Wenwu Liu

Neuroprotective effects of hydrogen saline in neonatal hypoxia-ischemia rat model.

Cerebral hypoxia-ischemia (HI) represents a major cause of brain damage in the term newborn. This study aimed to examine the short and long-term neuroprotective effect of hydrogen saline (H(2) saline) using an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 degrees C). H(2) saturated saline was administered by peritoneal injection (5 ml/kg) immediately and again at 8 h after HI insult. At 24 h after HI, the pups were decapitated and brain morphological injury was assessed by 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, and TUNEL staining. Acute cell death, inflammation and oxidative stress were evaluated at 24 h by studying caspase-3 activity, MDA measurement as well as Iba-1 immunochemistry in the brain. At 5 weeks after HI, spontaneous activity test and Morris water maze test were conducted. We observed that H(2) saline treatment reduced the caspase activity, MDA, Iba-1 levels, the infarct ratio, and improved the long-term neurological and neurobehavioral functions. H(2) saline has potentials in the clinical treatment of HI and other ischemia-related cerebral diseases.

Hydrogen-rich saline protects against intestinal ischemia/reperfusion injury in rats

Hydrogen gas was reported to reduce reactive oxygen species and alleviate cerebral, myocardial and hepatic ischemia/reperfusion (I/R) injuries. This paper studied the effect of hydrogen-rich saline, which was easier for clinical application, on the intestinal I/R injury. Model of intestinal I/R injury was induced in male Sprague-Dawley rats. Physiological saline, hydrogen-rich saline or nitrogen-rich saline (5 ml/kg) was administered via intravenous infusion at 10 min before reperfusion, respectively. The intestine damage was detected microscopically and was assessed by Chiu score system after I/R injury. In addition, serum DAO activity, TNF-α, IL-1β and IL-6 levels, tissue MDA, protein carbonyl and MPO activity were all increased significantly by I/R injury. Hydrogen-rich saline reduced these markers and relieved morphological intestinal injury, while no significant reduction was observed in the nitrogen-rich saline-treated animals. In conclusion, hydrogen-rich saline protected the small intestine against I/R injury, possibly by reduction of inflammation and oxidative stress.

Hydrogen-rich saline protects myocardium against ischemia/reperfusion injury in rats.

Protective effect of hydrogen (H2) gas on cardiac ischemia-reperfusion (I/R) injury has been demonstrated previously. This study was designed to test the hypothesis that hydrogen-rich saline (saline saturated with molecular hydrogen), which is easy to use, induces cardioprotection against ischemia (30 min) and reperfusion (24 h) injury in rats. Adult male Sprague-Dawley rats underwent 30-min occlusion of the left anterior descending (LAD) coronary artery and 24-h reperfusion. Intraperitoneal injection of hydrogen-rich saline before reperfusion significantly decreased plasma and myocardium malondialdehyde (MDA) concentration, decreased cardiac cell apoptosis, and myocardial 8-hydroxydeoxyguanosine (8-OHdG) in area at risk zones (AAR), suppressed the activity of caspase-3, and reduced infarct size. The heart function parameters including left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), +(dP/dt)max and −(dP/dt)max were also significantly improved 24 h after reperfusion. It is concluded that hydrogen-rich saline is a novel, simple, safe, and effective method to attenuate myocardial I/R injury.

Hyperbaric oxygen preconditioning induces tolerance against brain ischemia–reperfusion injury by upregulation of antioxidant enzymes in rats

The present study examined the hypothesis that cerebral ischemic tolerance induced by hyperbaric oxygen preconditioning (HBO-PC) is associated with an increase of antioxidant enzyme activity. Male Sprague-Dawley rats (250-280 g, n=74) were divided into sham, middle cerebral artery occlusion (MCAO) for 90 min, and MCAO plus HBO-PC groups. HBO-PC was conducted four times by given 100% oxygen at 2.5 atmosphere absolute (ATA), for 1 h at every 12 h interval for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function and Nissl Staining were performed to evaluate the effect of HBO-PC. Malondialdehyde (MDA) content, activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) sampled from the hippocampus, ischemic penumbra or core of cortex were measured. HBO-PC decreased mortality rate, improved neurological recovery, lessened neuronal injury, reduced the level of MDA and increased the antioxidant activity of CAT and SOD. These observations demonstrated that an upregulation of the antioxidant enzyme activity by HBO preconditioning plays an important role in the generation of tolerance against brain ischemia-reperfusion injury.

Sulforaphane protects brains against hypoxic-ischemic injury through induction of Nrf2-dependent phase 2 enzyme.

