 min after, and 30 min after administration of H2-enriched dialysate in three cases. Hydrogen concentrations just after and 15 min after administration were significantly higher than that before administration.

Breath hydrogen concentrations before, just after, 15 min after, and 30 min after administration of H2-enriched dialysate in three cases are shown in Figure 2C. Hydrogen concentrations were significantly higher just after and 15 min after administration (22.7 ± 5.7 and 15.3 ± 3.5 ppm, respectively) than before administration (4.0 ± 1.7 ppm).

Figure 3 shows the redox state of albumin in effluent fluid. The mean proportion of HMA (ƒ(HMA)) was significantly higher in H2-enriched dialysate (62.31 ± 11.10%) than in standard dialysate (54.70 ± 13.08%). In contrast, ƒ(HNA-1) was significantly lower in H2-enriched dialysate (34.26 ± 10.24%) than in standard dialysate (41.36 ± 12.04%). Like ƒ(HNA-1), ƒ(HNA-2) was significantly lower in H2-enriched dialysate (3.43 ± 0.92%) than in standard dialysate (3.94 ± 1.13%). These results suggest that the use of H2-enriched dialysate reduced peritoneal OS. Regarding the result of fast PET (D/P-Cre, drained volume) and effluent creatinine, albumin, interleukin 6 and carbohydrate antigen 125 levels, no differences were evident between standard and H2-enriched dialysate (Table 2).

An external file that holds a picture, illustration, etc. Object name is 2045-9912-3-14-3.jpg

Redox state of albumin in effluent fluid. Mean proportion of reduced albumin (ƒ(HMA)) was significantly higher (A), and that of oxidized albumin (ƒ(HNA-1) (B) and ƒ(HNA-2)) (C) was significantly lower in H2-enriched dialysate than in standard dialysate.

Table 2

The results of serum creatinine value, fast PET and effluent test

Standard dialysate H2-enriched dialysate
Creatinine mg/dL


10.53 ± 2.27


10.03 ± 2.19


Parameter of fast PET




   D/P-Cre


0.71 ± 0.12


0.66 ± 0.11


   Drained volume mL/4 hr


470 ± 184


442 ± 130


Effluent test




   Albumin mg/L


408 ± 175


402 ± 145


   Interleukin-6 pg/mL


6.0 ± 3.3


5.5 ± 2.3


   CA125 U/mL 18.8 ± 8.5 19.5 ± 5.0

Figure 4 shows the redox state of albumin in serum before and after fast PET. The serum ƒ(HMA) level after administration of H2-enriched dialysate (65.75 ± 7.52%) was significantly higher than that after standard dialysate (62.44 ± 7.66%). In contrast, ƒ(HNA-1) after administration of H2-enriched dialysate (31.12 ± 6.73%) was significantly lower than that of standard dialysate (34.73 ± 7.02%). These results suggest that use of H2-enriched dialysate reduced not only peritoneal, but also systemic OS. No significant difference was seen between effluent and serum ƒ(HMA) levels after administration of H2-enriched dialysate (65.31 ± 11.10% and 62.71 ± 7.52%, respectively), while effluent ƒ(HMA) after administration of standard dialysate was significantly lower than serum ƒ(HMA) before administration of standard dialysate (54.70 ± 13.08% and 62.96 ± 8.34%, respectively; P = 0.0339), suggesting that intraperitoneal oxidation of albumin was suppressed by H2-enriched dialysate.

An external file that holds a picture, illustration, etc. Object name is 2045-9912-3-14-4.jpg

Redox state of albumin in serum before and after fast PET. The mean proportion of reduced albumin (ƒ(HMA)) was significantly higher after fast PET using H2-enriched dialysate than after that using standard dialysate (A). Conversely, the mean proportion of reversibly oxidized albumin (ƒ(HNA-1)) was significantly lower after fast PET using H2-enriched dialysate than that after using standard dialysate (B). No significant changes were found in irreversibly oxidized albumin (ƒ(HNA-2)) in the both groups (C).

Discussion

Several reports have suggested that OS participates in peritoneal deterioration, with findings such as strong cytoplasmic staining of 8-hydroxy-2′-deoxyguanosine in peritoneal biopsy specimens of long-term PD patients [], amplified protein kinase C signaling and fibronectin expression due to enhanced ROS in cultured human mesothelial cells []. In terms of the central role of enhanced OS in PD peritoneal damage, Gunal et al. [] showed that oral supplementation with the anti-oxidative agent trimetazidine inhibited morphological and functional deterioration of the peritoneum in a PD rat model. However, regarding suppressing OS, no clinical approaches have been available for PD treatment so far.

The present study aimed to test the therapeutic possibility of using dissolved hydrogen in the dialysate to suppress intra-cavity OS in the clinical setting. This study examined the redox state of albumin as a marker of OS. Since the change in redox state of albumin is a physiological and direct reaction, it is appropriate when evaluating real-time OS and/or detecting rapid changes in OS, as compared to other OS markers such as 8-hydroxy-2′–deoxyguanosine, oxidized low-density lipoprotein and F2 isoprotanes, all of which are in vivo by-products during the process of oxidation.

