Molecular hydrogen alleviates motor deficits and muscle degeneration in mdx mice

Objective: Duchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD.

Methods: The pregnant mother started to take supersaturated hydrogen water (>5?ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed.

Results: Hydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks.

Discussion: Although molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.     

Protection against radiation oxidative damage in mice by Triphala

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.     

Hydrophobic proteins which bind cholinergic agonists from brains of lethargic mutant mice

An investigation of hydrophobic proteins of synaptosomes isolated from the CNS tissue of the lethargic mutant mouse, which exhibits a behavioral epilepsy as the major visible effect of the mutant gene (lh), has revealed that the binding affinity of these proteolipid components for acetylcholine was higher than that observed in control (+/+) animals, a finding that may be directly related to the behavioral epilepsy.Submitted in partial fulfillment of the requirements for the Ph.D. degree.     

Early memory deficits precede plaque deposition in APPswe/PS1dE9 mice: involvement of oxidative stress and cholinergic dysfunction

A large body of evidence has shown that cognitive deficits occur early, before amyloid plaque deposition, suggesting that soluble amyloid-β protein (Aβ) contributes to the development of early cognitive dysfunction in Alzheimer disease (AD). However, the underlying mechanism(s) through which soluble Aβ exerts its neurotoxicity responsible for cognitive dysfunction in the early stage of AD remains unclear so far. In this study, we used preplaque APPswe/PS1dE9 mice ages 2.5 and 3.5 months to examine alterations in cognitive function, oxidative stress, and cholinergic function. We found that only soluble Aβ, not insoluble Aβ, was detected in these preplaque APPswe/PS1dE9 mice. APPswe/PS1dE9 mice 2.5 months of age did not show any significant changes in the measures of cognitive function, oxidative stress, and cholinergic function, whereas 3.5-month-old APPswe/PS1dE9 mice exhibited spatial memory impairment in the Morris water maze, accompanied by significantly decreased acetylcholine (ACh), choline acetyltransferase (ChAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-px) as well as increased malondialdehyde (MDA) and protein carbonyls. In 3.5-month-old preplaque APPswe/PS1dE9 mice, correlational analyses revealed that the performance of impaired spatial memory was inversely correlated with soluble Aβ, MDA, and protein carbonyls, as well as being positively correlated with ACh, ChAT, SOD, and GSH-px; soluble Aβ level was inversely correlated with ACh, ChAT, SOD, and GSH-px, as well as being positively correlated with MDA and protein carbonyls; ACh level showed a significant positive correlation with ChAT, SOD, and GSH-px, as well as a significant inverse correlation with MDA and protein carbonyls. Collectively, this study provides direct evidence that increased oxidative damage and cholinergic dysfunction may be early pathological responses to soluble Aβ and involved in early memory deficits in the preplaque stage of AD. These findings suggest that early antioxidant therapy and improving cholinergic function may be a promising strategy to prevent or delay the onset and progression of AD.     

Formation of 7-methylguanine by dichlorvos in bacteria and mice

The methylating capability of dichlorvos, as assayed by the formation of labelled 7-methylguanine from dichlorvos radioactively labelled in the methyl groups, has been studied in an Escherichia coli test system and in mice. Labelled 7-methylguanine is present in both DNA and RNA isolated from E. coli exposed to dichlorvos. Mice exposed to dichlorvos by inhalation as well as by i.p. injection excrete in the urine labelled 7-methylguanine which most likely originates from the non-enzymatic methylation of guanine moieties. The methylating capability of dichlorvos is less, by a factor of 10 to 100, than that of strongly genotoxic methylating compounds.     

Myotubularin-related protein protects against neuronal degeneration mediated by oxidative stress or infection

Microbial infections have been linked to the onset and severity of neurodegenerative diseases such as amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer''s disease, but the underlying mechanisms remain largely unknown. Here, we used a genetic screen for genes involved in protection from infection-associated neurodegeneration and identified the gene mtm-10. We then validated the role of the encoded myotubularin-related protein, MTM-10, in protecting the dendrites of Caenorhabditis elegans from degeneration mediated by oxidative stress or Pseudomonas aeruginosa infection. Further experiments indicated that mtm-10 is expressed in the AWC neurons of C. elegans, where it functions in a cell-autonomous manner to protect the dendrite degeneration caused by pathogen infection. We also confirm that the changes observed in the dendrites of the animals were not because of premature death or overall sickness. Finally, our studies indicated that mtm-10 functions in AWC neurons to preserve chemosensation after pathogen infection. These results reveal an essential role for myotubularin-related protein 10 in the protection of dendrite morphology and function against the deleterious effects of oxidative stress or infection.     

