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The effects of dietary antioxidants on psychomotor performance in aged mice
Experimental Gerontology 34 (1999) 797– 808
The effects of dietary antioxidants on psychomotor performance in aged mice Barbara Shukitt–Hale*, Donald E. Smith, Mohsen Meydani, James A. Joseph United States Department of Agriculture-Agricultural Research Service, Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111 USA Received 15 December 1998; received in revised form 12 April 1999; accepted 12 April 1999
Abstract Male C57BL/6NIA mice were provided one of six different antioxidant diets: vitamin E, glutathione, vitamin E plus glutathione, melatonin, strawberry extract, or control, beginning at 18 months of age. A battery of motor tests—rod walk, wire hang, plank walk, and inclined screen—was administered either: 1) before dietary treatment and then 6 months later at 24 months of age; or 2) only after 6 months of dietary treatment at age 24 months. An untreated group of 4-month-old mice served as young controls. Psychomotor performance was lower in 18-month-old mice compared with 4-month-old mice in the rod walk, wire hang, and inclined screen tests; however, no further decline was seen from 18 to 24 months on any measure. Chronic dietary antioxidant treatments were not effective in reversing age-related deficits in psychomotor behavior, except for the glutathione diet on inclined screen performance. It seems that motor performance deteriorates profoundly with age, because deficits at 18 months of age were as severe as they were at 24 months, and these age-associated motor deficits may be difficult to reverse, even with antioxidant treatment. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Oxidative stress; Motor behavior; Aging; Chronic dietary treatment; Vitamin E; Glutathione; Melatonin; Strawberry
1. Introduction Oxidative stress (OS) is thought to be a contributing factor to the decrements in motor performance seen in aging. The “free radical hypothesis of aging” maintains that, with age, oxidative damage increases and endogenous antioxidant defense mechanisms become insufficient to detoxify the generation of reactive oxygen species (ROS), such as super* Corresponding author. Tel.: ⫹001-617-556-3118; fax: ⫹1-617-556-3222. E-mail address: [email protected] (B. Shukitt–Hale) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 3 9 - X
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oxide and hydroxyl radicals (Harman, 1992; Ames et al., 1993; Mo et al., 1995). The subsequent accumulation of these oxygen free radicals results in oxidative damage to critical biological molecules, particularly in the brain (Olanow, 1992), and contributes to the detrimental effects of aging (Harman, 1992; Ames et al., 1993; Carney et al., 1994). Manipulations shown to increase the presence of ROS in the brain increase oxidized protein and worsen performance deficits, whereas interventions designed to quench ROS decrease oxidized protein levels and normalize associated behavioral deficits (Carney et al., 1994). Therefore, if the generation and accumulation of ROS are important factors in causing age-related psychomotor decrements, chronic enhancement of antioxidant defenses could slow, or possibly reverse, this process by reducing OS, resulting in improved motor performance (Socci et al., 1995). It has been postulated that accumulation of oxidized proteins or lipid peroxidation in aging may be involved in the age-related functional neuronal loss contributing to deficits in motor performance (Joseph, 1992; Forster et al., 1996). Specifically, these alterations in motor function may include decreases in balance, muscle strength, and coordination. Both aged rats and mice show decrements in performance on several tasks requiring coordinated control of motor and reflexive responses, such as suspension time on a horizontal wire or inclined wire mesh screen and the length of time it takes for the animal to traverse a wooden rod or plank (Dean et al., 1981; Joseph et al., 1983a; Joseph and Lippa, 1986; Ingram et al., 1994b, Shukitt–Hale et al., 1998b). Age-related deficits in motor performance are thought to be the result of alterations in the striatal dopamine (DA) system; the striatum, a brain area which mediates several aspects of motor behavior (Joseph and Roth, 1988), shows marked neurodegenerative changes with age (Joseph, 1992). Antioxidant nutrients added to the diet are one defense strategy to prevent, intercept, or repair age-induced OS (Harman, 1992; Sies, 1993; Joseph