This pilot study of 6 patients clearly demonstrated that single administration of H2-enriched dialysate increased levels of both peritoneal and plasma ƒ(HMA) without any detrimental effects.

Intraperitoneal administration of H2 altered the local redox state, which may indicate the therapeutic potential of delivering H2 directly to the abdominal cavity in respect to the amelioration of peritoneal damage by PD treatment. On the other hand, interestingly, significant increases in serum ƒ(HMA) levels were seen on intraperitoneal administration of H2. Rapid changes in hydrogen concentration of expired gas after the administration of H2-enriched dialysate may mean that molecular hydrogen in dialysate is rapidly distributed to the body to suppress systemic OS. Another possibility is that increased ƒ(HMA) in the cavity may be recruited into systemic circulation through the abdominal lymphatic drainage. The exact mechanisms underlying increased serum ƒ(HMA) need to be addressed in the future.

In addition, the mechanisms of increased ƒ(HMA) and decreased ƒ(HMA1) by H2 have remained unclear in this study. However, molecular hydrogen is known to directly reduce levels of the cytotoxic hydroxyl radical [], through several possible mechanisms, such as regulation of particular metalloproteins by bonding, or metalloprotein-hydrogen interactions []. Whether H2 directly reacts with the mercapto-residue of albumin, or H2 indirectly modifies it, should be clarified in the future.

Satisfactory anti-oxidative capability of drinking H2-enriched water without any detrimental effects has been reported, in both experimental [] and clinical settings, e.g., type II diabetes mellitus [], metabolic syndrome [], myopathies (progressive muscular dystrophy and polymyositis/dermatomyositis) [], and rheumatoid arthritis []. In addition, we also reported the clinical feasibility of applying H2-enriched water as dialysate for hemodialysis treatment [,]. Given these reports and our present findings, H2-enriched peritoneal dialysate could be of interest in clinical trials with respect to peritoneal preservation. Furthermore, therapeutic effects seem plausible in terms of the prevention of cardiovascular events in patients, since low f(HMA) has been a significant risk factor for cardiovascular mortality among patients treated with PD [] and HD [].

In summary, single administration of H2-enriched dialysate reduced peritoneal and systemic OS without any detrimental effects. A longitudinal study is warranted to ensure clinically beneficial effects, such as suppression of peritoneal deterioration and cardiovascular damage.

 

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Link to Publisher's site
. 2013; 3: 14.
Published online 2013 Jul 1. doi: 10.1186/2045-9912-3-14
PMCID: PMC3734057
PMID: 23816239
Transperitoneal administration of dissolved hydrogen for peritoneal dialysis patients: a novel approach to suppress oxidative stress in the peritoneal cavity
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HT, YH, and WJZ carried out the selections of patients, and the sample collections. HT drafted the manuscript. YM, TT, and SE carried out the measurements of samples. SK, and TW contributed to the study as senior advisers. BS carried out the set-up of equipment system for study. MN organized the study project, and drafted the final manuscript. All authors read and approved the final manuscript.

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Molecular Hydrogen water effects on Mild Cognitive Impairment

Abstract

Background:

Oxidative stress is one of the causative factors in the pathogenesis of neuro-degenerative diseases including mild cognitive impairment (MCI) and dementia. We previously reported that molecular hydrogen (H2) acts as a therapeutic and preventive antioxidant.

Objective:

We assess the effects of drinking H2 hydrogen-water (water infused with hydrogen gas H2) on oxidative stress model mice and human subjects with MCI.

Methods:

Transgenic mice expressing a dominant-negative form of aldehyde dehydrogenase 2 were used as a dementia model. The mice with enhanced oxidative stress were allowed to drink hydrogen H2-water.

For a ran-domized double-blind placebo-controlled clinical study, 73 subjects with mild cognitive impairment MCI drank ~300 mL of hydrogen H2-water (H2-group) or placebo water (control group) per day, and the Alzheimer’s Disease Assessment Scale-cognitive subscale (ADAS-cog) scores were determined after 1 year.

Results:

In mice, drinking hydrogen H2-water decreased oxidative stress markers and suppressed the decline of memory impairment and neurodegeneration. Moreover, the mean lifespan in the hydrogen H2-water group was longer than that of the control group.’

In MCI subjects, although there was no significant difference between the hydrogen water H2- and control groups in ADAS-cog score after 1 year, carriers of the apolipoprotein E4 (APOE4) geno-type in the H2-group were improved significantly on total ADAS-cog score and word recall task score (one of the sub-scores in the ADAS-cog score).

Conclusion:

H2-water may have a potential for suppressing dementia in an oxidative stress model and in the APOE4 carriers with MCI.