Neonatal hypoxia-ischemia (HI) brain injury involves reactive oxygen species (ROS) and inflammatory responses. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, has cytoprotective effects against oxidative stress and its effect was mediated by NF-E2-related factor-2 (Nrf2), a transcription factor, and heme oxygenase 1 (HO-1) which is one of Nrf2 downstream target genes. This study was undertaken to investigate the neuroprotective mechanisms of SFN in a neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 degrees C) for 90 min. SFN (5mg/kg) was systemically administered 30 min before HI insult. Brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, TUNEL staining, malondialdehyde (MDA), 8OH-dG level, and caspase-3 activity in the cortex and hippocampus. SFN pretreatment increased the expression of Nrf2 and HO-1 in the brain and reduced infarct ratio at 24h after HI. The number of TUNEL-positive neurons as well as activated macroglia and the amount of 8OH-dG, were markedly reduced after SFN treatment, accompanied by suppressed caspase-3 activity and reduced lipid peroxidation (MDA) level. These results demonstrated that SFN could exert neuroprotective effects through increasing Nrf2 and HO-1 expression.

Hyperbaric oxygen preconditioning reduces ischemia–reperfusion injury by inhibition of apoptosis via mitochondrial pathway in rat brain

This study examined the hypothesis that apoptotic inhibition via mitochondrial pathway was involved in hyperbaric oxygen preconditioning (HBO-PC)-induced neuroprotection on ischemia-reperfusion injury in rat brain. Male Sprague-Dawley rats (250 approximately 280 g, n=144) were divided into control, middle cerebral artery occlusion (MCAO) for 90 min, and HBO-PC plus MCAO groups. HBO-PC was conducted four times by giving 100% oxygen at 2.5 atm absolute (ATA), for 1 h at 12 h intervals for 2 days. At 24 h after the last HBO-PC, MCAO was performed and at 24 h after MCAO, neurological function, brain water content, infarct volume, and cell death were evaluated. Enzymatic activity of capase-3 and -9, and expression of cytochrome c, Bcl-2 and Bax proteins were performed in the samples from hippocampus, ischemic penumbra and core of the brain cortex, respectively. HBO-PC reduced brain edema, decreased infarction volume, and improved neurological recovery. HBO-PC reduced cytoplasm cytochrome c levels, decreased caspase enzyme activity, upregulated the ratio of Bcl-2 and Bax expression, and abated the apoptosis of ischemic tissue. HBO-PC protects brain tissues from ischemia-reperfusion injury by suppressing mitochondrial apoptotic pathways.

Cyclooxygenase-2 Mediates Hyperbaric Oxygen Preconditioning in the Rat Model of Transient Global Cerebral Ischemia

Background and Purpose— Hyperbaric oxygen (HBO) preconditioning (PC) allows brain protection against transient global ischemia. In the present study, we hypothesize that the mechanism of HBO-PC involves the induction of cyclooxygenase-2 (COX-2) in cerebral tissues before ischemia, which leads to a suppression of COX-2 and its downstream targets after global ischemic insult. Methods— One hundred twenty-nine male Sprague Dawley rats (body weight 280–300 grams) were allocated to the naive control group and the sham operation group, and 3 groups of animals were subjected to 15-minute 4-vessel occlusion: untreated, preconditioned with HBO 2.5 atmospheres absolutes for 1 hour daily for 5 days, preconditioned as mentioned and administered with COX-2 inhibitor NS-398 (1 mg/kg body weight intraperitoneal) before each preconditioning session, and normal rats preconditioned with HBO without ischemia. The mortality, the incidence of seizures, and T-maze scores were recorded. The quantitative cell count in Nissl stain and TUNEL was conducted on day 7 after ischemia. The brain expression of COX-2 was analyzed with Western blotting and immunofluorescence staining. Results— HBO-PC increased the number of surviving neurons in the Cornu Ammonis area 1, which was associated with the reduced COX-2 expression in the hippocampus and in the cerebral cortex at 1 and 3 days after ischemia. HBO-PC improved functional performance and tended to decrease mortality and the frequency of seizures. These beneficial effects of HBO-PC were abolished by the COX-2 selective inhibitor NS-398. Conclusions— HBO-PC reduced COX-2 expression and provided brain protection after global ischemia. Administration of COX-2 inhibitor with HBO before ischemia abolished preconditioning effect, thereby implicating COX-2 as a mediator of HBO-PC in the ischemic brain.

Hydrogen gas reduced acute hyperglycemia-enhanced hemorrhagic transformation in a focal ischemia rat model.

