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Dietary antioxidant supplementation reverses age-related neuronal changes
Neurobiology of Aging, Vol. 19, No. 5, pp. 461– 467, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/98 $19.00 1 .00
PII:S0197-4580(98)00082-7
Dietary Antioxidant Supplementation Reverses Age-related Neuronal Changes E. O’DONNELL AND M. A. LYNCH1 Department of Physiology, Trinity College, Dublin, Ireland Received 11 May 1998; Revised July 31; Accepted 31 July 1998 O’DONNELL, E. AND M. A. LYNCH. Dietary antioxidant supplementation reverses age-related neuronal changes. NEUROBIOL AGING 19(5) 461– 467, 1998.—Evidence suggests that reactive oxygen species in brain may play a role in the development of age-related neuronal impairments, and that the increase in the concentration of the proinflammatory cytokine, interleukin-1b (IL-1b), in aged brain tissue, may also be a contributory factor. In this study, we have analyzed changes in enzymatic and nonenzymatic antioxidant levels, in parallel with interleukin-1b concentration, in cortical tissue prepared from young and aged rats. We report that there was an age-related increase in the activity of superoxide dismutase without concomitant changes in the activity of catalase or glutathione peroxidase and an age-related decrease in the concentrations of a-tocopherol and ascorbate. These observations, coupled with age-related increases in lipid peroxidation and interleukin-1b concentration, are consistent with a compromised antioxidant defense in cortex of aged rats, a proposal supported by the finding that these changes were not observed in cortical tissue prepared from rats fed on a diet supplemented with a-tocopherol and ascorbate for 12 weeks. © 1998 Elsevier Science Inc. Antioxidants
a-Tocopherol
IL-1b
Cortex
Lipid peroxidation
AGEING is associated with an array of changes in neuronal function, many of which have been attributed to accumulation of reactive oxygen species, arising from either increased formation of reactive oxygen species, compromised ability of the aged brain to cope with oxidative stress, or both (10,34,52). Neuronal tissue is particularly susceptible to oxidative damage due to high oxygen consumption coupled with modest antioxidant defense strategies (17), high concentrations of iron (4), and high concentrations of polyunsaturated fatty acids, which renders the tissue susceptible to oxygen radicals (35). Among the likely consequences of prolonged exposure to reactive oxygen species are changes in the lipid composition of membranes (52) which in turn are likely to impact on membraneassociated functions. Thus release of several neurotransmitters from cortical tissue, e.g., acetylcholine (18,25), 5-hydroxytryptamine (14), and glutamate [e.g., (2), but see (39)] decreases with age, and this is paralleled by age-related alterations in calcium handling by cells (13,19,48) and down-regulation of membrane associated enzymes like lipases (24) and kinases (3). Direct evidence has demonstrated that membrane composition alters with age (7,15,22,24) resulting in increased rigidity of neuronal membranes (10,52), which might, in turn, underlie age-related changes in membrane-associated functions. The trigger which induces the change in membrane composition has not been identified, but we have recently observed an age-related increase in the proinflammatory cytokine, interleukin 1b (IL-1b) in the hippocampus (28 –30) and have some evidence suggesting that IL-1b may contribute to the membrane changes. Thus we observed that the endogenous increase in IL-1b was accompanied by 1
Superoxide dismutase
Ascorbic acid
increased formation of reactive oxygen species, increased lipid peroxidation, and decreased arachidonic acid concentration (29, 30), whereas reactive oxygen species formation (41) and lipid peroxidation (30) were both increased by IL-1b in vitro. In this study we have investigated age-related changes in enzymatic and nonenzymatic antioxidant strategies in the cortex of the rat in parallel with age-related changes in lipid peroxidation and IL-1b. We have assessed the possibility that age-related changes might be reversed by dietary supplementation with antioxidant vitamins. EXPERIMENTAL PROCEDURES
Male Wistar rats, aged 4 months or 22 months (Charles River Laboratory, UK) were used in this study. Animals were housed in the BioResources Unit in Trinity College, Dublin in groups of four (young rats) or in pairs (aged rats) on a 12-h light:dark cycle, at an ambient temperature of 22–23°C. Twenty-four aged and twelve young animals were randomly divided into two subgroups; one aged and one young group were fed for 12 weeks on laboratory chow, to which dL-a-tocopheryl acetate (250 mg/rat/day; Beeline Healthcare, Ireland, UK) dissolved in corn oil was added. Ascorbic acid (250 mg/rat/day) was added to the water given to these rats. The second subgroup of aged and young rats were fed a control diet which was normal laboratory chow to which corn oil was added to ensure isocaloric intake. Sufficient diet was prepared for 1 week, divided into daily portions, and stored in sealed bags at 220°C until required. During the 2 weeks before the start of dietary manipulation, daily food and water intake was measured and during the period of dietary manipulation, rats were offered
Address correspondence to: M. A. Lynch, Faculty of Health Sciences, Department of Physiology, Trinity College, Dublin 2, Ireland; E-mail: [email protected]
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462 100% of their average daily food and average water intakes so that the full daily allowances of vitamins would be ingested. Average daily food intake was similar in young (34 g) and aged (35 g) rats, whereas daily water intake was slightly higher in young rats (53 mL) compared with aged rats (40 mL). While the average weight of young rats increased from 330 to 548 g during the 12 weeks of dietary manipulation, aged rats did not show any significant change in body weight; in the vitamin-treated group average weight increased from 572 to 589 g and in the control group average weight decreased from 637 to 626 g. Rats were monitored daily throughout the experimental period, and were maintained under veterinary supervision; two aged rats in the vitamin-treated group were euthanized during the first 2 weeks of the experiment due to apparent ill-health, whereas all other rats remained healthy for the duration of the experiment. At the end of the 12-week period of dietary manipulation, rats were anaesthetized with urethane (1.5 g/kg), analyzed for their ability to sustain long-term potentiation in hippocampus (not reported here), and at the end of a 1-h recording period were killed by decapitation. The cortex was dissected free and homogenized in either Krebs solution containing 2 mM CaCl2, or in 5% trichloroacetic acid for later analysis of ascorbic acid and glutathione. Aliquots of homogenate were transferred to microfuge tubes and stored at 280°C until required for analysis. For use, supernatant was prepared by centrifugation at 15,000 rpm for 6 min. Total glutathione was measured as described previously (44). Briefly, aliquots of supernatant prepared from homogenate (30 mL) were incubated for 1 min on ice with sample buffer (100 mM potassium phosphate containing 5 mM EDTA, pH 7.5; 450 mL), in the presence of glutathione reductase (100 mL; 5 U/mL) and 10 mM 5,59-dithiobis-2-nitrobenzoic acid (50 mL). NADPH (2.4 mm; 100 mL) was added and the absorbance monitored for 2 min at 412 nm. Results were expressed as mmol GSH/g of tissue. Glutathione peroxidase activity was measured according to the method of Lawrence and Burk (21). Samples of supernatant (100 mL) were added to incubation buffer (50 mM potassium phosphate (pH 7), containing 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase, 1 mM GSH glutathione, and 1.5 mM cumene hydroperoxide; 800 mL) and incubated for 5 min at room temperature. The reaction was initiated by addition of cumene hydroperoxide (15 mM; 100 mL). Absorbance was recorded at 340 nm for 5 min; activity was calculated from the slope (i.e., change in absorbance with time), and the results were expressed as mmols NADPH oxidised per minute per milligram of protein (6). Ascorbic acid concentrations were determined as previously described (32). Briefly, duplicate aliquots of supernatant (100 mL) were added to a 2,4-dinitrophenylhydrazine/thiourea/copper solution (50 mM thiourea, 2 mM copper sulfate and 150 mM dinitrophenylhydrazine in 9 N H2SO4; 20 mL) and incubated for 3 h at 37°C. Ice-cold H2SO4 (65%; 150 mL) was added to stop the reaction; samples were vortex-mixed and incubated at room temperature for 30 min before aliquots (100 mL) were transferred to 96-well plates for assessment by ultraviolet (UV) spectroscopy at 545 nm. Results were expressed as mmoles of ascorbic acid per gram of tissue. Ascorbic acid standards were prepared in 5% trichloroacetic acid. Superoxide dismutase activity was determined according to a method previously described (40). Aliquots (800 mL) of incubation buffer (50 mM potassium buffer (pH 7.8) containing 1.8 mM xanthine, 2.24 mM nitroblue tetrazolium, 40 U catalase, 7 mL/mL xanthine oxidase, and 1.33 mM diethylenetriaminepentacetic acid) were added to 1.5 mL microfuge tubes containing samples of supernatant (100 mL) at different dilutions (1:2, 1:5, 1:10, 1:20, 1:50, and 1:100) and analyzed by spectroscopy at 560 nm. Activity