1. INTRODUCTION

Oxidative stress is one of the causative factors in the pathogenesis of major neurodegenerative diseases including Alzheimer’s disease (AD), mild cognitive impairment (MCI), and Parkinson disease (PD) []. Moreover, the genotype of apolipoprotein E4 (APOE4) is a genetic risk for AD, and the increased oxidative stress in the APOE4 carriers is considered as one of the modifiers for the risk [].

To explore effective dietary antioxidants to mitigate age-dependent neurodegeneration, it may be useful to construct model mice in which AD phenotypes would progress in an age-dependent manner in response to oxidative stress. We constructed transgenic DAL101 mice expressing a polymorphism of the mitochondrial aldehyde dehydrogenase 2 gene (ALDH2*2) []. ALDH2*2 is responsible for a deficiency in ALDH2 activity and is specific to North-East Asians []. We reported previously that ALDH2 deficiency is a risk factor for late-onset AD in the Japanese population, [] which was reproduced by Chinese and Korean studies in their respective populations []. DAL101 mice exhibited a decreased ability to detoxify 4-hydroxy-2-nonenal (4-HNE) in cortical neurons, and consequently an age-dependent neurodegeneration, cognitive decline, and a shortened lifespan [].

We proposed that molecular hydrogen (H2) has potential as a novel antioxidant, [] and numerous studies have strongly suggested its potential for preventive and therapeutic applications []. In addition to extensive animal experiments, more than 25 clinical studies examining the efficacy of molecular hydrogen H2 have been reported, [] including double-blind clinical studies. Based on these studies, the field of hydrogen medicine is growing rapidly.

There are several methods to administer hydrogen H2, including inhaling hydrogen gas (H2-gas), drinking hydrogen H2-dissolved water (H2-water), and injecting hydrogen H2-dissolved saline (hydrogen-rich saline) []. Drinking hydrogen H2-water prevented the chronic stress-induced impairments in learning and memory by reducing oxidative stress in mice [] and protects neural cells by stimulating the hormonal expression of ghrelin []. Additionally, injection of hydrogen-rich saline improved memory function in a rat model of amyloid-β-induced dementia by reducing oxidative stress []. Moreover, hydrogen inhalation during normoxic resuscitation improved neurological outcome in a rat model of cardiac arrest independently of targeted temperature management [].

In this study, we examined whether drinking hydrogen H2-water could suppress aging-dependent memory impairment induced by oxidative stress in DAL101 mice. Next, in a randomized double-blind placebo-controlled study, we investigated whether H2-water could delay the progression of MCI as assessed by the scores on the Alzheimer’s Disease Assessment Scale-cognition sub-scale (ADAS-cog) [] from baseline at 1-year. We found a significant improvement in cognition at 1 year in carriers with the APOE4 genotype in the H2-group using sub- and total ADAS-cog scores.

2. MATERIALS AND METHODS

2.1. Ethical Approval and Consent to Participate

This animal study was approved by the Animal Care and Use Committee of Nippon Medical School. The methods were carried out in “accordance” with the relevant guidelines and regulations.

The clinical study protocol was approved by the ethics committees of University of Tsukuba, and registered in the university hospital medical information network (UMIN) as UMIN000002218 on July 17, 2009 at https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=history&action =list&type= summary&recptno= R000002-725&language=J.

Participants were enrolled from July 2009. All patients provided written informed consent prior to research investigations, which were conducted according to the Declaration of Helsinki and subsequent revisions.

2.2. Transgenic DAL101 Mice

Transgenic mice (DAL101) that express a transgene containing a mouse version of ALDH2*2 were constructed as described previously []. Since the number of mice used for each experiment was not consistent because of a breeding difficulty, the number of the mice used was specified. All mice were kept in a 12-hr light/dark cycle with ad libitum access to food and water. Examiners performed experiments in a blinded fashion. Since no significant decline was observed in cognitive impairment at the age of 18 months in wild-type mice with the same genetic background (C57BL/6), [] the effects of hydrogen H2-water were not assessed in this study.

2.3. Hydrogen Water

For animal experiments, saturated hydrogen H2-water was prepared as described previously []. In brief, hydrogen  H2 was dissolved in water under high pressure (0.4 MPa) to a supersaturated level, and the saturated H2-water was stored under atmospheric pressure in an aluminum bag with no headspace. As a control, H2-water was completely degassed by gentle stirring for one day. Mice were given water freely using closed glass vessels equipped with an outlet line containing two ball bearings, which kept the water from being degassed. The vessel was freshly refilled with H2-water 6 days per week at 2:00 pm. The hydrogen H2-concentration was still more than 0.3 mM on the next day.

For this clinical study, commercially available hydrogen H2-water was a gift from Blue Mercury, Inc. (Tokyo, Japan). The hydrogen H2-water (500 mL) was packed in an aluminum pouch with no headspace to maintain H2 concentration, and sterilized at 80°C for 30 min. The concentration of hydrogen H2 was measured using a hydrogen sensor (Unisense, Aarhus N, Denmark), and used if the value was more than 0.6 mM. Placebo water packed in an identical package (500 mL) was also provided by Blue Mercury Inc. This company played no role in collection of data, management, analysis, or interpretation of the data. One package with 500 mL of placebo or hydrogen H2-water per day was provided after showing previous empty packages, by which self-reported compliance rates in the intervention group were calculated as the volume of hydrogen  H2-water at 1-year.