et al., 1998). Vitamin E (vit E), glutathione (GSH), melatonin, and strawberry extract have all been found to have antioxidant properties. Vit E is a radical chain-breaking antioxidant (Sies, 1993) and is the most important free radical scavenger within membranes and lipoproteins (Halliwell, 1994). Vit E inhibits lipid peroxidation by scavenging peroxyl radicals (Halliwell, 1994) and severe deprivation of vit E in humans produces neurological damage (Muller and Goss–Sampson, 1990). GSH is an endogenous protective agent that plays a critical role in intracellular antioxidant defense. GSH acts to destroy ROS and is capable of neutralizing free radicals generated by OS (Pileblad and Magnusson, 1990); brain GSH is decreased in aging (Chen et al., 1989). Melatonin, the primary hormone of the pineal gland, has been shown to be a free radical scavenger and a general antioxidant (Reiter, 1995). Melatonin preserves macromolecules including DNA, protein, and lipids from oxidative damage and, compared with other antioxidants, melatonin seems to have a greater efficacy in protecting against cellular OS (Reiter, 1995). Fruits and vegetables are the main source of antioxidants in the diet (Ames et al., 1993). Strawberries have been shown to have a high total antioxidant capacity (Wang et al., 1996), possibly because of flavonoids, specifically the anthocyanins (Wang et al., 1996; Cao et al., 1997; Wang et al., 1997). Fruit and vegetable intake is associated with a lowered risk of degenerative disease, whereas the lack of adequate consumption of fruits and vegetables is linked to cancer incidence (Ames et al., 1993). Recent research from our laboratory demonstrated that strawberry, spinach, and vit E diets were effective in retarding age-associated deficits in muscarinic receptor sensitivity, as assessed via oxotremorine enhancement of striatal dopamine release; isoproteronal facilitation of
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Table 1 Mouse survival at 24 months by dietary group and behavioral testing group
Dietary Group
Control Vitamin E GSH E ⫹ GSH Melatonin Strawberry Overall survival
Behavioral Group Pre & Post
Post only
Survival per group
% Survived
9/19 14/20 14/20 14/19 12/19 12/20 75/117
13/20 12/20 17/19 13/19 15/19 14/18 84/115
22/39 26/40 31/39 27/38 27/38 26/38 159/232
56% 65% 79% 71% 71% 68% 69%
GABAergic inhibition of cerebellar Purkinje neurons; calcium regulation; and cognitive performance as measured by the Morris water maze (Joseph et al., 1998). The objective of the present study was to determine the efficacy of dietary supplementation with antioxidants in reversing/restoring age-related declines in motor performance in mice. Treatment of mice with one of six diets (control, vit E, GSH, E⫹GSH, melatonin, or strawberry) was begun at 18 months of age and carried through to 24 months of age. Motor performance was assessed on a battery of age-sensitive tests, both before and after treatment with these antioxidants in the diets. This investigation was part of a larger study designed to investigate the effects of dietary antioxidants in aged animals on oxidative stress, immune response, age-associated pathogenesis (Lipman et al., 1998), and survival; results from these other studies are forthcoming.
2. Materials and methods 2.1. Animals Male C57BL/6NIA mice (bred and raised at the HNRCA under barrier conditions, in four monthly onset groups) were used in this study (see Table 1). The 232 ad libitum chow-fed mice were 18 months of age before being started on a 6-month dietary treatment regimen consisting of a control diet (Meydani et al., 1987) or control diet supplemented with one of five antioxidants. From the original group, 159 mice (69%) survived until age 24 months. An untreated group (n ⫽ 11) of 4-month-old mice served as young, chow-fed, controls. Throughout the 6 months, the mice were individually housed in autoclaved, filtered, polycarbonate cages with wood shavings as bedding and free access to food and water, and maintained on a 12-h light/dark cycle. An ongoing health surveillance ensured that the animals remained viral antibody free. These animals were used in compliance with all applicable laws and regulations as well as principles expressed in the National Institutes of Health, United States Public Health Service Guide for the Care and Use of Laboratory Animals. This study was reviewed and approved by the Animal Care and Use Committee at the U.S. Department of Agriculture, Human Nutrition Research Center on Aging at Tufts University (an AAALAC accredited facility).