O’DONNELL AND LYNCH of superoxide dismutase was assessed as the rate of reduction of nitroblue tetrazolium, which was inhibited with increasing concentrations of protein. One unit of activity was defined as the amount of protein necessary to decrease the rate of the reduction of nitroblue tetrazolium by 50%. Results were expressed in units of superoxide dismutase activity per mg protein. Catalase activity was determined as previously described (11) in tissue prepared from different groups of aged (22 months) and young (4 months) rats; this was simply because of insufficient tissue remaining when all other analyses had been completed on tissue derived from the experiment in which dietary manipulations were made. Rats were killed by stunning and decapitation. The brain was removed, and the cortex was isolated and homogenized in Krebs solution containing 2 mM CaCl2. Samples of homogenate were stored at 280°C for analysis. Duplicate aliquots of supernatant (50 mL) were added to microfuge tubes containing buffer (10 mM potassium phosphate, pH 7.0; 800 mL) tubes and kept on ice. The reaction was initiated by the addition of ice-cold H2O2 (60 mM; 100 mL), samples were mixed and incubated on ice for 2 or 10 min after which time aliquots (100 mL) were removed and quenched by addition to H2SO4 (0.6 N; 4 mL) and 10 mM FeS4 (1 mL) at room temperature. Color was developed at room temperature by addition of 2.5 M potassium thiocyanate (400 mL). Aliquots (200 mL) were transferred immediately to 96-well plates, absorbance was read at 492 nm and results were expressed in terms of the first order reaction rate constant (k), corrected for protein using the formula: enzyme units 5 k/protein 5 [ln (A1/A2/t]/ protein, where A1 and A2 represented the absorbance at the two selected time points (i.e., 2 and 10 min), and t, the time difference between the two points (i.e., 8 min). Results are expressed as enzyme units per gram of protein. We used an ELISA for analysis of IL-1b (Genzyme Diagnostics). Briefly, 96-well plates were coated with 100 mL of capture antibody (2.0 mg/mL final concentration, diluted in 0.1 M sodium carbonate buffer, pH 9.5; monoclonal hamster anti-mouse IL-1b antibody) and incubated overnight at 4°C. Wells were washed several times with PBS containing 0.05% Tween 20 and then blocked for 2 h at 37°C with 250 mL of blocking buffer, (PBS, pH 7.3; 0.1 M with 4% bovine serum albumin (BSA)). Blocking buffer was aspirated and aliquots (50 mL) of samples or IL-1b standards (0 –1000 pg/mL) were added to each well and incubated for 1 h at 37°C. Plates were washed, secondary antibody (100 mL; final concentration 0.8 mg/mL in PBS containing 0.05% Tween 20 and 1% BSA; biotinylated polyclonal rabbit anti-mouse IL-1b antibody) was added to each well and incubation continued for 1 h at 37°C. The plates were washed, and 100 mL detection agent (horseradish peroxidase conjugated streptavidin; 1:1000 dilution in PBS containing 0.05% Tween 20 and 1% BSA) was added to each well, incubation continued for a further 15 min at 37°C, and plates were again washed. Aliquots of substrate (100 mL; tetramethylbenzidine liquid substrate; Sigma) were added, and the samples were incubated at room temperature for 10 min. Absorbance was read at 450 nm within 30 min. Results were expressed as pg IL-1b/mg of tissue. Lipid peroxidation was determined by a previously described method in which analysis of thiobarbiturate reactive substances was made (12). Aliquots (10 mL) of homogenate were incubated at 37°C for 60 min, at which time 8.1% (wt/vol) SDS (30 mL), 20% acetic acid (pH 3.5 with NaOH; 225 mL), and 0.8% (wt/vol) thiobarbituric acid (225 mL) were added. The volume was adjusted with H2O to 600 mL, the samples incubated for a further 60 min at 95°C, cooled at room temperature, and absorbance assessed at 532 nm with reference to a standard curve made using 1,1,3,3,tetramethoxypropane. In some experiments, tissue or IL-1b or buffer was added to samples and incubation continued as described
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FIG. 1. The age-related decreases in ascorbate and a-tocopherol concentration in rat cortical tissue are prevented by dietary supplementation with ascorbate and a-tocopherol. (A) Ascorbate concentration was significantly decreased in cortical tissue prepared from aged rats (22 months; 22 m) fed on control diet, compared with young rats (4 months; 4 m; * p , 0.05; Student’s t-test for independent means); dietary supplementation reversed this age-related change (1 p , 0.05; Student’s t-test for independent means; comparison of the two aged groups). (B) Glutathione concentration was not affected by age, but the concentration was increased in cortical tissue prepared from aged rats which has received a diet supplemented with ascorbate and a-tocopherol (1 p , 0.05; Student’s t-test for independent means; comparison of the 2 aged groups). (C) a-Tocopherol concentration was significantly decreased in cortical tissue prepared from aged, compared with young rats (* p , 0.05; Student’s t-test for independent means); dietary supplementation reversed this age-related change (1 p , 0.05; Student’s t-test for independent means; comparison of the two aged groups). In all cases, values are the means (6SEM).
above. Thiobarbiturate reactive substances were expressed as nmol malondialdehyde/mg of tissue. a-Tocopherol was analyzed as previously described (47). Briefly, aliquots of homogenate (150 mL) were incubated in the presence of ethanol containing 0.025% butyl-hydroxytoluene (150 mL), 25% ascorbic acid (70 mL), and 10% potassium hydroxide (135 mL) for 30 min at 60°C. Hexane (540 mL) containing 0.025% butyl-hydroxytoluene was added, samples were vortex-mixed for 1 min and centrifuged at 1500 rpm for 6 min. The hexane phase was removed and evaporated to dryness under nitrogen; the recovery of a-tocopherol using this procedure was between 70 and 80%. For high-performance liquid chromotography (HPLC) analysis, dried samples were resuspended in methanol (150 mL) containing 0.025% butyl-hydroxytoluene, and 30 mL volumes were injected onto an Intersil C18 column (maintained at 40°C). Separation of a-tocopherol was achieved using a mobile phase of 75% acetonitrile:25% methanol at a flow rate of 1.2 mL/min, and samples were detected by UV spectroscopy at 292 nm. a-Tocopherol concentration was estimated by the external standard method and expressed as mg/mg of tissue. Arachidonic acid concentration was assessed as previously described (26). Aliquots (150 mL) of cortical homogenate prepared from cortex were added to chloroform:methanol (2:1 v/v; 1 mL), and fatty acids were extracted by vigorous shaking for 10 min followed by centrifugation at 1000 g for 5 min to separate the phases. The aqueous layer was discarded, and the chloroform phase was evaporated under nitrogen and resuspended in ethanol for analysis. Arachidonic acid was analyzed as its 2-nitrophenylhydrazine (NPH) derivatives by reverse phase HPLC. Fatty acids were derivatized by adding 2-NPH-HCl solution (0.02 M 2-nitrophenylhydrazine-HCl in 40 mm HCl-ethanol (3:1, v/v)) and EDC solution (1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide hydro-
chloride; 0.25 M EDC in ethanol mixed in equal volumes with 3% ethanolic pyridine), and incubated at 60°C for 20 min. After addition of KOH (15% w/v in MeOH:H2O, 80:20) samples were incubated at 60°C for 15 min and cooled in running water. Fatty acid derivatives were concentrated; n-hexane and phosphate buffer (0.033 M, pH 6.4 in 0.5 M HCl) were added, samples were vortex mixed for 30 s, centrifuged for 5 min at 1500 g, and the hexane phase evaporated to dryness under nitrogen. For HPLC analysis, samples were resuspended in methanol, injected onto a Microsorb C18 column (maintained in a column oven at 30°C), and fatty acid derivatives were separated in isocratic mode with a mobile phase of 85% methanol:15% water (maintained at pH 4.5 with HCl) and detected by UV spectroscopy at 230 nm. Arachidonic acid concentration, expressed as mmol/mg of tissue, was estimated using the external standard method. RESULTS
There were no statistically significant differences in any of the parameters studied in cortical tissue prepared from young rats fed on the control and experimental diets; therefore the data presented are pooled results from the two groups of young rats. Figure 1 indicates that there was a significant age-related decrease in concentrations of both ascorbic acid and a-tocopherol (p , 0.05; Student’s t-test for independent data), whereas glutathione concentrations was similar in cortical tissue prepared from young rats and aged rats which had been given the control diet. Dietary manipulation with antioxidant vitamins reversed the agerelated decreases in both a-tocopherol and ascorbic acid, and there was a significant difference in these two measures in aged rats fed on the control and experimental diets (p , 0.05; Student’s t-test for independent data). We also observed that dietary manipulation
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FIG. 2. The activity of superoxide dismutase, but not glutathione or catalase, is increased in cortex of aged rats. (A) Activity of superoxide dismutase (SOD) was significantly increased in cortical tissue prepared from aged rats (22 m) fed on control diet, compared with young rats (4 m; * p , 0.05; Student’s t-test for independent means); dietary supplementation reversed this age-related change. (B) The activity of glutathione peroxidase (Gpx) was not affected by age, but there was a significant decrease in its activity in cortical tissue prepared from aged rats which were fed on the experimental diet compared with aged rats fed on the control diet (* p , 0.05; Student’s t-test for independent means). (C) No age-related change in catalase activity was observed. In all cases values are means (6SEM).
