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Neurotherapeutic and antioxidant response of D-ribose-L-Cysteine nutritional dietary supplements on Alzheimer-type hippocampal neurodegeneration induced by cuprizone in adult male Wistar rat model

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Abstract

Introduction: Cuprizone is a neurotoxicant causing neurodegeneration through enzyme inhibition and oxidative stress. D-Ribose-L-Cysteine (DRLC) is a powerful antioxidant with neuroprotective properties. This study explored the antioxidant response of DRLC against cuprizone-induced behavioral alterations, biochemical imbalance, and hippocampal neuronal damage in adult Wistar rats. Materials and methods: Thirty-two (32) adult male Wistar rats (150–200g) were divided into four groups (n = 8). Group A received normal saline only as placebo; Group B received 0.5% cuprizone diet only; Group C received a combination of 0.5% cuprizone diet and 100 mg/kg BW of DRLC and Group D received 100 mg/kg BW of DRLC only. The administration was done through oral gavage once daily for 45 days. After the last treatment, neurobehavioral tests (Morris Water Maze and Y maze) was conducted; animals sacrificed and brain harvested for histological analysis and biochemical estimations of levels of antioxidants, oxidative stress markers, neurotransmitters and enzyme activities. Results: The results showed significant memory decline, hippocampal alterations, decrease levels of antioxidant markers, enzyme and neurotransmitters activities with a concomitant increase in norepinephrine and oxidative stress markers in cuprizone induced rats relative to normal but were attenuated with DRLC administration. Conclusion: Cuprizone causes cognitive impairment and neurodegeneration through oxidative stress; however, administration of DRLC ameliorated neuropathological alteration induced by cuprizone.

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A broad spectrum of diseases is characterized by myelin abnormalities and/or oligodendrocyte pathology. In most, if not all, of these diseases, early activation of microglia occurs. Our knowledge regarding the factors triggering early microglia activation is, however, incomplete. In this study, we used the cuprizone model to investigate the temporal and causal relationship of oligodendrocyte apoptosis and early microglia activation. Genome-wide gene expression studies revealed the induction of distinct chemokines, among them Cxcl10, Ccl2, and Ccl3 in cuprizone-mediated oligodendrocyte apoptosis. Early microglia activation was unchanged in CCL2- and CCL3-deficient knockouts, but was significantly reduced in CXCL10-deficient mice, resulting in an amelioration of cuprizone toxicity at later time points. Subsequent in vitro experiments revealed that recombinant CXCL10 induced migration and a proinflammatory phenotype in cultured microglia, without affecting their phagocytic activity or proliferation. In situ hybridization analyses suggest that Cxcl10 mRNA is mainly expressed by astrocytes, but also oligodendrocytes, in short-term cuprizone-exposed mice. Our results show that CXCL10 actively participates in the initiation of microglial activation. These findings have implications for the role of CXCL10 as an important mediator during the initiation of neuroinflammatory processes associated with oligodendrocyte pathology. Copyright © 2015 by The American Association of Immunologists, Inc.
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Oxidative stress has been strongly implicated in both the inflammatory and neurodegenerative pathological mechanisms in multiple sclerosis (MS). In response to oxidative stress, cells increase and activate their cellular antioxidant mechanisms. Glutathione (GSH) is the major antioxidant in the brain, and as such plays a pivotal role in the detoxification of reactive oxidants. Previous research has shown that GSH homeostasis is altered in MS. In this review, we provide a comprehensive overview on GSH metabolism in brain cells, with a focus on its involvement in MS. The potential of GSH as an in vivo biomarker in MS is discussed, along with a short overview of improvements in imaging methods that allow non-invasive quantification of GSH in the brain. These methods might be instrumental in providing real-time measures of GSH, allowing the assessment of the oxidative state in MS patients and the monitoring of disease progression. Finally, the therapeutic potential of GSH in MS is discussed.