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2.2. Diets Initially, mice were weight matched and randomly assigned to one of six diet groups (38 – 40 mice in each group, see Table 1): control (a modified AIN76 diet with 30 ppm vit E), vit E (dl-␣-tocopheryl acetate, 500 ppm vit E), GSH (0.5% GSH), E⫹GSH (500 ppm vit E ⫹ 0.5% GSH), melatonin (11 ppm melatonin), or freeze-dried aqueous strawberry extract (1%); all doses were based on previous investigations [vit E: (Meydani et al., 1986); GSH: (Furukawa et al., 1987; Favilli et al., 1997); melatonin: (Pierpaoli and Regelson, 1994); strawberry: (Wang et al., 1996; Joseph et al., 1998)] that have shown increased levels in plasma and other tissues following feeding. The basal diet contained casein (18%), corn starch (33.55%), sucrose (33.4%), cellulose (5%), DL-methionine (0.3%), choline bitartrate (0.25%), AIN76A salt (3.5%) and vitamin mix (1%), plus 5% soybean oil (Meydani et al., 1987). Because melatonin is light-sensitive, the melatonin diet was provided in ceramic (lead-free) jars. For a more complete description of the diets, see Lipman et al. (1998). 2.3. Behavioral Testing Mice were randomly assigned to one of two behavioral groups: pre & post (motor testing before and after 6 months of dietary treatment) and post only (motor testing following 6 months of dietary treatment) (see Table 1). A young control group was tested one time, at age 4 months. For each test session, a battery of psychomotor behavioral tests was performed that consisted of complex movement tasks which have been shown to deteriorate with age (Joseph et al., 1983; Joseph, 1992; Ingram et al., 1994b; Shukitt–Hale et al., 1998b), including in C57BL/6J mice (Dean et al., 1981; Ingram, 1983; Ingram et al., 1993). These tests were administered one time at each test session in the following order, with a short break between them. Briefly, the tests included: 1) Rod walking, which measures psychomotor coordination and the integrity of the vestibular system by requiring the animal to balance on a stationary, horizontal rod (120 cm long, 12 mm in diameter, positioned 35 cm above the table surface); 2) Wire suspension/wire hanging, which measures muscle strength and the prehensile reflex, an animal’s ability to grasp a taut horizontal wire (2 mm in diameter, 62 cm above the table top) with its forepaws and to remain suspended; 3) Plank walking, which measures balance and coordination by exposing the mice to three different sizes of horizontal planks (wide ⫽ 20 mm; medium ⫽ 13 mm; narrow ⫽ 6 mm, each 100 cm long, placed 33 cm above the floor); and 4) Inclined screen, which measures muscle tone, strength, stamina, and balance by placing the mouse on a wire mesh screen that was tilted 60o or 85o to the horizontal plane of the floor. For a more complete description of the tests, see Shukitt–Hale et al. (1998b). 2.4. Statistics For each behavioral measure, between-subjects analyses of variance (ANOVA) models comparing the different diet groups were performed by using Biomedical Data Package (BMDP) to test for statistical significance at the p ⱕ 0.05 level; post hoc comparisons were performed by using Duncan’s multiple range test. Additionally, ANOVAs were run to compare differences between the age groups, by using data from the 4, 18 (pre & post), and 24 (post only) month control groups only; these groups were comparable because they were all naive at the time of testing.
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Fig. 1. Latency to fall (mean ⫹ SEM: s) in the rod walk, wire hang, and inclined screen (85o tilt) tests for two or three different age groups: 4-month (n ⫽ 11), 18-month (n ⫽ 19), and 24-month (n ⫽ 8). Asterisks indicate significant differences from 4 months (** p ⬍ 0.01).