with antioxidants resulted in an increase in glutathione concentration. Our data indicated that superoxide dismutase activity was increased in cortical tissue prepared from aged rats which had been fed on control diet, compared with cortical tissue prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 2). This increase was not observed in tissue prepared from aged rats which had received the experimental diet. There was no evidence of any age-related change in either glutathione peroxidase or catalase (Fig. 2) though the activity of glutathione peroxidase was significantly lower in cortical tissue prepared from aged rats which had received the experimental diet, compared with aged rats which had received the control diet (p , 0.05; Student’s t-test for independent data; Fig. 2). Analysis of malondialdehyde formation indicated that there was a significant increase in lipid peroxidation in cortical tissue prepared from aged rats which had been fed on the control diet compared with that in tissue prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 3A). However, there was no evidence of an increase in aged rats fed on the experimental diet suggesting that dietary supplementation with antioxidant vitamins prevented the age-related increase in lipid peroxidation; the value was similar to that observed in young rats and significantly reduced compared with that in the aged rats fed on the control diet. Concomitant with the age-related increase in lipid peroxidation was a significant decrease in arachidonic acid concentration (Fig. 3B; p , 0.05; Student’s t-test for paired samples), suggesting that one target lipid molecule for lipid peroxidation was arachidonic acid. Previous data from analysis of age-related changes in hippocampus revealed that increased lipid peroxidation was accompanied by an increase in endogenous IL-1b and that lipid peroxidation was stimulated by IL-1b in vitro, suggesting that IL-1b may trigger lipid peroxidation. We observed that there was a significant increase in endogenous IL-1b in cortex of aged rats fed on the
control diet compared with that prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 3C). In parallel with the observations in lipid peroxidation, there was no evidence of an increase in IL-1b in cortex of aged rats fed on the experimental diet; the value was similar to that observed in young rats and significantly reduced compared with that in the aged rats fed on the control diet. In vitro analysis revealed that exogenous IL-1b (3.5 ng/mL) significantly increased lipid peroxidation in cortical tissue prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 3D). DISCUSSION
The aim of this study was to compare enzymatic and nonenzymatic antioxidative levels in cortical tissue prepared from aged and young rats and to establish whether any age-related changes might be attenuated by dietary supplementation with antioxidant vitamins. Analysis revealed that: a) there was an age-related increase in superoxide dismutase activity and agerelated decreases in concentrations of a-tocopherol and ascorbate; b) these changes were accompanied by increased lipid peroxidation and increased IL-1b concentration; and c) these changes were not observed in cortical tissue prepared from aged rats which received a diet supplemented with vitamins C and E. We found that there was a significant age-related increase in superoxide dismutase activity in cortical tissue, but that no change was observed in activities of catalase or glutathione peroxidase. These data support previous findings indicating that activity of Mn-superoxide dismutase, but not Cu-superoxide dismutase, was increased in several regions of the aged brain (8), whereas catalase and glutathione peroxidase were unchanged (8,16,49). One consequence of an increased superoxide activity in the aged hippocampus in the absence of concurrent increased activities of catalase and glutathione peroxidase might be a decrease in the ability of the tissue to metabolise H2O2; this is consistent with the reported
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FIG. 3. Lipid peroxidation and IL-1b are increased in cortical tissue prepared from aged rats. (A and C) Lipid peroxidation (A) and IL-1b (C) were both significantly increased in cortical tissue prepared from aged rats fed on control diet compared with young rats (* p , 0.05; Student’s t-test for independent means); these changes were not observed in aged rats fed on the experimental diet (1p , 0.05; Student’s t-test for independent means). Values given are means 6 SEM. (B) Arachidonic acid concentration was significantly decreased in cortical tissue prepared from aged (22 m), compared with young (4 m), rats (* p , 0.05; Student’s t-test for independent means; n 5 6). (D) IL-1b (3.5 ng/mL) stimulated lipid peroxidation in vitro in cortical tissue prepared from young rats (* p , 0.05; Student’s t-test for paired means; n 5 6).
increase in formation of hydroxyl ions which has been observed in cortex and hippocampus of the aged gerbil (51). We also report here that there was a decrease in the concentrations of both ascorbate and a-tocopherol in cortical tissue prepared from aged rats compared with young rats. The age-related decrease in a-tocopherol concentration parallels the decrease we observed in hippocampus of aged rats (30) though an age-related increase has been reported in brain of the aged gerbil (51). Similarly, whereas the present findings support the previously described age-related decrease in ascorbate in rat whole brain (42), an age-related increase in ascorbate concentration has been observed in gerbil brain (51). These data suggest that age-related changes in antioxidant defense might be species-dependent although we observed that glutathione concentrations were similar in cortical samples prepared from young and aged rats, which is consistent with the reported findings in gerbil brain (51). As a major oxidant scavenger in the brain, the decrease in a-tocopherol concentration in the aged cortex is likely to contribute to an accumulation of oxidative species. This potential compromise in antioxidative capacity is compounded by the agerelated decrease in ascorbic acid concentration, which has a dual role; it functions as a potent antioxidant, and it also has the ability to recycle the tocopherol radical, generated through the action of a-tocopherol on reactive oxygen species, back to the non-radical
molecule (33,43). This age-related compromise in antioxidant defenses, arising from increased activity of superoxide dismutase and decreased ascorbate and a-tocopherol, might be expected to lead to oxidative damage and may therefore explain the increased lipid peroxidation in cortical tissue of aged rats observed here. Evidence indicating the role of a-tocopherol in maintenance of neuronal function has been gleaned by analysis of the consequences of vitamin E deficiency. Neurological sequelae such as axon damage, secondary demyelination, and neuropathological features are associated with vitamin E deficiency in man (27), whereas in the rat, prolonged vitamin E deficiency leads to gliosis and also to demyelination (46). Maintenance of rats on a vitamin E deficient diet has been shown to reduce the vitamin concentration in whole brain (45) and cortex (23) and to increase lipid peroxidation and susceptibility to oxidative stress (23,31,45), whereas protection against oxidative damage was provided by vitamin E administration (45). We report that the concentrations of a-tocopherol and ascorbate in cortex of aged rats which received dietary supplementation with vitamins were similar to those observed in cortex of young rats and significantly lower than those in cortex of aged rats fed on control diet. This data may be interpreted as an indication that dietary manipulation reversed age-related changes in vitamin concentrations or alternatively that dietary manipulation prevented
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the decreases which occurred in the 12 weeks leading up to the time of analysis. The time period of 12 weeks described here is consistent with the relatively slow incorporation of a-tocopherol into membranes which has been described previously following dietary manipulation (17). These diet-induced changes, together with the fact that superoxide dismutase activity in cortical tissue prepared from aged rats which received the experimental diet was similar to that in tissue prepared from young rats, are likely to be indicative of protection against oxidative damage to the aged brain. Thus whereas an increase in lipid peroxidation will be triggered by an accumulation of reactive oxygen species, and this arises from a compromise in antioxidant defenses, lipid peroxidation should be limited if antioxidant defense strategies are intact. The data presented indicate that lipid peroxidation in cortical tissue prepared from aged rats fed on the experimental diet was similar to that of young rats and, therefore, significantly reduced compared with aged rats fed on the control diet. We must conclude that dietary manipulation either reversed or prevented oxidative stress which induces lipid peroxidation. This result parallels earlier data in peripheral tissue which showed decreased lipid peroxidation following dietary supplementation with antioxidants (9,20,36). These findings are also consistent with previous data indicating that whereas lipid peroxidation was increased in neuronal tissue, particularly cortex, of vitamin E-deficient young rats (23), vitamin E prevented oxidative injury and alterations in the antioxidative defense system caused by the exposure of young rats to 100% O2 (45). It has been acknowledged for many years that lipid peroxidation will affect membrane composition because it results in depletion of polyunsaturated fatty acids (34), and data from this laboratory have coupled increased lipid peroxidation with decreased membrane arachidonic acid concentration in hippocampus (30). The present study indicated that the age-related increase in lipid peroxidation was paralleled by a decrease in arachidonic acid concentration in cortical tissue also; the coupling of these effects suggests that one target lipid molecule for lipid peroxidation is arachidonic acid. We have observed an age-related increase in endogenous IL-1b in hippocampus (28 –30) and have recently found that IL-1b increased lipid peroxidation and reactive oxygen species production in hippocampal tissue in vitro. These observations led us to
consider that IL-1b may be the trigger for increase lipid peroxidation reported in the aged brain, presumably by stimulating an increase in reactive oxygen species production, which has been described in peripheral tissues (41). We report that there was an increase in endogenous IL-1b concentration in parallel with the age-related increase in lipid peroxidation in cortical tissue, whereas dietary supplementation with antioxidant vitamins reversed both these age-related changes. These data provide further support for the hypothesis that IL-1b may trigger some of the oxidative changes associated with age. While the concentration of IL-1b in brain tissue is generally low, its expression is increased in response to injury or insult (38). IL-1b is synthesized by glia and neurons (50) and also by macrophages, which may cross the blood brain barrier, particularly during insult (37). It has been reported to upregulate Mn-superoxide dismutase gene expression in cultured rat hepatocytes (1), to increase Mn-superoxide dismutase mRNA (5), and to increase the activities of both the Mn and Cu/Zn-superoxide dismutases in rat pancreatic islets (5). Therefore, a chronic increase in endogenous IL-1b, which may occur with age, might trigger the increase in superoxide dismutase activity which we report here; the parallel decreases in IL-1b concentration and superoxide dismutase activity observed after dietary supplementation with a-tocopherol and ascorbate support this hypothesis. The data presented demonstrate a compromise in antioxidative defenses in cortical tissue prepared from aged rats and an associated increase in lipid peroxidation, which is considered to be an indicator of oxidative damage. Our evidence indicates that dietary supplementation of aged rats with antioxidant vitamins prevented or reversed the age-related changes in antioxidant defenses in the cortex and decreased oxidative stress as assessed by lipid peroxidation. The evidence presented is consistent with the view that IL-1b triggers lipid peroxidation and therefore contributes to oxidative stress in neuronal tissue, whereas previous evidence, which indicates the ability of IL-1b to upregulate superoxide dismutase, supports this hypothesis. ACKNOWLEDGEMENTS
We acknowledge the generous financial support of BioResearch, Ireland, UK. The vitamins were a kind gift from Beeline Ltd. Ireland.
REFERENCES 1. Antras–Ferry, J.; Maheo, K.; Morel, F.; Guillouzo, A.; Cillard, P.; Cillard, J. Dexamethasone differently modulates TNF-a- and IL-1 b-induced transcription of the hepatic Mn-superoxide dismutase gene. FEBS Lett. 403:100 –104; 1997. 2. Aprikyan, G. V.; Gekchyan, K. G. Release of neurotransmitter amino acids from rat brain synaptosomes and its regulation in aging. Gerontology 34:35– 40; 1988. 3. Battaini, F.; Elkabes, S.; Bergamaschi, S.; Ladisa, V.; Lucci, L.; De Grann, P. N. E.; Schuurman, T.; Wetsel, W. C.; Trabucchi, M.; Govoni, S. Protein kinase C activity, translocation, and conventional isoforms in the aging rat brain. Neurobiol. Aging 16:137– 148; 1995. 4. Benkovic, S. A.; Connor, J. R. Ferritin, transferrin and iron in selected regions of the adult and aged rat brain. J. Comp. Neurol. 338:97–113; 1993. 5. Borg, L. A.; Cagliero, E.; Sandler, S.; Welsh, N.; Eizirik, D. L. IL-1b increases the activity of superoxide dismutase in rat pancreatic islets. Endocrinology 130:2851–2857; 1992. 6. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilising the principle of protein dye binding. Anal. Biochem. 72:248 –254; 1976.