2.4. Measurement of Oxidative Stress

As an oxidative stress marker, 8-OHdG [] was measured using urine samples, which were collected between 9:00 and 10:00 am as described previously [], by using a competitive enzyme-linked immunoassay (New 8-OHdG check; Japan Institute for the Control of Aging, Shizuoka, Japan). The values were normalized by urinary creatinine concentration, which was assayed using a standard kit (Wako, Kyoto, Japan). As an additional oxidative stress marker in the brain, accumulated MDA was determined using a Bioxytech MDA-586 Assay Kit (Percipio Biosciences, CA, USA). Malondialdehyde(MDA)levels were normalized against protein concentrations.

2.5. Measurement of Memory Impairment: Object Recognition Task

Learning and memory abilities were examined using objection recognition task (ORT) []. A mouse was habituated in a cage for 4 h, and then two different-shaped objects were presented to the mouse for 10 min as training. The number of times of exploring and/or sniffing each object was counted for the first 5 min (Training test). The frequencies (%) in training test were considered as the backgrounds. To test memory retention after 1 day, one of the original objects was replaced with a novel one of a different shape and then times of exploration and/or sniffing was counted for the first 5 min (Retention test). When mice would lose learning and memory abilities, the frequencies of exploration and/or sniffing of each object should be equal (about 50%) in the training session, indicating that mice showed a similar interest in each object because of lack of memory for the objects. Learning and memory abilities were evaluated as the subtraction of the frequencies (%) in the retention test from each background (Training test).

2.6. Measurement of Memory Impairment: Passive Avoidance Task (PA)

The apparatus consisted of two compartments, one light and the other dark, separated by a vertical sliding door []. On day 1, we initially placed a mouse in the light compartment for 20 s. After the door was opened, the mouse could enter the dark compartment (mice instinctively prefer being in the dark). On day 2, the mouse was again placed in the light section to allow the mouse to move into the dark section. After the mouse entered the dark compartment, the door was closed. After 20 s, the mouse was given a 0.3 mA electric shock for 2 s. The mouse was allowed to recover for 10 s, and was then returned to the home cage. On day 3, 24h after the shock, the mouse was again placed in the light section with the door opened to allow the mouse to move into the dark section. We examined the latency time for stepping through the door. Learning and memory abilities were assessed as the subtraction of the latency times after the electric shock from each background (before).

2.7. Immunostaining of the Hippocampal CA1 Region

To examine neuronal loss and glial activation, the hippocampus region was stained with a pyramidal neuron-specific anti-NeuN antibody (clone A60; Merck Millipore, Darmstadt, Germany), an astrocyte-specific anti-glial fibrillary acidic protein (anti-GFAP) antibody (Thermo Scientific, MA, USA) or a microglia-specific anti-IbaI antibody (Wako). Mice were transcardially perfused to be fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) under anesthesia, and their brains were cryoprotected with 30% sucrose, and then frozen brain was sectioned at 8 μm thickness. After incubation with each primary antibody, sections were treated with secondary antibodies (Vector Laboratories, CA, USA) and their immunereactivity was visualized by the avidin-biotin complex method (Vector Laboratories).

2.8. Subjects of the Clinical Study

This study was a randomized, double-blind, placebo-controlled trial undertaken as a part of Tone project, an ongoing epidemiological study conducted in Tone Town, Ibaraki, Japan as described in detail previously []. This town is located approximately 40 km northeast of central Tokyo and consists of 22 districts. The baseline survey of the Tone project included 1,032 participants in July 2009, and subjects of the present study were recruited from these participants.

Eligibility criteria are age 67 years or older, being able to give written informed consent for participation in the present study, with a diagnosis of MCI, being able to observe the following requirement: good compliance with water consumption; participation in the scheduled examinations for assessment; keeping a log-diary recording consumption of the water, with a modified Hachinski Ischemic score of 4 or less and a 15-item Geriatric Depression Scale score of 6 or less. In brief, 3 months before this clinical study, all participants underwent a group assessment which used a set of 5 tests that measured the following cognitive domains: attention; memory; visuospatial function; language; and reasoning as described previously []. Objective impairment in at least 1 cognitive domain based on the average of the scores on the neuropsychological measures within that domain and 1 SD cut-off using normative corrections for age, years of education, and sex.

Exclusion criteria were having “The Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV TR” criteria for dementing illnesses, a serious or unstable illnesses, a history within the past 5 years of serious infectious disease affecting the brain and/or malignant diseases, a history of alcohol or drug abuse or dependence (on DSM-IV TR) within the past 5 years, and receiving any types of anti-Alzheimer drugs and recent (within 4 weeks) initiation of medications that affect the central nervous system. When the score of Mini Mental State Examination (MMSE) [] was less than 24, the subjects were excluded.