3. Results Initially, ANOVAs were run to compare motor performance between ages to determine if there were significant differences between 4, 18, and 24 months (controls only). Several tests showed deficits from 4 to 18 months; however, no tests showed additional performance decrements from 18 to 24 months. Specifically, in the rod walking test (Fig. 1), ANOVA showed a significant age difference [F(2, 35) ⫽ 8.11, p ⬍ 0.01]; post-hoc testing
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showed that latency to fall was longer for the young group than for the 18-month (p ⬍ 0.01) or the 24-month group (p ⬍ 0.01). There was also a significant age effect in the wire-hang test (Fig. 1) [F(2,35) ⫽ 12.77, p ⬍ 0.001]; again, latency to fall was longer for the 4-month group when compared with the 18-month (p ⬍ 0.01) or 24-month group (p ⬍ 0.01). Only data from the 4- and 24-month groups were comparable for the inclined screen, as both of these groups were tested at the 85o angle (the pre & post group animals were tested at the 60o angle, but this angle proved too easy for the mice as they all achieved the 600-s max time, regardless of age or diet). There was a significant age effect on the inclined screen (Fig. 1) [F(1, 17) ⫽ 115.93, p ⬍ 0.001], with the 24-month-old animals having a shorter latency to fall than the 4-month-group. There were no age differences for plank walk latency, distance, or turns; again, this test proved too easy for the mice, as almost all of them achieved the 60-s max score for latency, regardless of age. In summary, although some deficits were seen from 4 to 18 months, motor performance for the 18-month group on all measures was never different from the 24-month group. Additionally, there was a significant effect of age on body weight [F(2, 35) ⫽ 130.74, p ⬍ 0.001] for these control groups. The 4-month group weighed, on average, 26.1 ⫾ 0.26 g (mean ⫾ SEM), the 18 month group weighed 40.6 ⫾ 0.77 g, and the 24 month group weighed 51.1 ⫾ 1.88 g. Body weight increased with age, with both the 18- and 24-month groups weighing more than the 4-month group (p ⬍ 0.01) and the 24 month group weighing more than the 18-month group (p ⬍ 0.01). However, Ingram et al. (1983, 1994b) found that even though body weight in mice is correlated with motor performance, the age-related decline in motor function is still intact after adjusting for this factor, i.e., weight is not the main factor contributing to age-related performance impairments. Because motor performance was not decreased from 18 to 24 months in the control group, and because initial analysis of the data from the group tested at 18 and 24 months showed no difference between the diet groups on any test of motor performance (because of variation and practice effects), we primarily looked at the effect for the antioxidant diets in the 24-month post only group. This group, tested for the first time at 24 months of age after 6 months on the various diets, was naive to the tests; prior practice was therefore not a factor (as it was in the pre & post group). An initial ANOVA revealed a difference among the diet groups with respect to weight [F(5, 78) ⫽ 2.67, p ⬍ 0.05] and inclined screen performance [F(5, 78) ⫽ 2.96, p ⬍ 0.05]; the GSH group weighed significantly (p ⬍ 0.05) less than the control, vit E, and melatonin groups and performed significantly (p ⬍ 0.05) better than these groups on the inclined screen. Therefore, to control for this difference in body weight and prevent its influence on the behavioral tests, subsequent ANOVAs were run on a subset of the animals by discarding mice with a weight of greater or less than one standard deviation away from the mean weight of all the mice. This weight-matching strategy eliminated 32 mice from the sample: 5 in the control group, 3 in the vit E group, 8 in the GSH group, 7 in the E⫹GSH group, 5 in the melatonin group, and 4 in the strawberry group; the group effect for weight by using this subset of animals was now nonsignificant (p ⫽ 0.26). A diet effect was seen for only one parameter (when analyzing the weight-matched dataset), inclined screen latency [F(5, 46) ⫽ 5.00, p ⬍ 0.001]. The GSH group had a longer latency to fall and performed significantly (p ⬍ 0.05) better than all the other groups on this measure (Fig. 2). Interestingly, this parameter was the only one that showed a diet effect before the groups were weight-matched, although using the weight-matched dataset revealed that the performance of the GSH group was improved relative to all other groups, not just the ones in which the weights differed. It was also found that the GSH group had a significantly higher survivability rate (79% vs. 56%) than the control group
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Fig. 2. Latency to fall (mean ⫹ SEM: s) in the inclined screen test (85o tilt) for six different diet groups: control (n ⫽ 8), vit E (n ⫽ 9), GSH (n ⫽ 9), melatonin (n ⫽ 10), E⫹GSH (n ⫽ 6), and strawberry (n ⫽ 10). The GSH group is significantly (p ⬍ 0.05) different from all other groups.