7. Calderini, G.; Boneti, A. C.; Battistella, A.; Crews, F. T.; Toffano, G. Biochemical changes of rat brain membranes with aging. Neurochem. Res. 8:483– 492; 1983. 8. Carrillo, M. C.; Kanai, S.; Sato, Y.; Kitani, K. Age-related changes in antioxidant enzyme activities are region and organ, as well as sex selective in the rat. Mech. Aging Dev. 65:187–198; 1992. 9. Chen, H.; Tappel, A. Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate. Lipids 31:47–50; 1996. 10. Choi, J. H.; Yu, B. P. Brain synaptosomal aging: Free radicals and membrane fluidity. Free Radic. Biol. Med. 18:133–139; 1995. 11. Cohen, G.; Kim, M.; Ogwu, V. A modified catalase assay suitable for a plate reader and for the analysis of brain cell cultures. J. Neurosci. Meth. 67:53–56; 1996. 12. Dexter, D. T.; Carter, C. J.; Wells, F. R.; Javoy–Agid, F.; Agid, Y., Lees, A.; Jenner, P.; Marsden, C. D. Basal lipid peroxidation in substantia nigra is increased in Parkinson’s disease. J. Neurochem. 52:381–389; 1989. 13. Disterhoft, J. F.; Moyer, Jr., J. R.; Thompson, L. T. The calcium rationale in aging and Alzheimer’s disease: Evidence from an animals model of normal aging. Ann. N.Y. Acad. Sci. 747:382– 406; 1994. 14. Friedman, E.; Wang, H.-Y. Effect of age on brain cortical protein
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15. 16.
17. 18.
19.
20. 21. 22. 23. 24. 25. 26. 27. 28.
29.
30.
31.
kinase C and its mediation of 5-hydroxytryptamine release. J. Neurochem. 52:187–192; 1989. Gaiti, A. The aging brain. A normal phenomenon with not-so-normal arachidonic acid metabolism. Ann. N.Y. Acad. Sci. 559:365–373; 1989. Geremia, E.; Baratta, D.; Zafarana, S.; Giordano, R.; Pinizzotto, M. R.; La Rosa, M. G.; Garozzo, A. Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging. Neurochem. Res. 15:719 –723; 1990. Halliwell, B. Reactive oxygen species and the central nervous system. J. Neurochem. 59:1609 –1623; 1992, 1992. Huidobro, A.; Blanco, P.; Villalba, M.; Gomez–Puertas, P.; Villa, A.; Periera, R.; Bogonez, E.; Martinez–Serrano, A.; Aparicio, J. J.; Satrustegui, J. Age-related changes in calcium homeostatic mechanisms in synaptosomes in relation with working memory deficiency. Neurobiol. Aging 14:479 –186; 1993. Kirischuk, S.; Voitenko, N.; Kostyuk, P.; Verkhratsky, A. Ageassociated changes of cytoplasmic calcium homeostasis in cerebellar granule neurons in situ: Investigation on thin cerebellar slices. Exp. Gerontol. 31:475– 487; 1996. Knudsen, C. A.; Tappel, A. L.; North, J. A. Multiple antioxidants protect against heme protein and lipid oxidation in kidney tissue. Free Radic. Biol. Med. 20:165–173; 1996. Lawrence, R. A.; Burk, R. F. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Comm. 71:952– 959; 1976. Lynch, M. A.; Voss, K. L. Membrane arachidonic acid concentration correlates with age and induction of long-term potentiation in the dentate gyrus of the rat. Eur. J. Neurosci. 6:1008 –1014; 1994. MacEvilly, C. J.; Muller, D. P. R. Lipid peroxidation in neural tissues and fractions from vitamin E deficient rats. Free Radic. Biol. Med. 20:639 – 648; 1996. McGahon B.; Clements, M. P.; Lynch, M. A. The ability of aged rats to sustain LTP is restored when the age-related increase in membrane arachidonic acid is reversed. Neuroscience 81:9 –16; 1997. Meyer, E. M.; Crews, F. T.; Otero, D. H.; Larsen, K. Aging decreases the sensitivity of rat cortical synaptosomes to calcium ionophoreinduced acetylcholine release. J. Neurochem. 47:1244 –1246; 1986. Miwa H.; Yamamoto M.; Nishida T. Assay of free and total fatty acids (as 2-nitrophenylhydrazides) by high-performance liquid chromatography (HPLC). Clinica Chimica Acta 155:95–102; 1986. Muller, D. P. R. Neurological Disease. In: Holbrook, N. J., Martin, G. R.; Lockshin, R. A., eds. Cellular Aging and Cell Death. New York: Wiley-Liss; 1996:557–580. Murray, C. A.; McGahon, B.; McBennett, S.; Lynch M. A. IL-1b inhibits glutamate release in hippocampal synaptosomes prepared from young adult but not aged rats. Neurobiol. Aging 18:343–348; 1997. Murray, C. A.; Lynch, M. A. Evidence that increased hippocampal expression of the cytokine, IL-1b, is a common trigger for age and stress-induced impairments in long-term potentiation. J. Neurosci. 18:2974 –2981; 1998. Murray, C. A.; Lynch, M. A. Analysis of the mechanism by which dietary supplementation with Vitamin E and Vitamin C restores ability of aged animals to sustain long-term potentiation in dentate gyrus. J. Biol. Chem. 273:12161–12168; 1998. Noda, Y.; McGeer, P. L.; McGeer, E. G. Lipid peroxides in brain during aging and vitamin E deficiency: Possible relations to changes in neurotransmitter indices. Neurobiol. Aging 3:173–178; 1982.
467 32. Omaye, S. T.; Turnbull, J. D.; Sauberlich, H. E. Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Methods Enzymol. 62: 3–13; 1979. 33. Packer, J. E.; Slater, T. F.; Willson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737– 738; 1979. 34. Reiter, R. J. Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J. 9:526 –533; 1995. 35. Rice–Evans, C.; Burdon, R. Free radical-lipid peroxidations and their pathological consequences. Prog. Lipid Res. 32:71–110; 1993. 36. Rojas, C.; Cadenas, S.; Lopez–Torres, M.; Perez–Campo, R.; Barja, G. Increase in heart tathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin E dietary supplementation in guinea pigs. Free Radic. Biol. Med. 21:907–915; 1996. 37. Rothwell, N. J.; Strijbos, P. J. L. M. Cytokines in neurodegeneration and repair. Int. J. Dev. Neurosci. 13:179 –185; 1995. 38. Rothwell, N. J.; Hopkins, S. J. Cytokines and the nervous system 11: Actions and mechanisms of action. TINS 18:130 –136; 1995. 39. Sanchez–Prieto, J.; Herrero, I.; Miras–Portugal, M. T.; Mora, F. Unchanged exocytotic release of glutamic acid in cortex and neostriatum of the rat during aging. Brain Res. Bull. 33:357–359; 1994. 40. Spitz, D. R.; Oberley, L. W. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem. 179:8 –18; 1989. 41. Sumoski, W.; Baquerizo, H.; Rabinovitch, A. Oxygen free radical scavengers protect rat islet cells from damage by cytokines. Diabetologia 32:792–796; 1989. 42. Svensson, L.; Wu, C.; Hulthe, P.; Johannessen, K.; Engel, J. A. Effect of ageing on extracellular ascorbate concentration in rat brain. Brain Res. 609:36 – 40; 1993. 43. Tappel, A. L. Will antioxidant nutrients slow the ageing process? Geriatrics 23: 97–105: 1968. 44. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27:502– 522; 1969. 45. Urano, S.; Asai, Y.; Makabe, S.; Matsuo, M.; Izumiyama, N.; Ohtsubo, K.; Endo, T. Oxidative injury of synapse and alteration of antioxidative defence systems in rats, and its prevention by vitamin E. Eur. J. Biochem. 245:64 –70; 1997. 46. Vatassery, G. T. Vitamin E: Neurochemistry and implications for neurodegeneration in Parkinson’s disease. Ann. N.Y. Acad. Sci. 669:97–110; 1992. 47. Vatassery, G. T. In vitro oxidation of vitamins C and E, cholesterol and thiols in rat brain synaptosomes. Lipids 30:1007–1013; 1994. 48. Verkhratsky, A.; Shmigol, A.; Kirischuk, S.; Pronchuk, N.; Kostyuk, P. Age-dependent changes in calcium currents and calcium homeostasis in mammalian neurons. Ann. N.Y. Acad. Sci. 747:365–381; 1994. 49. Vertechy, M.; Cooper, M. B.; Ghirardi, O.; Ramacci, M. T. The effect of age on the activity of enzymes of peroxide metabolism in rat brain. Exp. Gerontol. 28:77– 85; 1993. 50. Yao, J.; Keri, J.; Taffs, R. E.; Colton, C. A. Characterization of IL-1 production by microglia in culture. Brain Res. 591:88 –93; 1992. 51. Zhang, J.-R.; Andrus, P. K.; Hall, E. D. Age-related regional changes in hydroxyl radical stress and antioxidants in gerbil brain. J. Neurochem. 61:1640 –1647; 1993. 52. Zs–Nagy, I. The Membrane Hypothesis of Aging. CRC Press Inc.; 1994.