In this study, subjects were randomly assigned to either to an intervention group, who received H2-water every-day for 1 year, or a control group, who received placebo water. The allocation sequence was determined by computer-generated random numbers that were concealed from the investigators and subjects. Drs. Nakajima and Ikejima generated the random allocation sequence, enrolled participants, and assigned participants to interventions. Any participants and care providers were blindly masked.

In the original protocol, we planed to administer H2-water for 2 years and assess the secondary outcomes; however, we had to stop the project in 2011 by the Tsunami-disaster and could not obtained the 2-year data and secondary outcomes.

The APOE4 genotype was determined as described [].

2.9. Statistical Considerations

All statistical analyses were performed by an academic biostatistician using SAS software version 9.2 (SAS Institute Inc, Cary, NC, USA). Results were considered significant at p < 0.05.

For the comparison of two groups in learning and memory abilities, and lifespans, unpaired two-tailed Student’s t-test was used for the comparison of H2-group with control group. For the other animal experiments, one-way analysis of variance (ANOVA) with Tukey-Kramer or Dunnett post hoc analysis was applied unless otherwise mentioned.

For the clinical trial, we planned to recruit a total of 120 patients, which would provide 90% power to detect an effect size of 0.6 using a two-sided test with a 5% significance level, but the actual sample size for the primary analysis was 73, leading to 70% power in the same setting. End-points were scores in the Japanese version of ADAS-cog at 1-year, and the changes were evaluated by Mann-Whitney’s U test (non-parametric analysis) as well as Student’s t-test (parametric analysis).

3. RESULTS

3.1. Hydrogen-water Reduced Oxidative Stress in DAL Mice

Male DAL101 mice were given H2– or control water to drink ad libitum from the age of 1 month, and continued until the age of 18 months. The H2-water DAL101 group showed a significant decrease in the level of an oxidative stress marker, urinary 8-hydroxy-2’-deoxyguanosine (8-OHdG)[] at the age of 14months (Suppl. Fig. S1A). Moreover, DAL101 mice increased oxidative stress in the brain as measured by the level of MDA as an alternative oxidative stress marker, and H2-water showed a significant recovery of this increased level of MDA in DAL101 mice (Suppl. Fig. S1B).

3.2. Hydrogen Water Suppressed a Decline in Learning and Memory Impairment

We examined learning and memory abilities using ORT []. As described in MATERIALS AND METHODS, learning and memory abilities were evaluated as the subtraction of the frequency (%) in Retention test from each background (Training test). Mice were provided with control or H2-water from the age of 1 month. At the age of 14 months, the H2-group significantly memorized the original objects and showed the preference for the novel object more than the control group (Fig. 1A1A 14-month-old).

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Hydrogen water prevented cognitive decline. H2-water was provided from the age of 1 month (A, C), and from the age of 8 months (B). The mice were subjected to the first objection recognition task (ORT) at the age of 14 months (A, B, 14-month-old) and the second ORT at the age of 18 months (A, B, 18-month-old).

The recognition indexes were obtained as the frequency (%) of exploring and/or sniffing the object that would be replaced or the novel one that had been replaced. ΔRecognition index (%) indicates the frequencies in Retention test of ORT after the subtraction of those in Training test (background). WT, wild-type; (DAL, H2-),

DAL101 mice drinking degassed control water; (DAL, H2+), DAL101 mice drinking hydrogen water. Data are shown as the mean ± SEM. n = 9, *p < 0.05, **p < 0.01 by Student’s t-test. (C) The mice were subjected to a passive avoidance task. Step-through latencies before and after the electric shock are obtained and ΔStep-through latency (s) indicates the subtraction of Step-through latencies after from before the electric shock. WT, wild-type (n = 10); DAL, H2-, DAL101 mice receiving degassed control water (n = 8); and DAL, H2+, DAL101 mice receiving H2-water (n = 8). Data are shown as the mean ± SEM. *p < 0.05.

At the age of 18 months, the mice were subjected to the second ORT, which can be done by using different objects at the age of 18 months []. The aged DAL101 mice drinking H2-water still significantly memorized the original objects and preferred the novel one more than the control group (Fig. 1A1A 18-month-old).

Next, to test the drinking effects of H2-water from the later stage, we started giving H2-water to male DAL101 mice at the age of 8 months instead of 1 month, and subjected to ORT at the age of 14 months (Fig. 1B1B 14-month-old) and the second ORT at the age of 18 months (Fig. 1B1B 18-month-old). Even when the mice began to drink at the age of 8 months, H2-water significantly suppressed the decline in the learning and memory abilities at the age of 18 months as well as at the age of 14 months (Fig. 1B1B).

Moreover, we subjected the mice to PA [] at the age 18 months as an alternative method. One day after a 0.3 mA electric shock for 2 s was given, wild-type C57BL/6 mice memorized the shock as evaluated by the subtraction of the latency time (s) to re-enter the dark compartment from each background (Fig. 1C1C). The H2-water group significantly suppressed the decline in learning and memory more than the control group (Fig. 1C1C).