(Mann–Whitney U(1) ⫽ 585.00, p ⬍ 0.05; there were no other significant differences between groups in survivability). No effect of diet was seen for rod latency, wire latency, plank walk latency, distance, or turns (Fig. 3).
4. Discussion This study showed that motor performance was lower in 18-month-old animals compared with 4-month-old animals, however, no further decline was seen from 18 to 24 months. Dietary antioxidant supplementation was not effective in reversing age-related deficits in psychomotor behavior, except for the GSH diet on inclined screen performance. One possible reason why the diets were not efficacious could be related to the fact that motor performance deteriorates rapidly and profoundly with age (Shukitt–Hale et al., 1998b); this hypothesis is supported by the result that no decline in motor performance was seen past 18 months in the control group. Behaviors requiring a greater degree of motor coordination, balance, or strength have been shown to decline systematically and quickly with aging, all dropping sharply beginning in mid-life (Wallace et al., 1980; Shukitt–Hale et al., 1998b). Consistent with another investigation (Ingram et al., 1981), C57BL/6J mice show motor behavioral decrements from 4 to 18 months on the rod walk and wire-hang tests, but no further decrement at 24 months. These age-associated motor deficits may be difficult to reverse, even with antioxidant treatment. It could be that it is much more difficult to reverse the effects of aging than to try to prevent or protect against them, which could be accomplished by beginning dietary treatment at an earlier age. These results agree with those of other studies which showed no beneficial effect of various chronic treatments on motor performance in older animals. One previous study (Ingram et al., 1993) found no beneficial effects of chronic L-deprenyl treatment (beginning at 18 months of age) on motor performance in mice, with one exception—performance on the rotodrum in mice treated for 9 months. L-Deprenyl is a potent, irreversible monoamine oxidase Type B (MAO-B) inhibitor thought to have a general effect on reducing impairment caused by ROS by decreasing MAO-B activity in the brain. A
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Fig. 3. Latency to fall (mean ⫹ SEM: s) in the rod walk, wire hang, and plank walk, as well as distance traveled and number of turns in the plank walk test for six different diet groups: control (n ⫽ 8), vit E (n ⫽ 9), GSH (n ⫽ 9), melatonin (n ⫽ 10), E⫹GSH (n ⫽ 6), and strawberry (n ⫽ 10).
second study (Ingram et al., 1994a) found that chronic nimodipine (a calcium channel blocker) had no significant effects on motor performance (inclined screen, rotorod, and rod hang) when treatment was initiated at 24 months of age. Another study found that four months of antioxidant treatment (phenyl-␣-tert-butyl nitrone, ␣-tocopherol, and ascorbate) did not improve motor activity, but did improve cognitive performance, of aged rats (Socci et al., 1995). Finally, one study (Pitsikas et al., 1990) tested psychomotor perfor-
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mance of rats who had been kept on a life-long hypocaloric diet from the age of 3 weeks, because dietary restriction has been shown to have a sparing effect on age-related deficits. No differences were found between aged (24 month) animals who had been fed a standard versus a hypocaloric diet on motor tests (plank walking and rotarod test), yet beneficial dietary effects were found in adult (12 month) rats. Old rats performed very poorly, independently of their diet, particularly on tests calling for balance, suggesting that mechanisms controlling body balance are those that deteriorate most in old age (Pitsikas et al., 1990). Some studies have shown reduction of motor behavioral deficits in old animals. Treatments that increased striatal DA receptor density, e.g., haloperidol (Joseph et al., 1983a) and prolactin (Joseph and Lippa, 1986), or retarded their loss, e.g., dietary restriction (Joseph et al., 1983b), improved motor performance in senescent animals, suggesting the importance of the functional integrity of the striatum in these behaviors (Joseph and Roth, 1988). Further, another study (Ingram et al., 1987) found that the age-related declines in rotorod performance were attenuated in 31- to 35-month-old mice when dietary restriction began at