NIH Swiss and Black Swiss mice have retinal degeneration and performance deficits in cognitive tests

Swiss mice are among the most commonly used outbred strains in biomedical research. Because prior knowledge of the baseline phenotypes of mouse strains will allow informed selection of strains for particular experiments, we sought to characterize the behavior of two previously untested outbred Swiss strains--NIH Swiss and Black Swiss--in the two most widely used paradigms for evaluating the cognitive abilities of mice. Unlike the C57BL/6J and C57BL/6J-Tyr(c-2J) controls, animals of both outbred Swiss strains were unable to demonstrate learning in the Morris water maze and contextual fear conditioning paradigms. A polymerase chain reaction assay revealed that all of the NIH Swiss and Black Swiss mice tested were homozygous for the recessive retinal degeneration 1 mutation of the Pde6b gene. Histological examination of NIH Swiss and Black Swiss mouse eyes confirmed the presence of retinal degeneration, which causes visual image blindness. These findings indicate that NIH Swiss and Black Swiss mice are visually im paired and thus may be unsuitable for use in some experiments.     

Kainate-induced mitochondrial oxidative stress contributes to hippocampal degeneration in senescence-accelerated mice

We have demonstrated that kainate (KA) induces a reduction in mitochondrial Mn-superoxide dismutase (Mn-SOD) expression in the rat hippocampus and that KA-induced oxidative damage is more prominent in senile-prone (SAM-P8) than senile-resistant (SAM-R1) mice. To extend this, we examined whether KA seizure sensitivity contributed to mitochondrial degeneration in these mouse strains. KA-induced seizure susceptibility in SAM-P8 mice paralleled prominent increases in lipid peroxidation and protein oxidation and was accompanied by significant impairment in glutathione homeostasis in the hippocampus. These findings were more pronounced in the mitochondrial fraction than in the hippocampal homogenate. Consistently, KA-induced decreases in Mn-SOD protein expression, mitochondrial transmembrane potential, and uncoupling protein (UCP)-2 expression were more prominent in SAM-P8 than SAM-R1 mice. Marked release of cytochrome c from mitochondria into the cytosol and a higher level of caspase-3 cleavage were observed in KA-treated SAM-P8 mice. Additionally, electron microscopic evaluation indicated that KA-induced increases in mitochondrial damage and lipofuscin-like substances were more pronounced in SAM-P8 than SAM-R1 animals. These results suggest that KA-mediated mitochondrial oxidative stress contributed to hippocampal degeneration in the senile-prone mouse.     

Peroxiredoxin 4 protects against ovarian ageing by ameliorating d-galactose-induced oxidative damage in mice

Peroxiredoxin 4 (Prdx4), a member of the Prdx family, is a vital ER-resident antioxidant in cells. As revealed in our previous study, Prdx4 expression was detected in ovarian granulosa cells and was closely related to ovarian function. This research aimed to explore the effect and underlying molecular mechanism of the protective role of Prdx4 against d-gal-induced ovarian ageing in mice. The d-gal-induced ovarian ageing model has been extensively used to study the mechanisms of premature ovarian failure (POF). In this study, adult Prdx4^−/− and wild-type mice were intraperitoneally injected with d-gal (150 mg/kg/day) daily for 6 weeks. Ovarian function, granulosa cell apoptosis, oxidative damage and ER stress in the ovaries were evaluated in the two groups. Ovarian weight was significantly lower, the HPO axis was more strongly disrupted, and the numbers of atretic follicles and apoptotic granulosa cells were obviously higher in Prdx4^−/− mice. In addition, Prdx4^−/− mice showed increased expression of oxidative damage-related factors and the ovarian senescence-related protein P16. Moreover, the levels of the proapoptotic factors CHOP and activated caspase-12 protein, which are involved in the ER stress pathway, and the level of the apoptosis-related BAX protein were elevated in the ovaries of Prdx4^−/− mice. Thus, d-gal-induced ovarian ageing is accelerated in Prdx4^−/− mice due to granulosa cell apoptosis via oxidative damage and ER stress-related pathways, suggesting that Prdx4 is a protective agent against POF.Subject terms: Infertility, Experimental models of disease     

Countermeasures against space radiation induced oxidative stress in mice

Of particular concern for the health of astronauts during space travel is radiation from protons and high atomic number (Z), high energy particles (HZE particles). Space radiation is known to induce oxidative stress in astronauts after extended space flight. In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by proton and HZE particle radiation in the plasma of CBA mice and the protective effect of dietary supplement agents. The results indicate that exposure to proton and HZE particle radiation significantly decreased the plasma level of total antioxidants in the irradiated CBA mice. Dietary supplementation with l-selenomethionine (SeM) or a combination of selected antioxidant agents (which included SeM) could partially or completely prevent the decrease in the total antioxidant status in the plasma of animals exposed to proton or HZE particle radiation. These findings suggest that exposure to space radiation may compromise the capacity of the host antioxidant defense system; this adverse biological effect can be prevented at least partially by dietary supplementation with agents expected to have effects on antioxidant activities.     