Hyperglycemia is one of the major factors for hemorrhagic transformation after ischemic stroke. In this study, we tested the effect of hydrogen gas on hemorrhagic transformation in a rat focal cerebral ischemia model. Sprague-Dawley rats (n=72) were divided into the following groups: sham; sham treated with hydrogen gas (H(2)); Middle Cerebral Artery Occlusion (MCAO); and MCAO treated with H(2) (MCAO+H(2)). All rats received an injection of 50% dextrose (6 ml/kg i.p.) and underwent MCAO 15 min later. Following a 90 min ischemic period, hydrogen was inhaled for 2 h during reperfusion. We measured the level of blood glucose at 0 h, 0.5 h, 4 h, and 6 h after dextrose injection. Infarct and hemorrhagic volumes, neurologic score, oxidative stress (evaluated by measuring the level of 8 Hydroxyguanosine (8OHG), 4-Hydroxy-2-Nonenal (HNE) and nitrotyrosine), and matrix metalloproteinase (MMP)-2/MMP-9 activity were measured at 24 h after ischemia. We found that hydrogen inhalation for 2 h reduced infarct and hemorrhagic volumes and improved neurological functions. This effect of hydrogen was accompanied by a reduction of the expression of 8OHG, HNE, and nitrotyrosine and the activity of MMP-9. Furthermore, a reduction of the blood glucose level from 500+/-32.51 to 366+/-68.22 mg/dl at 4 h after dextrose injection was observed in hydrogen treated animals. However, the treatment had no significant effect on the expression of ZO-1, occludin, collagen IV or aquaporin4 (AQP4). In conclusion, hydrogen gas reduced brain infarction, hemorrhagic transformation, and improved neurological function in rats. The potential mechanisms of decreased oxidative stress and glucose levels after hydrogen treatment warrant further investigation.

Methane attenuates myocardial ischemia injury in rats through anti-oxidative, anti-apoptotic and anti-inflammatory actions.

Myocardial infarction (MI) remains the most frequent cardiovascular disease with high mortality. Recently, methane has been shown protective effects on small intestinal ischemia-reperfusion injury. We hypothesized that methane-rich saline (MS) could protect the myocardium again MI via its anti-oxidative, anti-apoptotic and anti-inflammatory effects. In experiment 1, tetrazolium chloride staining and detection of myocardial enzymes and oxidative and inflammatory parameters were performed at 12h after MI to determine the optimal dose at which intraperitoneal MS exerted the best protective effects on MI. In experiment 2, rats were treated with 10 ml/kg MS. Myocyte apoptosis was detected 72 h after MI, and cardiac function and myocardial remodeling were evaluated 4 weeks after MI. Results showed different dose of MS reduced infarct area, decreased myocardial enzymes, inhibited inflammation and oxidative stress following MI. The optimal dose of MS was 10 mg/kg. Moreover, treatment with 10mg/kg MS for 3 days significantly reduced myocyte apoptosis, improved cardiac function and inhibited myocardial remodeling (reduced anterior wall thickness, attenuated myocyte hypertrophy, and decreased myocardial collagen). MS protects the myocardium of MI rats via its anti-oxidative, anti-inflammatory, anti-apoptotic and anti-remodeling activities. Thus, MS provides a novel and promising strategy for the treatment of ischemic heart diseases.

Lactulose ameliorates cerebral ischemia–reperfusion injury in ratsby inducing hydrogen by activating Nrf2 expression

Molecular hydrogen has been proven effective in ameliorating cerebral ischemia/reperfusion (I/R) injury by selectively neutralizing reactive oxygen species. Lactulose can produce a considerable amount of hydrogen through fermentation by the bacteria in the gastrointestinal tract. To determine the neuroprotective effects of lactulose against cerebral I/R injury in rats and explore the probable mechanisms, we carried out this study. The stroke model was produced in Sprague-Dawley rats through middle cerebral artery occlusion. Intragastric administration of lactulose substantially increased breath hydrogen concentration. Behavioral and histopathological verifications matched biochemical findings. Behaviorally, rats in the lactulose administration group won higher neurological scores and showed shorter escape latency time in the Morris test. Morphologically, 2,3,5-triphenyltetrazolium chloride showed smaller infarction volume; Nissl staining manifested relatively clear and intact neurons and TUNEL staining showed fewer apoptotic neurons. Biochemically, lactulose decreased brain malondialdehyde content, caspase-3 activity, and 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine concentration and increased superoxide dismutase activity. The effects of lactulose were superior to those of edaravone. Lactulose orally administered activated the expression of NF-E2-related factor 2 (Nrf2) in the brain as verified by RT-PCR and Western blot. The antibiotics suppressed the neuroprotective effects of lactulose by reducing hydrogen production. Our study for the first time demonstrates a novel therapeutic effect of lactulose on cerebral ischemia/reperfusion injury and the probable underlying mechanisms. Lactulose intragastrically administered possessed neuroprotective effects on cerebral I/R injury in rats, which could be attributed to hydrogen production by the fermentation of lactulose through intestinal bacteria and Nrf2 activation.