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Acetylcholinesterase is a member of the α/β hydrolase protein super family, with a significant role in acetylcholine-mediated neurotransmission. Research in the modulators of AChEs has moved from a potent poison (Sarin, Soman) in war times to the potent medicine (physostigmine) in peaceful times. Natural anti-AChE includes carbamates, glycoalkaloids, anatoxins derived from green algae; synthetic anti-AChE includes highly poisonous organophosphates used as nerve gases and insecticides. Recently, the role of anti-AChE was reassessed from neurotoxins to neuron-protective in the diseases characterized by impaired acetylcholine-mediated neurotransmission like Alzheimer's disease (AD). So, the AChE has been proven to be the most viable therapeutic target for the symptomatic treatment of AD. This review article gives a spectrum of strategies to design AChE inhibitors used in the Alzheimer therapy.
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In this review the differences in pathology and disease mechanisms between early and late stages of multiple sclerosis are discussed. The data suggest that affection of the brain is different, depending on the location of lesions, on the stage of the disease, when lesions arise, and on inter-individual differences between patients. We suggest that in the early stage of the disease new lesions are formed by new waves of inflammatory cells, entering the central nervous system from the circulation and giving rise to focal demyelinated plaques in the white and gray matter. In contrast, at late stages of the disease inflammation decreases, but the susceptibility of the target tissue for neurodegeneration increases. New data suggest that mitochondrial injury, mediated through oxidative injury, is in the center of the pathogenetic events leading to brain damage in multiple sclerosis patients.
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The oral ingestion of soluble nickel compounds leads to neurological symptoms in humans. Deficiencies in aerobic metabolism induced by neurotoxic stimulus can cause an energy crisis in the brain that results in a variety of neurotoxic effects. In the present study, we focused on the aerobic metabolic states to investigate whether disturbance of aerobic metabolism was involved in nickel-induced neurological effects in mice. Mice were orally administered nickel chloride, and neurobehavioral performance was evaluated using the Morris water maze and open field tests at different time points. Aerobic metabolic states in the cerebral cortex were analyzed at the same time points at which neurobehavioral changes were evident. We found that nickel exposure caused deficits in both spatial memory and exploring activity in mice and that nickel was deposited in their cerebral cortex. Deficient aerobic metabolism manifested as decreased O2 consumption and ATP concentrations, lactate and NADH accumulation, and oxidative stress. Meanwhile, the activity of prototypical iron-sulfur clusters (ISCs) containing enzymes that are known to control aerobic metabolism, including complex I and aconitase, and the expression of ISC assembly scaffold protein (ISCU) were inhibited following nickel deposition. Overall, these data suggest that aerobic metabolic disturbances, which accompanied the neurobehavioral changes, may participate in nickel-induced neurologic effects. The inactivation of ISC containing metabolic enzymes may result in the disturbance of aerobic metabolism. A better understanding of how nickel impacts the energy metabolic processes may provide insight into the prevention of nickel neurotoxicity.
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Remyelination is disrupted in demyelinating diseases such as multiple sclerosis, but the underlying pathogenetic mechanisms are unclear. In this study, we employed the murine cuprizone model of demyelination, in which remyelination occurs after removal of the toxin from the diet, to examine the cellular and molecular changes during demyelination and remyelination. Microglia accumulated in the corpus callosum during weeks 2-4 of the cuprizone diet, and these cells remained activated 2 weeks after the change to the normal diet. To examine the role of microglia in remyelination, mice were treated with minocycline to inactivate these cells after cuprizone-induced demyelination. Minocycline treatment reduced the number of CC1-positive oligodendrocytes, as well as levels of MBP and CNPase in the remyelination phase. The expression of CNTF mRNA in the corpus callosum increased after 4 weeks on the cuprizone diet and remained high 2 weeks after the change to the normal diet. Minocycline suppressed CNTF expression during the remyelination phase on the normal diet. Primary culture experiments showed that CNTF was produced by microglia in addition to astrocytes. In vitro, CNTF directly affected the differentiation of oligodendrocytic cells. These findings suggest that minocycline reduces remyelination by suppressing CNTF expression by microglia after cuprizone-induced demyelination. This article is protected by copyright. All rights reserved.