Ergothioneine and melatonin attenuate oxidative stress and protect against learning and memory deficits in C57BL/6J mice treated with D-galactose

Abstract

Male C57BL/6J mice treated with D-galactose (DG) were used to examine the effects of ergothioneine (EGT), melatonin (MEL), or their combination (EGT+MEL) on learning and memory abilities. The mice were divided into five groups and injected subcutaneously with DG (0.3 mL of 1% DG/mouse) except for group 1 (normal controls). Group 3 was orally supplemented with EGT [0.5 mg/kg body weight (bw)], group 4 with MEL (10 mg/kg bw, p.o.), and group 5 with EGT+MEL. EGT and MEL were provided daily for 88 days, while DG was provided between days 7 to 56. Active avoidance task and Morris water-maze task were used to evaluate learning and memory abilities. DG treatment markedly increased escape latency and decreased the number of avoidance in the active avoidance test, whereas EGT and MEL alone significantly improved the performance. DG also impaired the learning and memory abilities in the water-maze task, and EGT and MEL alone also significantly improved the performance. EGT+MEL produced the strongest effects in both tasks. EGT and MEL alone markedly decreased β-amyloid protein accumulation in the hippocampus and significantly inhibited lipid peroxidation and maintained glutathione/glutathione disulfide ratio and superoxide dismutase activity in brain tissues of DG-treated mice. MEL alone completely prevented the rise in brain acetylcholine esterase activity induced by DG, whereas EGT and EGT+MEL were only partially effective. Overall, EGT, MEL, and, in particular, the combination of EGT and MEL effectively protect against learning and memory deficits in C57BL/6J mice treated with DG, possibly through attenuation of oxidative damage.     

Teratogenic potential of dichlorvos given by inhalation and gavage to mice and rabbits

Dichlorvos (2,2-dichlorovinyl dimethyl phosphate) is an important organophosphate insecticide and anthelmintic with widespread use. The purpose of this study was to evaluate the teratogenic potential of dichlorvos given orally at the maximum tolerated dose to mice (60 mg/kg/day) and rabbits (5 mg/kg/day) and by inhalation in both species at a concentration of 4 microgram/l seven hours daily. Dichlorvos was not found to be teratogenic in either species by either route of administration.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015-15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg(9) to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

Prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice

The present study investigated the prophylactic influence of melatonin against cyclophosphamide-induced oxidative stress in mouse tissues. Lipid peroxidation, reduced glutathione (GSH), glutathione disulphide (GSSG), glutathione peroxidase (GSH-Px) and serum phosphatase levels were analyzed in brain, spleen liver, lungs, kidney and testes. Fifteen days oral administration with melatonin (0.1 mg/kg bw per day) before treatment checked the augmentation of the level of lipid peroxidation, blood GSSG and acid phosphatase caused by an acute treatment with a radiomimetic drug, cyclophosphamide (75 mg/kg bw). Cyclophosphamide-induced depletion in the level of GSH, GSH-Px and alkaline phosphatase was made up statistically significant by chronic melatonin administration given orally. The results indicate the antioxidative properties of melatonin resulting into its prophylactic property against the cyclophosphamide-induced biochemical alterations. The finding support the idea that melatonin is a potent free-radical scavenger and antioxidant.     

Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein

Although minor abnormalities have been reported in prion protein (PrP) knock-out (Prnp-/-) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp-/- as compared to wild-type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin-protein conjugates were also observed in Prnp-/- mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.     