weaning. It is interesting that only the GSH diet in the present study improved motor behavior, specifically inclined screen performance; the GSH group also had the highest survival rate—79%—which was significantly higher than the control group. Brain GSH is decreased in aging (Chen et al., 1989) and the GSH/GSSG (oxidized GSH) ratio falls with age in C57BJ mice (Vina et al., 1992); these age-related changes can be prevented with oral administration of GSH (Vina et al., 1992). Additionally, administration of GSH also improved the performance of these mice, in that they did not lose their ability to pass a motor coordination test at 18 months of age (Vina et al., 1992). In the same manner, GSH supplementation in this study reversed the age-related effect on inclined screen performance, possibly by destroying ROS and neutralizing free radicals generated by OS through increased brain levels of GSH. This improvement in the GSH group at 24 months of age was not due to differences in body weight, as the changes were still evident after adjusting for body weight. In this study, the 44% mortality rate in the control group, while not unusual for C57BL/6 mice, may account for the lack of a difference in motor performance between 18 and 24 months (i.e., mice that may have been impaired at 24 months died before they could be tested). This attrition is a problem with aging studies in that only animals that survive can be tested. However, additional analyses run on the 117 original mice in the Pre & Post behavioral group showed that there were no differences on any measure of performance at 18 months between survivors (those mice who lived until 24 months, n ⫽ 75) and casualties (those who died, n ⫽ 42), showing that those animals who die earlier do not initially have worse motor performance. This study showed that age-related deficits in motor behavior appear early in life and are fairly resistant to change. Further research should continue to explore the protective effects of antioxidants implemented at an earlier age so as to prevent OS effects. Our laboratory has shown beneficial effects of strawberry, spinach, and vit E diets on agerelated neuronal signal-transduction and cognitive behavioral deficits in animals fed supplemented diets from age 6 months to 15 months (Joseph et al., 1998), and we are conducting additional studies along these lines. Preliminary results show that a blueberry diet (similar to the strawberry diet in the present study) reversed deficits on two measures of motor performance (rod walk and accelerating rotarod) and reversed age-related changes in neuronal signal transduction (Bickford et al., 1998; Shukitt–Hale et al., 1998a).
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Age-related deficits in motor performance are thought to be the result of OS-induced alterations in the striatum, which shows marked neurodegenerative changes beginning at an early age (Joseph, 1992). A recent study that justifies this antioxidant approach found that the age-related loss of motor coordination in mice (aged 22 months) was correlated with OS damage within the cerebellum (Forster et al., 1996). OS damage within the hippocampus and striatum was not predictive of functional impairment; however the authors believe that the relatively high level of damage in these areas may indicate a more advanced stage of aging in which oxidative damage is no longer a reliable indication of functional status (Forster et al., 1996). Therefore, these results (Forster et al., 1996) support the view that OS is a causal factor in brain senescence and possibly age-related behavioral changes, justifying the continued exploration of the potential for prevention or reversal by antioxidant treatments as assessed in this study.
Acknowledgments The authors thank Simin Nikbin Meydani for providing the mice used in this study. We also thank George Mouzakis for graphical assistance and Ippolita Cantuti–Castelvetri for assisting with data collection. Additionally, we thank Sung Nim Han and Dayong Wu for preparing the diets, Ronald L. Prior and Guohua Cao for supplying the Strawberry extract, and the animal carestaff (Department of Comparative Biology and Medicine) of the U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University for the feeding and maintenance of the mice used in this study. We also thank Kyowa Hakko Kogyo, Tokyo, Japan, for providing the glutathione.
References Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA, 90, 7915–7922. Bickford, P. C., Shukitt–Hale, B., Gould, T. J., Briedrick, L., Denisova, N., Bielinski, D., & Joseph, J. A. (1998). Reversal of age-related declines in CNS neurotransmission with diets supplemented with fruit or vegetable extracts. Soc Neurosci Abst, 24, 2157. Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med, 22, 749 –760. Carney, J. M., Smith, C. D., Carney, A. M., & Butterfield, D. A. (1994). Aging- and oxygen-induced modifications in brain biochemistry and behavior. Ann N Y Acad Sci, 738, 44 –53. Chen, T. S., Richie, J. P., & Lang, C. A. (1989). The effect of aging on glutathione and cysteine levels in different regions of the mouse brain. Proc Soc Exp Biol Med, 190, 399 – 402. Dean, R. L., Scozzafava, J., Goas, J. A., Regan, B., Beer, B., & Bartus, R. T. (1981). Age-related differences in behavior across the life span of the C57BL/6J mouse. Exp Aging Res, 7, 427– 451. Favilli, F., Marraccini, P., Iantomasi, T., & Vincenzizi, M. T. (1997). Effect of orally administered glutathione on glutathione levels in some organs of rats: role of specific transporters. Br J Nutr, 78, 293–300. Forster, M. J., Dubey, A., Dawson, K. M., Stutts, W. A., Lal, H., & Sohal, R. S. (1996). Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain. Proc Natl Acad Sci, USA 93, 4765– 4769. Furukawa, T., Meydani, S. N., & Blumberg, J. B. (1987). Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech Ageing Dev, 38, 107–117. Halliwell, B. (1994). Free radicals and antioxidants: a personal view. Nutr Rev, 52, 253–265. Harman, D. (1992). Role of free radicals in aging and disease. Ann NY Acad Sci, 673, 126 –141. Ingram, D. K. (1983). Toward the behavioral assessment of biological aging in the laboratory mouse: concepts, terminology, and objectives. Exp Aging Res, 9, 225–238.
B. Shukitt–Hale et al. / Experimental Gerontology 34 (1999) 797– 808
807
Ingram, D. K., Joseph, J. A., Spangler, E. L., Roberts, D., Hengemihle, J., & Fanelli, R. J. (1994a). Chronic nimodipine treatment in aged rats: analysis of motor and cognitive effects and muscarinic-induced striatal dopamine release. Neurobiol Aging, 15, 55– 61. Ingram, D. K., Jucker, M., & Spangler, E. L. (1994b). Behavioral manifestations of aging. In: U. Mohr, D.L. Cungworth, C.C. Capen (Eds.), Pathobiology of the Aging Rat, vol. 2 (pp. 149 –170). Washington, DC: ILSI Press. Ingram, D. K., London, E. D., Reynolds, M. A., Waller, S. B., & Goodrick, C. L. (1981). Differential effects of age on motor performance in two mouse strains. Neurobiol Aging, 2, 221–227. Ingram, D. K., Weindruch, R., Spangler, E. L., Freeman, J. R., & Walford, R. L. (1987). Dietary restriction benefits learning and motor performance of aged mice. J Gerontol, 42, 78 – 81. Ingram, D. K., Wiener, H. L., Chachich, M. E., Long, J. M., Hengemihle, J., & Gupta, M. (1993). Chronic treatment of aged mice with L-deprenyl produces marked striatal MAO-B inhibition but no beneficial effects on survival, motor performance, or nigral lipofuscin accumulation. Neurobiol Aging, 14, 431– 440. Joseph, J. A. (1992). The putative role of free radicals in the loss of neuronal functioning in senescence. Integ Physiol Behav Sci, 27, 216 –227. Joseph, J. A., Bartus, R. T., Clody, D., Morgan, D., Finch, C., Beer, B., & Sesack, S. (1983a). Psychomotor performance in the senescent rodent: reduction of deficits via striatal dopamine receptor up-regulation. Neurobiol Aging, 4, 313–319. Joseph, J. A. & Lippa, A. S. (1986). Reduction of motor behavioral deficits in senescent animals via chronic prolactin administration - II. Non-stereotypic behaviors. Neurobiol Aging, 7, 37– 40. Joseph, J. A. & Roth, G. S. (1988). Altered striatal dopaminergic and cholinergic reciprocal inhibitory control and motor behavioral decrements in senescence. Ann NY Acad Sci, 521, 110 –122. Joseph, J. A., Shukitt–Hale, B., Denisova, N. A., Prior, R. L., Cao, G., Martin, A., Taglialatela, G., & Bickford, P. C. (1998). Long-term