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The in vivo and in vitro effects of Al on the cholinergic system of rat brain were studied. The amount of Al accumulated after the chronic, intraperitoneal administration of aluminium gluconate (Al-G) or AlCl3, both at a dose of 1 mg/ml/100 g of body weight, increased in the frontal and parietal cortices, the hippocampus, and the striatum. Significantly decreased choline acetyltransferase activities after chronic Al treatment were measured in the parietal cortex, the hippocampus, and the striatum, but not in the frontal cortex. The acetylcholinesterase activity was not changed significantly in any brain area investigated. Both Al-G and AlCl3 administrations resulted in a general decrease (to 40-70% of the control values) in the specific l-[3H]nicotine binding, involving all brain areas studied. The specific (-)-[3H]quinuclidinyl benzilate binding was reduced (to 40-60% of the control values) only after 25 days of Al treatment. Al-G and AlCl3 were equivalent in eliciting these reductions in vitro studies revealed different alterations of the cholinergic system in response to Al treatment. No changes were observed either in choline acetyltransferase activity or in cholinergic receptor bindings. Both Al-G and Al2(SO4)3 treatments, however, exhibited a biphasic effect on the acetylcholinesterase activity. At low Al concentrations (10(-8)-10(-6) M), the activity was slightly increased, whereas at higher concentrations (10(-6)-10(-4) M), it was inhibited by a maximum of 25% as compared to the controls. Thus, these cholinotoxic effects are probably due not to a direct interaction between the metal and the cholinergic marker proteins, but rather to a manifestation and consequence of its neurodegenerative effects.
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Multiple sclerosis (MS) is defined as a chronic inflammatory disease of the central nervous system, which leads to focal inflammatory demyelinated lesions with secondary neurodegeneration. However, this concept has recently been challenged by several observations suggesting that in this disease neurodegeneration might occur independently of inflammation. Here, these new findings are critically discussed and evidence that active neurodegeneration in MS is invariably associated with inflammation is provided. The present review shows, however, that the inflammatory reaction is much more complex, as thought before, and that in the progressive stage of the disease it might become trapped in the central nervous system behind a repaired blood–brain barrier. Future therapeutic options for this disease are discussed on the basis of recent knowledge of the mechanisms of inflammation and neurodegeneration. (Clin. Exp. Neuroimmunol. doi: 10.1111/ j.1759-1961.2009.00003.x, January 2010)
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INTRODUCTION Hematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years because it works well with a variety of fixatives and displays a broad range of cytoplasmic, nuclear, and extracellular matrix features. Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast. The Golgi zone can be tentatively identified by the absence of staining in a region next to the nucleus. Thus, the stain discloses abundant structural information, with specific functional implications. A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed. Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.
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Growing evidence indicates that oxidative stress and mitochondrial dysfunction plays a critical role in brain aging. Chronic injection of D-galactose can cause gradual deterioration in learning and memory capacity and serve as an animal model of aging. Recently, potential therapeutic effect of carvedilol (CAR) has been reported by virtue of which its antioxidant and mitochondrial permeability transitional property. The present study has been designed to explore the CAR effect against D-galactose-induced behavioral, biochemical, and mitochondrial dysfunction in mice. Systemic administration of D-galactose for 6 weeks significantly impaired behavioral (learning and memory and locomotor activity), biochemical parameters (raised lipid peroxidation, nitrite concentration, depletion of reduced glutathione, and catalase activity), and mitochondrial enzymes (decreased complex I, II and III enzymes levels) as compared to sham group. CAR (2.5 and 5 mg/kg) treatment significantly improved behavioral abnormalities and biochemical and cellular alterations as compared to control. Chronic administration of D-galactose for a period of 6 week results into a significant increase of acetylcholine esterase enzyme level. CAR (2.5 and 5 mg/kg) treatment significantly attenuated the elevated level of acetylcholine esterase of mice. In conclusion, present studies highlight the protective effects of CAR against D-galactose-induced behavioral, biochemical, and mitochondrial dysfunction in mice. The study further provides a hope that CAR could be used in the management of cognitive dysfunction and related symptoms during aging.