PII:S0197-4580(98)00082-7
Dietary Antioxidant Supplementation Reverses Age-related Neuronal Changes E. O’DONNELL AND M. A. LYNCH1 Department of Physiology, Trinity College, Dublin, Ireland Received 11 May 1998; Revised July 31; Accepted 31 July 1998 O’DONNELL, E. AND M. A. LYNCH. Dietary antioxidant supplementation reverses age-related neuronal changes. NEUROBIOL AGING 19(5) 461– 467, 1998.—Evidence suggests that reactive oxygen species in brain may play a role in the development of age-related neuronal impairments, and that the increase in the concentration of the proinflammatory cytokine, interleukin-1b (IL-1b), in aged brain tissue, may also be a contributory factor. In this study, we have analyzed changes in enzymatic and nonenzymatic antioxidant levels, in parallel with interleukin-1b concentration, in cortical tissue prepared from young and aged rats. We report that there was an age-related increase in the activity of superoxide dismutase without concomitant changes in the activity of catalase or glutathione peroxidase and an age-related decrease in the concentrations of a-tocopherol and ascorbate. These observations, coupled with age-related increases in lipid peroxidation and interleukin-1b concentration, are consistent with a compromised antioxidant defense in cortex of aged rats, a proposal supported by the finding that these changes were not observed in cortical tissue prepared from rats fed on a diet supplemented with a-tocopherol and ascorbate for 12 weeks. © 1998 Elsevier Science Inc. Antioxidants
a-Tocopherol
IL-1b
Cortex
Lipid peroxidation
AGEING is associated with an array of changes in neuronal function, many of which have been attributed to accumulation of reactive oxygen species, arising from either increased formation of reactive oxygen species, compromised ability of the aged brain to cope with oxidative stress, or both (10,34,52). Neuronal tissue is particularly susceptible to oxidative damage due to high oxygen consumption coupled with modest antioxidant defense strategies (17), high concentrations of iron (4), and high concentrations of polyunsaturated fatty acids, which renders the tissue susceptible to oxygen radicals (35). Among the likely consequences of prolonged exposure to reactive oxygen species are changes in the lipid composition of membranes (52) which in turn are likely to impact on membraneassociated functions. Thus release of several neurotransmitters from cortical tissue, e.g., acetylcholine (18,25), 5-hydroxytryptamine (14), and glutamate [e.g., (2), but see (39)] decreases with age, and this is paralleled by age-related alterations in calcium handling by cells (13,19,48) and down-regulation of membrane associated enzymes like lipases (24) and kinases (3). Direct evidence has demonstrated that membrane composition alters with age (7,15,22,24) resulting in increased rigidity of neuronal membranes (10,52), which might, in turn, underlie age-related changes in membrane-associated functions. The trigger which induces the change in membrane composition has not been identified, but we have recently observed an age-related increase in the proinflammatory cytokine, interleukin 1b (IL-1b) in the hippocampus (28 –30) and have some evidence suggesting that IL-1b may contribute to the membrane changes. Thus we observed that the endogenous increase in IL-1b was accompanied by 1
Superoxide dismutase
Ascorbic acid
increased formation of reactive oxygen species, increased lipid peroxidation, and decreased arachidonic acid concentration (29, 30), whereas reactive oxygen species formation (41) and lipid peroxidation (30) were both increased by IL-1b in vitro. In this study we have investigated age-related changes in enzymatic and nonenzymatic antioxidant strategies in the cortex of the rat in parallel with age-related changes in lipid peroxidation and IL-1b. We have assessed the possibility that age-related changes might be reversed by dietary supplementation with antioxidant vitamins. EXPERIMENTAL PROCEDURES
Male Wistar rats, aged 4 months or 22 months (Charles River Laboratory, UK) were used in this study. Animals were housed in the BioResources Unit in Trinity College, Dublin in groups of four (young rats) or in pairs (aged rats) on a 12-h light:dark cycle, at an ambient temperature of 22–23°C. Twenty-four aged and twelve young animals were randomly divided into two subgroups; one aged and one young group were fed for 12 weeks on laboratory chow, to which dL-a-tocopheryl acetate (250 mg/rat/day; Beeline Healthcare, Ireland, UK) dissolved in corn oil was added. Ascorbic acid (250 mg/rat/day) was added to the water given to these rats. The second subgroup of aged and young rats were fed a control diet which was normal laboratory chow to which corn oil was added to ensure isocaloric intake. Sufficient diet was prepared for 1 week, divided into daily portions, and stored in sealed bags at 220°C until required. During the 2 weeks before the start of dietary manipulation, daily food and water intake was measured and during the period of dietary manipulation, rats were offered
Address correspondence to: M. A. Lynch, Faculty of Health Sciences, Department of Physiology, Trinity College, Dublin 2, Ireland; E-mail: [email protected]
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462 100% of their average daily food and average water intakes so that the full daily allowances of vitamins would be ingested. Average daily food intake was similar in young (34 g) and aged (35 g) rats, whereas daily water intake was slightly higher in young rats (53 mL) compared with aged rats (40 mL). While the average weight of young rats increased from 330 to 548 g during the 12 weeks of dietary manipulation, aged rats did not show any significant change in body weight; in the vitamin-treated group average weight increased from 572 to 589 g and in the control group average weight decreased from 637 to 626 g. Rats were monitored daily throughout the experimental period, and were maintained under veterinary supervision; two aged rats in the vitamin-treated group were euthanized during the first 2 weeks of the experiment due to apparent ill-health, whereas all other rats remained healthy for the duration of the experiment. At the end of the 12-week period of dietary manipulation, rats were anaesthetized with urethane (1.5 g/kg), analyzed for their ability to sustain long-term potentiation in hippocampus (not reported here), and at the end of a 1-h recording period were killed by decapitation. The cortex was dissected free and homogenized in either Krebs solution containing 2 mM CaCl2, or in 5% trichloroacetic acid for later analysis of ascorbic acid and glutathione. Aliquots of homogenate were transferred to microfuge tubes and stored at 280°C until required for analysis. For use, supernatant was prepared by centrifugation at 15,000 rpm for 6 min. Total glutathione was measured as described previously (44). Briefly, aliquots of supernatant prepared from homogenate (30 mL) were incubated for 1 min on ice with sample buffer (100 mM potassium phosphate containing 5 mM EDTA, pH 7.5; 450 mL), in the presence of glutathione reductase (100 mL; 5 U/mL) and 10 mM 5,59-dithiobis-2-nitrobenzoic acid (50 mL). NADPH (2.4 mm; 100 mL) was added and the absorbance monitored for 2 min at 412 nm. Results were expressed as mmol GSH/g of tissue. Glutathione peroxidase activity was measured according to the method of Lawrence and Burk (21). Samples of supernatant (100 mL) were added to incubation buffer (50 mM potassium phosphate (pH 7), containing 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase, 1 mM GSH glutathione, and 1.5 mM cumene hydroperoxide; 800 mL) and incubated for 5 min at room temperature. The reaction was initiated by addition of cumene hydroperoxide (15 mM; 100 mL). Absorbance was recorded at 340 nm for 5 min; activity was calculated from the slope (i.e., change in absorbance with time), and the results were expressed as mmols NADPH oxidised per minute per milligram of protein (6). Ascorbic acid concentrations were determined as previously described (32). Briefly, duplicate aliquots of supernatant (100 mL) were added to a 2,4-dinitrophenylhydrazine/thiourea/copper solution (50 mM thiourea, 2 mM copper sulfate and 150 mM dinitrophenylhydrazine in 9 N H2SO4; 20 mL) and incubated for 3 h at 37°C. Ice-cold H2SO4 (65%; 150 mL) was added to stop the reaction; samples were vortex-mixed and incubated at room temperature for 30 min before aliquots (100 mL) were transferred to 96-well plates for assessment by ultraviolet (UV) spectroscopy at 545 nm. Results were expressed as mmoles of ascorbic acid per gram of tissue. Ascorbic acid standards were prepared in 5% trichloroacetic acid. Superoxide dismutase activity was determined according to a method previously described (40). Aliquots (800 mL) of incubation buffer (50 mM potassium buffer (pH 7.8) containing 1.8 mM xanthine, 2.24 mM nitroblue tetrazolium, 40 U catalase, 7 mL/mL xanthine oxidase, and 1.33 mM diethylenetriaminepentacetic acid) were added to 1.5 mL microfuge tubes containing samples of supernatant (100 mL) at different dilutions (1:2, 1:5, 1:10, 1:20, 1:50, and 1:100) and analyzed by spectroscopy at 560 nm. Activity