Thus, drinking hydrogen H2-water suppressed the learning and memory impairment in the oxidative stress mice.

3.3. Hydrogen-water Suppressed Neurodegeneration

To examine whether hydrogen H2-water could prevent neurodegeneration in aged DAL101 mice, we stained the hippocampus with a neuron-specific anti-NeuN antibody (Fig. 2A2A). Neurodegeneration was evaluated by glial activations using an anti-GFAP antibody and a microglia-specific anti-Iba-I antibody. Immune-positive cells per field of view (FOV) were counted in the CA1 region (Fig. 2B2B).

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Hydrogen water suppressed neurodegeneration. (A) The hippocampal CA1 region was stained with antibodies against NeuN (a neuronal marker), GFAP (an astrocytic marker) or Iba-1 (a microglial marker) (Scale bars: 50 µm). Right panels show magnified images of the squares in the left panels (Scale bars: 10 µm). (B) Cells positive for anti-NeuN, anti-GFAP and anti-Iba-I antibodies per field of view (FOV) were counted in the CA1 region (n = 5). Data are shown as the mean ± SD. *p < 0.05, **p < 0.01 (wild-type vs DAL), #p < 0.05 (H2-water vs. control water in DAL).

The number of neurons was decreased in the control DAL101 group as the comparison with wild type group, and the H2-DAL101 group showed a trend in recovery of the decrease (Fig. 2A2A). As has been described previously, [] the control DAL101 mice exhibited an increase in glial activation, and the H2-water group suppressed the enhanced glial activation in the CA1 region (Fig. 22, GFAP and Iba-I).

3.4. Hydrogen-water Extended the Average Lifespan of Mice

DAL101 mice showed a shorter lifespan, which has also been described previously []. To examine whether consumption of hydrogen H2-water attenuated the shortened lifespan, female DAL101 mice started drinking control or H2-water at the age of 1 month. Although hydrogen H2-water did not extend the maximum lifespan (Fig. 3A3A), hydrogen H2-water significantly extended the mean of lifespan of DAL101 mice (Fig. 3B3B).

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Extension of the average lifespan by continuous drinking H2-water. (A) Kaplan-Meier curve representing the survival of female C57BL/6 mice (wild-type), female DAL101 mice drinking control water (control water) and H2-water (H2-water). (B) Each dot indicates the lifespan of each mouse. The bars indicate the average lifespan of each group. *p < 0.05 (p = 0.036) by Student’s t-test.

3.5. A Randomized, Placebo Controlled Clinical Study

Fig. (44) shows the profile on the recruitment, randomization, and follow-up of this study. A total of 81 subjects of the 1,032 participants were randomized; however, 3 in the control group and 5 in the intervention group were diagnosed as ineligible after randomization and not included in this analysis. Baseline characteristics and lifestyle factors were balanced between the study groups (Table 11). Random assignment was stratified by age of ~74 years and MMSE score of ~28 points. The average compliance rate of drinking water was estimated as 64% in both groups at 1-year, meaning the subjects drank 320 mL/day on the average. The mean total ADAS-cog scores in the H2– and control groups were 8.04 and 7.89, respectively, with no significance.

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Profile of the recruitment, randomization, and follow-up of this study. This study was a randomized, double-blind, placebo-controlled trial undertaken as a part of Tone project, an ongoing epidemiological study conducted in Tone Town, Ibaraki, Japan [].

Table 1

Background characteristics of 73 subjects with mild cognitive impairment.

Control (n=38) Intervention (n=35)
Mean SD or % Mean SD or %
Woman * 20 (52.6%) 19 (54.3%)
Age (years) 74.45 5.44 73.97 5.11
Body mass index (kg/m2) 23.55 2.59 23.19 4.08
Systolic blood pressure (mmHg) 131.26 12.35 135.14 13.31
Diastolic blood pressure (mmHg) 77.92 7.13 78.89 9.53
Education (years) 11.26 2.71 11.57 2.83
Current alcohol drinker * 19 (50.0%) 14 (40.0%)
Current smoker * 4 (10.5%) 5 (14.3%)
Current exercise habit * 27 (71.1%) 22 (62.9%)
APOE4 carrier * 6 (15.7%) 7 (20.0%)
Family history * 2 (5.3%) 2 (5.7%)
Comorbidity *
Hypertension 15 (39.5%) 14 (40.0%)
Diabetes mellitus 4 (10.5%) 5 (14.3%)
Dyslipidemia 4 (10.5%) 4 (11.4%)
Stroke 2 (5.3%) 1 (2.9%)
Depression 1 (2.6%) 2 (5.7%)
MMSE 28.08 1.66 27.83 1.74
ADAS-cog 7.89 3.19 8.04 3.47

* indicates frequency (%).