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This study examined the hypothesis that l-cysteine supplementation can lower insulin resistance, glycemia, oxidative stress, and markers of vascular inflammation in type 2 diabetes using Zucker diabetic fatty (ZDF) rats as a model. Starting at the age of 6 weeks, ZDF rats were supplemented orally (daily gavage, 8 weeks) with saline placebo (D) or l-cysteine (LC; 1 mg/kg bw) and fed a high-calorie diet. Six-week-old rats without any supplementation were considered baseline (BL) rats. D rats showed elevated fasting blood glucose, glycated Hb, CRP, and MCP-1 compared with BL rats in which there was no onset of diabetes. LC supplementation significantly lowered blood levels of glucose (18%, p= 0.05), glycated Hb (8%, p= 0.02), CRP (23%, p= 0.02), MCP-1 (32%, p= 0.01), and insulin resistance (25%) compared with levels seen in saline-supplemented D rats. There was a decrease in plasma protein oxidation levels (p< 0.01); however, GSH levels were similar in LC and D groups. Although LC did not change blood hematocrit or levels of transaminases, it did lower alkaline phosphatase (29%, p= 0.01) levels in comparison to D. Western blotting analyses of liver showed increased activation of NF-kappaB and Akt (50% pNF-kappaB and 20% pAkt) in D compared with BL rats. LC supplementation inhibited these effects (17% pAkt, 18% pNF-kappaB). This is the first report showing that l-cysteine supplementation can lower glycemia and markers of vascular inflammation in diabetes apparently by preventing NF-kappaB activation in a diabetic animal model.
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Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated chain-propagating autoxidation of epinephrine. The enzyme assay based on adrenochrome absorption at 480 nm has been improved by measuring the absorption change at 320 nm. This alternative procedure was found to be 6 to 10 times more sensitive and more consistent than that measured at 480 nm.
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The techniques of microscopic histochemistry, successfully applied to problems in histopathology, necessarily result in increased objectivity. Combined with procedures for quantification the increase may be such as to remove subjective judgment altogether. Extended to the ultrastructural level the quality of the information derived is greatly enhanced in terms of localization but there is usually no increase in objectivity. Of all branches of technology capable of conferring on histopathology an increase in objectivity, histochemistry must take first place.
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This laboratory has previously postulated that bromobenzene-induced hepatic necrosis results from the formation of a reactive metabolite that arylates vital cellular macromolecules. Accordingly, the severity of liver necrosis has been compared with the formation of metabolites of bromobenzene and with covalent binding of metabolites in vivo and in vitro after various pretreatment regimens that alter hepatotoxicity. These data provide direct kinetic evidence that 3,4-bromobenzene oxide is the reactive hepatotoxic metabolite. The studies also demonstrate that the hepatotoxic metabolite is preferentially conjugated (detoxified) with glutathione, thereby depleting glutathione from the liver. Liver necrosis and arylation of cellular macromolecules occur only when glutathione is no longer available. Thus, a dose threshold exists for bromobenzene-induced hepatic necrosis.
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A method is described for the rapid dissection of seventeen areas of the rat brain. Regions from fresh unfrozen brain tissue are dissected from coronal brain slices obtained with use of a cutting block. This method is applicable to pharmacological and behavioral studies which require the dissection of numerous brains during short time intervals.
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Focus on the biomedical aspects of AD in research and clinical care has resulted in the relative neglect of its psychological and social dimensions. An adequate clinical response to individuals diagnosed with AD must include attention to preserving continuity with the past and quality of life in the present. As AD is diagnosed earlier, care models that incorporate the experiences and perceptions of the diagnosed are not only feasible, but essential.
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The cholinotoxic effect of aluminium has been widely reported. In vitro aluminium has a biphasic effect on acetylcholinesterase activity. The present study analyses its in vivo effect in brain regions. Rats were exposed to aluminium chloride by the oral route at a dose of 320 mg/kg body weight for shorter (4 and 14 days) and longer (60 days) duration. Acetylcholinesterase activity in olfactory bulb, striatum and hypothalamus brain regions increased after 4 and 14 days and decreased after 60 days of aluminium exposure. Aluminium level in the brain regions studied increased significantly. No significant change in body weight of rats exposed to aluminium was found. The biphasic change in acetylcholinesterase activity may be due to slow accumulation of aluminium in the brain regions and its effect on the enzyme.