O’DONNELL AND LYNCH of superoxide dismutase was assessed as the rate of reduction of nitroblue tetrazolium, which was inhibited with increasing concentrations of protein. One unit of activity was defined as the amount of protein necessary to decrease the rate of the reduction of nitroblue tetrazolium by 50%. Results were expressed in units of superoxide dismutase activity per mg protein. Catalase activity was determined as previously described (11) in tissue prepared from different groups of aged (22 months) and young (4 months) rats; this was simply because of insufficient tissue remaining when all other analyses had been completed on tissue derived from the experiment in which dietary manipulations were made. Rats were killed by stunning and decapitation. The brain was removed, and the cortex was isolated and homogenized in Krebs solution containing 2 mM CaCl2. Samples of homogenate were stored at 280°C for analysis. Duplicate aliquots of supernatant (50 mL) were added to microfuge tubes containing buffer (10 mM potassium phosphate, pH 7.0; 800 mL) tubes and kept on ice. The reaction was initiated by the addition of ice-cold H2O2 (60 mM; 100 mL), samples were mixed and incubated on ice for 2 or 10 min after which time aliquots (100 mL) were removed and quenched by addition to H2SO4 (0.6 N; 4 mL) and 10 mM FeS4 (1 mL) at room temperature. Color was developed at room temperature by addition of 2.5 M potassium thiocyanate (400 mL). Aliquots (200 mL) were transferred immediately to 96-well plates, absorbance was read at 492 nm and results were expressed in terms of the first order reaction rate constant (k), corrected for protein using the formula: enzyme units 5 k/protein 5 [ln (A1/A2/t]/ protein, where A1 and A2 represented the absorbance at the two selected time points (i.e., 2 and 10 min), and t, the time difference between the two points (i.e., 8 min). Results are expressed as enzyme units per gram of protein. We used an ELISA for analysis of IL-1b (Genzyme Diagnostics). Briefly, 96-well plates were coated with 100 mL of capture antibody (2.0 mg/mL final concentration, diluted in 0.1 M sodium carbonate buffer, pH 9.5; monoclonal hamster anti-mouse IL-1b antibody) and incubated overnight at 4°C. Wells were washed several times with PBS containing 0.05% Tween 20 and then blocked for 2 h at 37°C with 250 mL of blocking buffer, (PBS, pH 7.3; 0.1 M with 4% bovine serum albumin (BSA)). Blocking buffer was aspirated and aliquots (50 mL) of samples or IL-1b standards (0 –1000 pg/mL) were added to each well and incubated for 1 h at 37°C. Plates were washed, secondary antibody (100 mL; final concentration 0.8 mg/mL in PBS containing 0.05% Tween 20 and 1% BSA; biotinylated polyclonal rabbit anti-mouse IL-1b antibody) was added to each well and incubation continued for 1 h at 37°C. The plates were washed, and 100 mL detection agent (horseradish peroxidase conjugated streptavidin; 1:1000 dilution in PBS containing 0.05% Tween 20 and 1% BSA) was added to each well, incubation continued for a further 15 min at 37°C, and plates were again washed. Aliquots of substrate (100 mL; tetramethylbenzidine liquid substrate; Sigma) were added, and the samples were incubated at room temperature for 10 min. Absorbance was read at 450 nm within 30 min. Results were expressed as pg IL-1b/mg of tissue. Lipid peroxidation was determined by a previously described method in which analysis of thiobarbiturate reactive substances was made (12). Aliquots (10 mL) of homogenate were incubated at 37°C for 60 min, at which time 8.1% (wt/vol) SDS (30 mL), 20% acetic acid (pH 3.5 with NaOH; 225 mL), and 0.8% (wt/vol) thiobarbituric acid (225 mL) were added. The volume was adjusted with H2O to 600 mL, the samples incubated for a further 60 min at 95°C, cooled at room temperature, and absorbance assessed at 532 nm with reference to a standard curve made using 1,1,3,3,tetramethoxypropane. In some experiments, tissue or IL-1b or buffer was added to samples and incubation continued as described
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FIG. 1. The age-related decreases in ascorbate and a-tocopherol concentration in rat cortical tissue are prevented by dietary supplementation with ascorbate and a-tocopherol. (A) Ascorbate concentration was significantly decreased in cortical tissue prepared from aged rats (22 months; 22 m) fed on control diet, compared with young rats (4 months; 4 m; * p , 0.05; Student’s t-test for independent means); dietary supplementation reversed this age-related change (1 p , 0.05; Student’s t-test for independent means; comparison of the two aged groups). (B) Glutathione concentration was not affected by age, but the concentration was increased in cortical tissue prepared from aged rats which has received a diet supplemented with ascorbate and a-tocopherol (1 p , 0.05; Student’s t-test for independent means; comparison of the 2 aged groups). (C) a-Tocopherol concentration was significantly decreased in cortical tissue prepared from aged, compared with young rats (* p , 0.05; Student’s t-test for independent means); dietary supplementation reversed this age-related change (1 p , 0.05; Student’s t-test for independent means; comparison of the two aged groups). In all cases, values are the means (6SEM).
above. Thiobarbiturate reactive substances were expressed as nmol malondialdehyde/mg of tissue. a-Tocopherol was analyzed as previously described (47). Briefly, aliquots of homogenate (150 mL) were incubated in the presence of ethanol containing 0.025% butyl-hydroxytoluene (150 mL), 25% ascorbic acid (70 mL), and 10% potassium hydroxide (135 mL) for 30 min at 60°C. Hexane (540 mL) containing 0.025% butyl-hydroxytoluene was added, samples were vortex-mixed for 1 min and centrifuged at 1500 rpm for 6 min. The hexane phase was removed and evaporated to dryness under nitrogen; the recovery of a-tocopherol using this procedure was between 70 and 80%. For high-performance liquid chromotography (HPLC) analysis, dried samples were resuspended in methanol (150 mL) containing 0.025% butyl-hydroxytoluene, and 30 mL volumes were injected onto an Intersil C18 column (maintained at 40°C). Separation of a-tocopherol was achieved using a mobile phase of 75% acetonitrile:25% methanol at a flow rate of 1.2 mL/min, and samples were detected by UV spectroscopy at 292 nm. a-Tocopherol concentration was estimated by the external standard method and expressed as mg/mg of tissue. Arachidonic acid concentration was assessed as previously described (26). Aliquots (150 mL) of cortical homogenate prepared from cortex were added to chloroform:methanol (2:1 v/v; 1 mL), and fatty acids were extracted by vigorous shaking for 10 min followed by centrifugation at 1000 g for 5 min to separate the phases. The aqueous layer was discarded, and the chloroform phase was evaporated under nitrogen and resuspended in ethanol for analysis. Arachidonic acid was analyzed as its 2-nitrophenylhydrazine (NPH) derivatives by reverse phase HPLC. Fatty acids were derivatized by adding 2-NPH-HCl solution (0.02 M 2-nitrophenylhydrazine-HCl in 40 mm HCl-ethanol (3:1, v/v)) and EDC solution (1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide hydro-
chloride; 0.25 M EDC in ethanol mixed in equal volumes with 3% ethanolic pyridine), and incubated at 60°C for 20 min. After addition of KOH (15% w/v in MeOH:H2O, 80:20) samples were incubated at 60°C for 15 min and cooled in running water. Fatty acid derivatives were concentrated; n-hexane and phosphate buffer (0.033 M, pH 6.4 in 0.5 M HCl) were added, samples were vortex mixed for 30 s, centrifuged for 5 min at 1500 g, and the hexane phase evaporated to dryness under nitrogen. For HPLC analysis, samples were resuspended in methanol, injected onto a Microsorb C18 column (maintained in a column oven at 30°C), and fatty acid derivatives were separated in isocratic mode with a mobile phase of 85% methanol:15% water (maintained at pH 4.5 with HCl) and detected by UV spectroscopy at 230 nm. Arachidonic acid concentration, expressed as mmol/mg of tissue, was estimated using the external standard method. RESULTS
There were no statistically significant differences in any of the parameters studied in cortical tissue prepared from young rats fed on the control and experimental diets; therefore the data presented are pooled results from the two groups of young rats. Figure 1 indicates that there was a significant age-related decrease in concentrations of both ascorbic acid and a-tocopherol (p , 0.05; Student’s t-test for independent data), whereas glutathione concentrations was similar in cortical tissue prepared from young rats and aged rats which had been given the control diet. Dietary manipulation with antioxidant vitamins reversed the agerelated decreases in both a-tocopherol and ascorbic acid, and there was a significant difference in these two measures in aged rats fed on the control and experimental diets (p , 0.05; Student’s t-test for independent data). We also observed that dietary manipulation
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FIG. 2. The activity of superoxide dismutase, but not glutathione or catalase, is increased in cortex of aged rats. (A) Activity of superoxide dismutase (SOD) was significantly increased in cortical tissue prepared from aged rats (22 m) fed on control diet, compared with young rats (4 m; * p , 0.05; Student’s t-test for independent means); dietary supplementation reversed this age-related change. (B) The activity of glutathione peroxidase (Gpx) was not affected by age, but there was a significant decrease in its activity in cortical tissue prepared from aged rats which were fed on the experimental diet compared with aged rats fed on the control diet (* p , 0.05; Student’s t-test for independent means). (C) No age-related change in catalase activity was observed. In all cases values are means (6SEM).