Constituents of bile, bilirubin and TUDCA, protect against oxidative stress-induced retinal degeneration

Two constituents of bile, bilirubin and tauroursodeoxycholic acid (TUDCA), have antioxidant activity. However, bilirubin can also cause damage to some neurons and glial cells, particularly immature neurons. In this study, we tested the effects of bilirubin and TUDCA in two models in which oxidative stress contributes to photoreceptor cell death, prolonged light exposure and rd10+/+ mice. In albino BALB/c mice, intraperitoneal injection of 5 mg/kg of bilirubin or 500 mg/kg of TUDCA prior to exposure to 5000 lux of white light for 8 h significantly reduced loss of rod and cone function assessed by electroretinograms. Both treatments also reduced light-induced accumulation of superoxide radicals in the outer retina, rod cell death assessed by outer nuclear layer thickness, and disruption of cone inner and outer segments. In rd10+/+ mice, intraperitoneal injections of 5 or 50 mg/kg of bilirubin or 500 mg/kg of TUDCA every 3 days starting at postnatal day (P) 6, caused significant preservation of cone cell number and cone function at P50. Rods were not protected at P50, but both bilirubin and TUDCA provided modest preservation of outer nuclear layer thickness and rod function at P30. These data suggest that correlation of serum bilirubin levels with rate of vision loss in patients with retinitis pigmentosa could provide a useful strategy to test the hypothesis that cones die from oxidative damage in patients with retinitis pigmentosa. If proof-of-concept is established, manipulation of bilirubin levels and administration of TUDCA could be tested in interventional trials.     

Protective effects of autophagy against blue light-induced retinal degeneration in aged mice

The aim of this study was to explore the role of autophagy in response to blue light damage in aged mice and in human retinal pigmented epithelium(hRPE) cells. Blue light damage to the retina was induced in 10-month-old(10 mo) C57 mice and hRPE cells. Flash electroretinography was used to assess retinal function. Retinal structure changes were observed by electron microscopy. Western blot was conducted to determine the expression levels of the following proteins: cleaved caspase-3, p38 mitogen-activated protein kinases, protein kinase R-like endoplasmic reticulum kinase(PERK), autophagy marker light chain 3(LC3), P62, and Beclin-1. On day 1 after light damage to the 10 mo mice, retinal function was changed. The latent periods of awave and b-wave were delayed, and amplitude was reduced. The electron microscopy results revealed mitochondria damage in the retinal pigmented epithelium and a disorganized photoreceptor outer segment(OS). PERK, LC3, and Beclin-1 were upregulated, whereas P62 was not. On day 5 after the blue light damage, restoration of electroretinography and OS was observed.PERK, LC3, and Beclin-1 were downregulated, whereas P62 was not. Protein changes in vitro were consistent with in vivo. The present study provided structural and functional evidence that autophagy plays an important role in the response to blue lightinduced retinal damage.     

Elevated oxidative stress and sensorimotor deficits but normal cognition in mice that cannot synthesize ascorbic acid

Oxidative stress is implicated in the cognitive deterioration associated with normal aging as well as neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. We investigated the effect of ascorbic acid (vitamin C) on oxidative stress, cognition, and motor abilities in mice null for gulono-γ-lactone oxidase (Gulo). Gulo−/− mice are unable to synthesize ascorbic acid and depend on dietary ascorbic acid for survival. Gulo−/− mice were given supplements that provided them either with ascorbic acid levels equal to- or slightly higher than wild-type mice (Gulo-sufficient), or lower than physiological levels (Gulo-low) that were just enough to prevent scurvy. Ascorbic acid is a major anti-oxidant in mice and any reduction in ascorbic acid level is therefore likely to result in increased oxidative stress. Ascorbic acid levels in the brain and liver were higher in Gulo-sufficient mice than in Gulo-low mice. F₄-neuroprostanes were elevated in cortex and cerebellum in Gulo-low mice and in the cortex of Gulo-sufficient mice. All Gulo−/− mice were cognitively normal but had a strength and agility deficit that was worse in Gulo-low mice. This suggests that low levels of ascorbic acid and elevated oxidative stress as measured by F₄-neuroprostanes alone are insufficient to impair memory in the knockouts but may be responsible for the exacerbated motor deficits in Gulo-low mice, and ascorbic acid may have a vital role in maintaining motor abilities.     

Endorphins in brains of decapitated and microwave-killed mice

The size distribution of opioid peptides (endorphins) in mouse brain was determined by gel filtration, using the inhibition of stereospecific ³H-etorphine binding to guinea pig brain membranes as a measure of endorphin activity. The largest endorphin was about the same size as β-LPH-(61–91), the next largest was about half this size and the smallest was the size of the pentapeptide enkephalins. There was no important difference in the size distribution between brains of mice killed by rapid microwave irradiation and those killed by decapitation. Thus, opioid peptides of all three size classes appear to be present in the brain $\text{in}$$\text{vivo}$.