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Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.
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The effect of kolaviron, a mixture of Garcinia biflavonoid 1 (GB1), Garcinia biflavonoid 2 (GB2) and kolaflavanone, used in the treatment of various ailments in southern Nigeria on hepatotoxicity and lipid peroxidation induced by 2-acetylaminofluorene (2-AAF) in rats was investigated. The ability of butylated hydroxyanisole (BHA) to attenuate the toxic effect of 2-AAF was also examined. Kolaviron administered orally to rats at a dose of 100mg/kg body weight twice a day for 1 week before challenge with 2-AAF (200mg/kg feed) and continuously for 3 weeks at a single dose of 200mg/kg body weight reversed the 2-AAF-mediated decrease in final body weight and relative organ weights, especially the liver. BHA was administered at a dose of 7.5g/kg feed to the animals for 4 weeks. The extract decreased significantly the 2-AAF-mediated increase in the activity of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase and ornithine carbamyl transferase by 58%, 62%, 60% and 67%, respectively. BHA elicited respectively 55%, 63%, 57% and 65% reduction in the 2-AAF induced-increase in the activities of these enzymes. Histological examination of the liver slices correlated with the changes in serum enzyme alterations. Similarly, kolaviron decreased the 2-AAF reduction of 5'-nucleotidase and glucose-6-phosphatase activities by 63% and 60%, respectively while BHA elicited 59% and 61% decrease in the activities of these enzymes. Simultaneous administration of kolaviron with 2-AAF inhibited microsomal lipid peroxidation as assessed by the thiobarbituric acid reacting substances (TBARS) formation by 66%. BHA produced a 64% reduction in TBARS formation. In the present study, kolaviron appears to act as an in vivo natural antioxidant and an effective hepatoprotective agent and is as effective as BHA.
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The work presented here describes an optimised, reversed-phase high-performance liquid chromatographic (RP-HPLC) method for separating 46 biogenic compounds, which, as neurotoxins or as their precursors or derivatives, may be relevant in the pathomechanism of Parkinson's disease. In some cases, the physico-chemical properties of these substances are very similar, in other cases they differ greatly. In order to facilitate their detection in one chromatographic run, ion-pair chromatography was uniquely combined with a gradient elution. A diode array or a dual wavelength detector was used in combination with a fluorescence detector to verify the identity of the compounds.
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Prolonged separation from the mother can interfere with normal growth and development and is a significant risk factor for adult psychopathology. In rodents, separation of a pup from its mother increases the behavioral and endocrine responses to stress for the lifetime of the animal. Here we investigated whether maternal deprivation could affect brain development of infant rats via changes in the rate of cell death as measured by labeling the 3' end of DNA fragments using terminal transferase (ApopTag). At postnatal day 12 (P12), the number of cells undergoing cell death approximately doubled in the cerebral cortex, cerebellar cortex and in several white matter tracts following 24 h of maternal deprivation. Deprivation strongly increased the number of ApopTag-labeled cells at P6 but not at P20. Stroking the infant rats only partially reversed the effects of maternal deprivation. Increased cell death in white matter tracts correlated with an induction of nerve growth factor which has been previously associated with oligodendrocyte cell death. Cell birth was either unchanged or decreased in response to deprivation. These results indicate that maternal deprivation can alter normal brain development by increasing cell death of neurons and glia, and provides a potential mechanism by which early environmental stressors may influence subsequent behavior.
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A water-soluble (at pH 8) aromatic disulfide [5,5′-dithiobis(2-nitrobenzoic acid)] has been synthesized and shown to be useful for determination of sulfhydryl groups.Several applications have been made to show its usefulness for biological materials.A study of the reaction of this disulfide with blood has produced some evidence for the splitting of disulfide bonds by reduced heme.