with antioxidants resulted in an increase in glutathione concentration. Our data indicated that superoxide dismutase activity was increased in cortical tissue prepared from aged rats which had been fed on control diet, compared with cortical tissue prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 2). This increase was not observed in tissue prepared from aged rats which had received the experimental diet. There was no evidence of any age-related change in either glutathione peroxidase or catalase (Fig. 2) though the activity of glutathione peroxidase was significantly lower in cortical tissue prepared from aged rats which had received the experimental diet, compared with aged rats which had received the control diet (p , 0.05; Student’s t-test for independent data; Fig. 2). Analysis of malondialdehyde formation indicated that there was a significant increase in lipid peroxidation in cortical tissue prepared from aged rats which had been fed on the control diet compared with that in tissue prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 3A). However, there was no evidence of an increase in aged rats fed on the experimental diet suggesting that dietary supplementation with antioxidant vitamins prevented the age-related increase in lipid peroxidation; the value was similar to that observed in young rats and significantly reduced compared with that in the aged rats fed on the control diet. Concomitant with the age-related increase in lipid peroxidation was a significant decrease in arachidonic acid concentration (Fig. 3B; p , 0.05; Student’s t-test for paired samples), suggesting that one target lipid molecule for lipid peroxidation was arachidonic acid. Previous data from analysis of age-related changes in hippocampus revealed that increased lipid peroxidation was accompanied by an increase in endogenous IL-1b and that lipid peroxidation was stimulated by IL-1b in vitro, suggesting that IL-1b may trigger lipid peroxidation. We observed that there was a significant increase in endogenous IL-1b in cortex of aged rats fed on the
control diet compared with that prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 3C). In parallel with the observations in lipid peroxidation, there was no evidence of an increase in IL-1b in cortex of aged rats fed on the experimental diet; the value was similar to that observed in young rats and significantly reduced compared with that in the aged rats fed on the control diet. In vitro analysis revealed that exogenous IL-1b (3.5 ng/mL) significantly increased lipid peroxidation in cortical tissue prepared from young rats (p , 0.05; Student’s t-test for independent data; Fig. 3D). DISCUSSION
The aim of this study was to compare enzymatic and nonenzymatic antioxidative levels in cortical tissue prepared from aged and young rats and to establish whether any age-related changes might be attenuated by dietary supplementation with antioxidant vitamins. Analysis revealed that: a) there was an age-related increase in superoxide dismutase activity and agerelated decreases in concentrations of a-tocopherol and ascorbate; b) these changes were accompanied by increased lipid peroxidation and increased IL-1b concentration; and c) these changes were not observed in cortical tissue prepared from aged rats which received a diet supplemented with vitamins C and E. We found that there was a significant age-related increase in superoxide dismutase activity in cortical tissue, but that no change was observed in activities of catalase or glutathione peroxidase. These data support previous findings indicating that activity of Mn-superoxide dismutase, but not Cu-superoxide dismutase, was increased in several regions of the aged brain (8), whereas catalase and glutathione peroxidase were unchanged (8,16,49). One consequence of an increased superoxide activity in the aged hippocampus in the absence of concurrent increased activities of catalase and glutathione peroxidase might be a decrease in the ability of the tissue to metabolise H2O2; this is consistent with the reported
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FIG. 3. Lipid peroxidation and IL-1b are increased in cortical tissue prepared from aged rats. (A and C) Lipid peroxidation (A) and IL-1b (C) were both significantly increased in cortical tissue prepared from aged rats fed on control diet compared with young rats (* p , 0.05; Student’s t-test for independent means); these changes were not observed in aged rats fed on the experimental diet (1p , 0.05; Student’s t-test for independent means). Values given are means 6 SEM. (B) Arachidonic acid concentration was significantly decreased in cortical tissue prepared from aged (22 m), compared with young (4 m), rats (* p , 0.05; Student’s t-test for independent means; n 5 6). (D) IL-1b (3.5 ng/mL) stimulated lipid peroxidation in vitro in cortical tissue prepared from young rats (* p , 0.05; Student’s t-test for paired means; n 5 6).
increase in formation of hydroxyl ions which has been observed in cortex and hippocampus of the aged gerbil (51). We also report here that there was a decrease in the concentrations of both ascorbate and a-tocopherol in cortical tissue prepared from aged rats compared with young rats. The age-related decrease in a-tocopherol concentration parallels the decrease we observed in hippocampus of aged rats (30) though an age-related increase has been reported in brain of the aged gerbil (51). Similarly, whereas the present findings support the previously described age-related decrease in ascorbate in rat whole brain (42), an age-related increase in ascorbate concentration has been observed in gerbil brain (51). These data suggest that age-related changes in antioxidant defense might be species-dependent although we observed that glutathione concentrations were similar in cortical samples prepared from young and aged rats, which is consistent with the reported findings in gerbil brain (51). As a major oxidant scavenger in the brain, the decrease in a-tocopherol concentration in the aged cortex is likely to contribute to an accumulation of oxidative species. This potential compromise in antioxidative capacity is compounded by the agerelated decrease in ascorbic acid concentration, which has a dual role; it functions as a potent antioxidant, and it also has the ability to recycle the tocopherol radical, generated through the action of a-tocopherol on reactive oxygen species, back to the non-radical
molecule (33,43). This age-related compromise in antioxidant defenses, arising from increased activity of superoxide dismutase and decreased ascorbate and a-tocopherol, might be expected to lead to oxidative damage and may therefore explain the increased lipid peroxidation in cortical tissue of aged rats observed here. Evidence indicating the role of a-tocopherol in maintenance of neuronal function has been gleaned by analysis of the consequences of vitamin E deficiency. Neurological sequelae such as axon damage, secondary demyelination, and neuropathological features are associated with vitamin E deficiency in man (27), whereas in the rat, prolonged vitamin E deficiency leads to gliosis and also to demyelination (46). Maintenance of rats on a vitamin E deficient diet has been shown to reduce the vitamin concentration in whole brain (45) and cortex (23) and to increase lipid peroxidation and susceptibility to oxidative stress (23,31,45), whereas protection against oxidative damage was provided by vitamin E administration (45). We report that the concentrations of a-tocopherol and ascorbate in cortex of aged rats which received dietary supplementation with vitamins were similar to those observed in cortex of young rats and significantly lower than those in cortex of aged rats fed on control diet. This data may be interpreted as an indication that dietary manipulation reversed age-related changes in vitamin concentrations or alternatively that dietary manipulation prevented
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the decreases which occurred in the 12 weeks leading up to the time of analysis. The time period of 12 weeks described here is consistent with the relatively slow incorporation of a-tocopherol into membranes which has been described previously following dietary manipulation (17). These diet-induced changes, together with the fact that superoxide dismutase activity in cortical tissue prepared from aged rats which received the experimental diet was similar to that in tissue prepared from young rats, are likely to be indicative of protection against oxidative damage to the aged brain. Thus whereas an increase in lipid peroxidation will be triggered by an accumulation of reactive oxygen species, and this arises from a compromise in antioxidant defenses, lipid peroxidation should be limited if antioxidant defense strategies are intact. The data presented indicate that lipid peroxidation in cortical tissue prepared from aged rats fed on the experimental diet was similar to that of young rats and, therefore, significantly reduced compared with aged rats fed on the control diet. We must conclude that dietary manipulation either reversed or prevented oxidative stress which induces lipid peroxidation. This result parallels earlier data in peripheral tissue which showed decreased lipid peroxidation following dietary supplementation with antioxidants (9,20,36). These findings are also consistent with previous data indicating that whereas lipid peroxidation was increased in neuronal tissue, particularly cortex, of vitamin E-deficient young rats (23), vitamin E prevented oxidative injury and alterations in the antioxidative defense system caused by the exposure of young rats to 100% O2 (45). It has been acknowledged for many years that lipid peroxidation will affect membrane composition because it results in depletion of polyunsaturated fatty acids (34), and data from this laboratory have coupled increased lipid peroxidation with decreased membrane arachidonic acid concentration in hippocampus (30). The present study indicated that the age-related increase in lipid peroxidation was paralleled by a decrease in arachidonic acid concentration in cortical tissue also; the coupling of these effects suggests that one target lipid molecule for lipid peroxidation is arachidonic acid. We have observed an age-related increase in endogenous IL-1b in hippocampus (28 –30) and have recently found that IL-1b increased lipid peroxidation and reactive oxygen species production in hippocampal tissue in vitro. These observations led us to
consider that IL-1b may be the trigger for increase lipid peroxidation reported in the aged brain, presumably by stimulating an increase in reactive oxygen species production, which has been described in peripheral tissues (41). We report that there was an increase in endogenous IL-1b concentration in parallel with the age-related increase in lipid peroxidation in cortical tissue, whereas dietary supplementation with antioxidant vitamins reversed both these age-related changes. These data provide further support for the hypothesis that IL-1b may trigger some of the oxidative changes associated with age. While the concentration of IL-1b in brain tissue is generally low, its expression is increased in response to injury or insult (38). IL-1b is synthesized by glia and neurons (50) and also by macrophages, which may cross the blood brain barrier, particularly during insult (37). It has been reported to upregulate Mn-superoxide dismutase gene expression in cultured rat hepatocytes (1), to increase Mn-superoxide dismutase mRNA (5), and to increase the activities of both the Mn and Cu/Zn-superoxide dismutases in rat pancreatic islets (5). Therefore, a chronic increase in endogenous IL-1b, which may occur with age, might trigger the increase in superoxide dismutase activity which we report here; the parallel decreases in IL-1b concentration and superoxide dismutase activity observed after dietary supplementation with a-tocopherol and ascorbate support this hypothesis. The data presented demonstrate a compromise in antioxidative defenses in cortical tissue prepared from aged rats and an associated increase in lipid peroxidation, which is considered to be an indicator of oxidative damage. Our evidence indicates that dietary supplementation of aged rats with antioxidant vitamins prevented or reversed the age-related changes in antioxidant defenses in the cortex and decreased oxidative stress as assessed by lipid peroxidation. The evidence presented is consistent with the view that IL-1b triggers lipid peroxidation and therefore contributes to oxidative stress in neuronal tissue, whereas previous evidence, which indicates the ability of IL-1b to upregulate superoxide dismutase, supports this hypothesis. ACKNOWLEDGEMENTS
We acknowledge the generous financial support of BioResearch, Ireland, UK. The vitamins were a kind gift from Beeline Ltd. Ireland.
REFERENCES 1. Antras–Ferry, J.; Maheo, K.; Morel, F.; Guillouzo, A.; Cillard, P.; Cillard, J. Dexamethasone differently modulates TNF-a- and IL-1 b-induced transcription of the hepatic Mn-superoxide dismutase gene. FEBS Lett. 403:100 –104; 1997. 2. Aprikyan, G. V.; Gekchyan, K. G. Release of neurotransmitter amino acids from rat brain synaptosomes and its regulation in aging. Gerontology 34:35– 40; 1988. 3. Battaini, F.; Elkabes, S.; Bergamaschi, S.; Ladisa, V.; Lucci, L.; De Grann, P. N. E.; Schuurman, T.; Wetsel, W. C.; Trabucchi, M.; Govoni, S. Protein kinase C activity, translocation, and conventional isoforms in the aging rat brain. Neurobiol. Aging 16:137– 148; 1995. 4. Benkovic, S. A.; Connor, J. R. Ferritin, transferrin and iron in selected regions of the adult and aged rat brain. J. Comp. Neurol. 338:97–113; 1993. 5. Borg, L. A.; Cagliero, E.; Sandler, S.; Welsh, N.; Eizirik, D. L. IL-1b increases the activity of superoxide dismutase in rat pancreatic islets. Endocrinology 130:2851–2857; 1992. 6. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of proteins utilising the principle of protein dye binding. Anal. Biochem. 72:248 –254; 1976.