After 1 year, no observable harms or unintended effects in each group were found, and there was a trend to improve total ADA-cog score both in the H2– and control-groups (Suppl. Table S1), probably because of interventions such as moderate exercise by the Tone project. Moreover, the subjects in the H2-group had more trends for the improvement than those in the control-groups although there was no significance (Suppl. Table S1). However, when we pay attention to score-changes in carriers of the APOE4 genotype, the total ADAS-cogs and word recall task scores (one of the sub-scores) significantly improved as assessed by the distribution of the score change in each subject (Fig. 55). In the APOE4 carriers, the hydrogen  water H2-group significantly improved, whereas the control group slightly worsened. Moreover, Fig. (66) shows the score change of each subject as an alternative presentation. Although the subjects in the control group did not improved, six and five out of 7 subjects improved on the total ADAS score and word recall task scores, respectively, in the hydrogen water H2-group of the APOE4 carriers.

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Distribution of changes of sub- and total-ADAS-cog score. Distribution of change of word recall task score (A), a sub-score of ADAS-cog, and (B) total ADAS-cogs score in APOE4 non-carriers (left) and APOE4 carriers (right). Each dot indicates the change of individual subjects. The difference between the H2- and control groups was significant in APOE4 carriers by a non-parametric analysis as well as a parametric analysis. (Ap = 0.036 (by Student’s t-test) and p =0.047 (by Mann-Whitney’s U test) and (Bp = 0.037 (by Student’s t-test) and p = 0.044 (by Mann-Whitney’s U test) for (A) and (B), respectively. Middle bars in lozenges indicate median values.

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Changes in a sub-sore and total ADAS-cog score of each subject in the APOE4 carriers. Each line indicates the 1-year change in the word recall task score (A) and total ADAS-cog score (B) of a subject in the APOE4 carriers. * indicates p < 0.05 as shown in the legend of Fig. 5.

DISCUSSION

Age-dependent neurodegenerative disorders are involved in oxidative stress. In this study, we showed that drinking hydrogen H2-water suppressed the biochemical, behavioral, and pathological decline in oxidative stress mice. The score of ADAS-cog [] is the most widely used general cognitive measure in clinical trials of AD []. The ADAS-cog score assesses multiple cognitive domains including memory, language, praxis, and orientation. Overall, the ADAS-cog has proven successful for its intended purpose. The present clinical study shows that drinking hydrogen H2-water significantly improved the ADAS-cog score of APOE4 genotype-carriers.

We have previously showed that DAL101 mice show age-dependent neurodegeneration and cognitive decline and the shorten lifespan []. DAL101 mice exhibit dementia phenotypes in an age-dependent manner in response to an increasing amount of oxidative stress []. Oxidative stress enhances lipid peroxidation, leading to the formation of highly reactive α, β-unsaturated aldehydes, such as MDA and 4-HNE []. The accumulation of 4-HNE-adducted proteins in pyramidal neurons has been observed in the brains of patients with AD and PD []. The decline of ALDH2*2 ability failed to detoxify cytotoxic aldehydes, and consequently increases in oxidative stress [].

Moreover, double-transgenic mice were constructed by crossing DAL101 mice with Tg2576 mice, which express a mutant form of human amyloid precursor protein (APP). They showed accelerated amyloid deposition, tau phosphorylation, and gliosis, as well as impaired learning and memory abilities. The lifespan of APP/DAL mice was significantly shorter than that of APP and DAL101 mice []. Thus, these model animals may be helpful to explore antioxidants that could be able to prevent age-dependent dementia. Indeed, a diet containing Chlorella showed mitigated effects on cognitive decline in DAL101 [].

One of the most potent risk factors for AD is carrier status of the APOE4 genotype, and the roles of APOE4 on the progression of AD have been extensively examined from various aspects []. APOE4 also increase the number of atherogenic lipoproteins, and accelerate atherogenesis []. The increased oxidative stress in APOE4 carriers is considered as one of the modifiers for the risk []. A combination of antioxidants improved cognitive function of aged subjects after 3 years, especially in APOE4 carriers []. This previous clinical result agrees with the present study. hydrogen H2 acts as an efficient antioxidant inside cells owing to its ability to rapidly diffuse across membranes []. Moreover, as a secondary anti-oxidative function, H2 seems to activate NF-E2-related factor 2 (Nrf2), [] which reduces oxidative stress by expression a variety of antioxidant enzymes []. We reported that drinking hydrogen H2-water prevented arteriosclerosis using APOE knockout mice, a model of the spontaneous development of atherosclerosis accompanying a decrease in oxidative stress []. Thus, it is possible that drinking H2-water improves vascular damage by decreasing oxidative stress as a direct or indirect antioxidant, leading to the improvement of a demintia model and MCI subjects. In this study, we focused on the genotype of APOE-isoforms; however, the polymorphism of the APOE gene in the promoter region influences the expression of the APOE gene []. Thus, it will be important to examine the effect of hydrogen H2-water under this polymorphism.