7. Calderini, G.; Boneti, A. C.; Battistella, A.; Crews, F. T.; Toffano, G. Biochemical changes of rat brain membranes with aging. Neurochem. Res. 8:483– 492; 1983. 8. Carrillo, M. C.; Kanai, S.; Sato, Y.; Kitani, K. Age-related changes in antioxidant enzyme activities are region and organ, as well as sex selective in the rat. Mech. Aging Dev. 65:187–198; 1992. 9. Chen, H.; Tappel, A. Protection by multiple antioxidants against lipid peroxidation in rat liver homogenate. Lipids 31:47–50; 1996. 10. Choi, J. H.; Yu, B. P. Brain synaptosomal aging: Free radicals and membrane fluidity. Free Radic. Biol. Med. 18:133–139; 1995. 11. Cohen, G.; Kim, M.; Ogwu, V. A modified catalase assay suitable for a plate reader and for the analysis of brain cell cultures. J. Neurosci. Meth. 67:53–56; 1996. 12. Dexter, D. T.; Carter, C. J.; Wells, F. R.; Javoy–Agid, F.; Agid, Y., Lees, A.; Jenner, P.; Marsden, C. D. Basal lipid peroxidation in substantia nigra is increased in Parkinson’s disease. J. Neurochem. 52:381–389; 1989. 13. Disterhoft, J. F.; Moyer, Jr., J. R.; Thompson, L. T. The calcium rationale in aging and Alzheimer’s disease: Evidence from an animals model of normal aging. Ann. N.Y. Acad. Sci. 747:382– 406; 1994. 14. Friedman, E.; Wang, H.-Y. Effect of age on brain cortical protein
ANTIOXIDANTS REVERSE AGE-RELATED CHANGES
15. 16.
17. 18.
19.
20. 21. 22. 23. 24. 25. 26. 27. 28.
29.
30.
31.
kinase C and its mediation of 5-hydroxytryptamine release. J. Neurochem. 52:187–192; 1989. Gaiti, A. The aging brain. A normal phenomenon with not-so-normal arachidonic acid metabolism. Ann. N.Y. Acad. Sci. 559:365–373; 1989. Geremia, E.; Baratta, D.; Zafarana, S.; Giordano, R.; Pinizzotto, M. R.; La Rosa, M. G.; Garozzo, A. Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging. Neurochem. Res. 15:719 –723; 1990. Halliwell, B. Reactive oxygen species and the central nervous system. J. Neurochem. 59:1609 –1623; 1992, 1992. Huidobro, A.; Blanco, P.; Villalba, M.; Gomez–Puertas, P.; Villa, A.; Periera, R.; Bogonez, E.; Martinez–Serrano, A.; Aparicio, J. J.; Satrustegui, J. Age-related changes in calcium homeostatic mechanisms in synaptosomes in relation with working memory deficiency. Neurobiol. Aging 14:479 –186; 1993. Kirischuk, S.; Voitenko, N.; Kostyuk, P.; Verkhratsky, A. Ageassociated changes of cytoplasmic calcium homeostasis in cerebellar granule neurons in situ: Investigation on thin cerebellar slices. Exp. Gerontol. 31:475– 487; 1996. Knudsen, C. A.; Tappel, A. L.; North, J. A. Multiple antioxidants protect against heme protein and lipid oxidation in kidney tissue. Free Radic. Biol. Med. 20:165–173; 1996. Lawrence, R. A.; Burk, R. F. Glutathione peroxidase activity in selenium-deficient rat liver. Biochem. Biophys. Res. Comm. 71:952– 959; 1976. Lynch, M. A.; Voss, K. L. Membrane arachidonic acid concentration correlates with age and induction of long-term potentiation in the dentate gyrus of the rat. Eur. J. Neurosci. 6:1008 –1014; 1994. MacEvilly, C. J.; Muller, D. P. R. Lipid peroxidation in neural tissues and fractions from vitamin E deficient rats. Free Radic. Biol. Med. 20:639 – 648; 1996. McGahon B.; Clements, M. P.; Lynch, M. A. The ability of aged rats to sustain LTP is restored when the age-related increase in membrane arachidonic acid is reversed. Neuroscience 81:9 –16; 1997. Meyer, E. M.; Crews, F. T.; Otero, D. H.; Larsen, K. Aging decreases the sensitivity of rat cortical synaptosomes to calcium ionophoreinduced acetylcholine release. J. Neurochem. 47:1244 –1246; 1986. Miwa H.; Yamamoto M.; Nishida T. Assay of free and total fatty acids (as 2-nitrophenylhydrazides) by high-performance liquid chromatography (HPLC). Clinica Chimica Acta 155:95–102; 1986. Muller, D. P. R. Neurological Disease. In: Holbrook, N. J., Martin, G. R.; Lockshin, R. A., eds. Cellular Aging and Cell Death. New York: Wiley-Liss; 1996:557–580. Murray, C. A.; McGahon, B.; McBennett, S.; Lynch M. A. IL-1b inhibits glutamate release in hippocampal synaptosomes prepared from young adult but not aged rats. Neurobiol. Aging 18:343–348; 1997. Murray, C. A.; Lynch, M. A. Evidence that increased hippocampal expression of the cytokine, IL-1b, is a common trigger for age and stress-induced impairments in long-term potentiation. J. Neurosci. 18:2974 –2981; 1998. Murray, C. A.; Lynch, M. A. Analysis of the mechanism by which dietary supplementation with Vitamin E and Vitamin C restores ability of aged animals to sustain long-term potentiation in dentate gyrus. J. Biol. Chem. 273:12161–12168; 1998. Noda, Y.; McGeer, P. L.; McGeer, E. G. Lipid peroxides in brain during aging and vitamin E deficiency: Possible relations to changes in neurotransmitter indices. Neurobiol. Aging 3:173–178; 1982.
467 32. Omaye, S. T.; Turnbull, J. D.; Sauberlich, H. E. Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids. Methods Enzymol. 62: 3–13; 1979. 33. Packer, J. E.; Slater, T. F.; Willson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 278:737– 738; 1979. 34. Reiter, R. J. Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J. 9:526 –533; 1995. 35. Rice–Evans, C.; Burdon, R. Free radical-lipid peroxidations and their pathological consequences. Prog. Lipid Res. 32:71–110; 1993. 36. Rojas, C.; Cadenas, S.; Lopez–Torres, M.; Perez–Campo, R.; Barja, G. Increase in heart tathione redox ratio and total antioxidant capacity and decrease in lipid peroxidation after vitamin E dietary supplementation in guinea pigs. Free Radic. Biol. Med. 21:907–915; 1996. 37. Rothwell, N. J.; Strijbos, P. J. L. M. Cytokines in neurodegeneration and repair. Int. J. Dev. Neurosci. 13:179 –185; 1995. 38. Rothwell, N. J.; Hopkins, S. J. Cytokines and the nervous system 11: Actions and mechanisms of action. TINS 18:130 –136; 1995. 39. Sanchez–Prieto, J.; Herrero, I.; Miras–Portugal, M. T.; Mora, F. Unchanged exocytotic release of glutamic acid in cortex and neostriatum of the rat during aging. Brain Res. Bull. 33:357–359; 1994. 40. Spitz, D. R.; Oberley, L. W. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem. 179:8 –18; 1989. 41. Sumoski, W.; Baquerizo, H.; Rabinovitch, A. Oxygen free radical scavengers protect rat islet cells from damage by cytokines. Diabetologia 32:792–796; 1989. 42. Svensson, L.; Wu, C.; Hulthe, P.; Johannessen, K.; Engel, J. A. Effect of ageing on extracellular ascorbate concentration in rat brain. Brain Res. 609:36 – 40; 1993. 43. Tappel, A. L. Will antioxidant nutrients slow the ageing process? Geriatrics 23: 97–105: 1968. 44. Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal. Biochem. 27:502– 522; 1969. 45. Urano, S.; Asai, Y.; Makabe, S.; Matsuo, M.; Izumiyama, N.; Ohtsubo, K.; Endo, T. Oxidative injury of synapse and alteration of antioxidative defence systems in rats, and its prevention by vitamin E. Eur. J. Biochem. 245:64 –70; 1997. 46. Vatassery, G. T. Vitamin E: Neurochemistry and implications for neurodegeneration in Parkinson’s disease. Ann. N.Y. Acad. Sci. 669:97–110; 1992. 47. Vatassery, G. T. In vitro oxidation of vitamins C and E, cholesterol and thiols in rat brain synaptosomes. Lipids 30:1007–1013; 1994. 48. Verkhratsky, A.; Shmigol, A.; Kirischuk, S.; Pronchuk, N.; Kostyuk, P. Age-dependent changes in calcium currents and calcium homeostasis in mammalian neurons. Ann. N.Y. Acad. Sci. 747:365–381; 1994. 49. Vertechy, M.; Cooper, M. B.; Ghirardi, O.; Ramacci, M. T. The effect of age on the activity of enzymes of peroxide metabolism in rat brain. Exp. Gerontol. 28:77– 85; 1993. 50. Yao, J.; Keri, J.; Taffs, R. E.; Colton, C. A. Characterization of IL-1 production by microglia in culture. Brain Res. 591:88 –93; 1992. 51. Zhang, J.-R.; Andrus, P. K.; Hall, E. D. Age-related regional changes in hydroxyl radical stress and antioxidants in gerbil brain. J. Neurochem. 61:1640 –1647; 1993. 52. Zs–Nagy, I. The Membrane Hypothesis of Aging. CRC Press Inc.; 1994.