For mitigating AD, significant attention has been given to regular, moderate exercise to help reduce the risk of dementia and prevent MCI from developing in aging patients [ – ]. Moderate exercise enhances energy metabolism and suppresses the expression of pro-inflammatory cytokines, [] and protects vascular systems [].molecular hydrogen H2 exhibits multiple functions by a decrease in the levels of pro-inflammatory cytokines and an increase in energy metabolism in addition to anti-oxidative roles. To exert multiple functions, molecular hydrogen H2 regulates various signal transduction pathways and the expression of many genes []. For examples,molecular hydrogen H2 protects neural cells and stimulates energy metabolism by stimulating the hormonal expression of ghrelin [] and fibroblast growth factor 21, [] respectively. In contrast, molecular hydrogen H2 relieves inflammation by decreasing pro-inflammatory cytokines []. Thus, the combination of these functions of molecular hydrogen H2 on anti-inflammation and energy metabolism-stimulation might prevent the decline in brain function, [] both of which are improved by regular and moderate exercise. Thus, it is possible that the multiple functions of molecular hydrogen H2, including energy metabolism-stimulation and anti-inflammation, may contribute to the improvement of the dementia model and the MCI subjects.

As an alternative aspect, molecular hydrogen H2 suppresses the nuclear factor of activated T cell (NFAT) transcription pathway to regulate various gene expression patterns []. NFAT signaling is altered in AD and plays an important role in driving amyloid β-mediated neurodegeneration []. Moreover, the NFAT transcriptional cascade contributes to amyloid β synaptotoxicity []. Additionally, an active involvement of the NFAT-mediated signaling pathway in α-syn-mediated degeneration of neurons in PD []. Indeed, patients with PD improved by drinking molecular hydrogen H2-water as revealed by a double-blind, placebo-controlled clinical study, [] and a larger scale of a clinical trial is under investigation []. Thus, the beneficial effects of molecular hydrogen H2 on the neurodegenerative diseases may be explained by the suppression of NFAT transcriptional regulation.

CONCLUSION

The present study suggests a possibility for slowing the progress of dementia by drinking molecular hydrogen H2-water by means of animal experiments and a clinical intervention study for APOE4 carriers; however, a longer and larger scale of trials will be necessary to clarify the effect of H2-water on MCI.

PMCID: PMC5872374
PMID: 29110615
Effects of Molecular Hydrogen Assessed by an Animal Model and a Randomized Clinical Study on Mild Cognitive Impairment
This is an open access article licensed under the terms of the Creative Commons Attribution-Non-Commercial 4.0 International Public License (CC BY-NC 4.0) (https://creativecommons.org/licenses/by-nc/4.0/legalcode), which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.

Associated Data

Supplementary Materials

ACKNOWLEDGEMENTS

We thank Blue Mercury, Inc. (Tokyo, Japan) for providing H2-water and placebo water, Ms. Hiroe Murakoshi for technical assistance and Ms. Suga Kato for secretarial work. Financial support for this study was provided by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23300257, 24651055, and 26282198 to S.O.; 23500971 and 25350907 to K.N.). Financial support for this study was provided by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23300257, 24651055, and 26282198 to S.O.; 23500971 and 25350907 to K.N.).

LIST OF ABBREVIATIONS

APOE4 Apolipoprotein E4
MCI Mild cognitive Impairment
ALDH2 Aldehyde Dehydrogenase 2
ADAS-cog Alzheimer’s Disease Assessment Scale-cognitive subscale
AD Alzheimer’s Disease
PD Parkinson’s Disease
DAL101 Dominant Negative Type 101 of the ALDH2 Mutant Polymorphism (ALDH2*2)
4-HNE 4-Hydroxy-2-nonenal
8-OHdG 8-Hydroxy-2’-deoxyguanosine
MDA Malondialdehyde
ORT Object Recognition Task
PA Passive Avoidance Task
GFAP Glial Fibrillary Acidic Protein
PBS Phosphate-buffered Saline
ANOVA One-way Analysis of Variance
CI Confidence Interval
MMSE Mini Mental State Examination
FOV Field of View
APP Amyloid Precursor Protein
Nrf2 NF-E2-related Factor 2
NFAT Nuclear Factor of Activated T Cell

 

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s web site along with the published article.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The animal study was approved by the Animal Care and Use Committee of Nippon Medical School.

The human clinical study protocol was approved by the ethics committees of University of Tsukuba.

HUMAN AND ANIMAL RIGHTS

All animal research procedures followed were in accordance with the standards set forth in the eighth edition of Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences, The National Academies Press, Washington, D.C.).

All human material was obtained in accordance with the standards set forth in the Declaration of Helsinkiprinciples of 1975, as revised in 2008 (http://www.wma.net/en/10ethics/10helsinki/<http://www.wma.net/en/10ethics/10helsinki/>).

Consent for Publication

All the patients provided written informed consent priority to research investigations.

CONFLICT OF INTEREST

We declare that there is no actual and potential conflict of interest on this study. Although SO was a scientific advisor of Blue Mercury, Inc. (Tokyo, Japan) from 2,005 to 2,008, there was no involvement during this study.

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