dietary strawberry, spinach, or vitamin E supplementation retards the onset of age-related neuronal signal-transduction and cognitive behavioral deficits. J Neurosci, 18, 8047– 8055. Joseph, J. A., Whitaker, J., Roth, G. S., & Ingram, D. K. (1983b). Life-long dietary restriction affects striatally-mediated behavioral responses in aged rats. Neurobiol Aging, 4, 191–196. Lipman, R. D., Bronson, R. T., Wu, D., Smith, D. E., Prior, R., Cao, G., Han, S. N., Martin, K. R., Meydani, S. N., & Meydani, M. (1998). Disease incidence and longevity are unaltered by dietary antioxidant supplementation initiated during middle age in C57BL/6 mice. Mech Ageing Dev, 103, 269 –284. Meydani, S. N., Meydani, M., Verdon, C. P., Shapiro, A. A., Blumberg, J. B., & Hayes, K. C. (1986). Vitamin E supplementation suppresses prostaglandin E2 synthesis and enhances the immune response of aged mice. Mech Ageing Dev, 34, 191–201. Meydani, S. N., Shapiro, A. C., Meydani, M., Macauley, J. B., & Blumberg, J. B. (1987). Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BL/6Nia mice. Lipids, 22, 345–350. Mo, J. Q., Hom, D. G., & Andersen, J. K. (1995). Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain. Mech Ageing Dev, 81, 73– 82. Muller, D. P. R. & Goss–Sampson, M. A. (1990). Neurochemical, neurophysiological, and neuropathological studies in Vitamin E deficiency. Crit Rev Neurobiol, 5, 239 –263. Olanow, C. W. (1992). An introduction to the free radical hypothesis in Parkinson’s disease. Ann Neurol, 32, S2–S9. Pierpaoli, W. & Regelson, W. (1994). Pineal control of aging: Effect of melatonin and pineal grafting on aging mice. Proc Natl Acad Sci USA, 91, 787–791. Pileblad, E. & Magnusson, T. (1990). Effective depletion of glutathione in rat striatum and substantia nigra by L-buthionine sulfoximine in combination with 2-Cyclohexene-1-one. Life Sci, 47, 2333–2342. Pitsikas, N., Carli, M., Fidecka, S., & Algeri, S. (1990). Effect of life-long hypocaloric diet on age-related changes in motor and cognitive behavior in a rat population. Neurobiol Aging, 11, 417– 423. Reiter, R. J. (1995). The role of the neurohormone melatonin as a buffer against macromolecular oxidative damage. Neurochem Int, 27, 453– 460. Shukitt–Hale, B., Bickford, P. C., McEwen, J. J., Cao, G., Prior, R. L., & Joseph, J. A. (1998a). Reversal of age-related motor and cognitive behavioral deficits with diets supplemented with fruit or vegetable extracts. Soc Neurosci Abst, 24, 2157. Shukitt–Hale, B., Mouzakis, G., & Joseph, J. A. (1998b). Psychomotor and spatial memory performance in aging male Fischer 344 rats. Exp Geron, 33, 615– 624. Sies, H. (1993). Strategies of antioxidant defense. Eur J Biochem, 215, 213–219. Socci, D. J., Crandall, B. M., & Arendash, G. W. (1995). Chronic antioxidant treatment improves the cognitive performance of aged rats. Brain Res, 693, 88 –94.
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Vina, J., Sastre, J., Anton, V., Bruseghini, L., Esteras, A., & Asensi, M. (1992). Effect of aging on glutathione metabolism: Protection by antioxidants. In: Emerit, I., Chance, B. (Eds.), Free Radicals and Aging (pp. 136 –144). Basel, Switzerland: Birkhauser Verlag. Wallace, J. E., Krauter, E. E., & Campbell, B. A. (1980). Motor and reflexive behavior in the aging rat. J Gerontol, 35, 364 –370. Wang, H., Cao, G., & Prior, R. L. (1996). Total antioxidant capacity of fruits. J Agric Food Chem, 44, 701–705. Wang, H., Cao, G., & Prior, R. L. (1997). Oxygen radical absorbing capacity of anthocyanins. J Agric Food Chem, 45, 304 –309.
The effects of dietary antioxidants on psychomotor performance in aged mice Barbara Shukitt–Hale*, Donald E. Smith, Mohsen Meydani, James A. Joseph United States Department of Agriculture-Agricultural Research Service, Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111 USA Received 15 December 1998; received in revised form 12 April 1999; accepted 12 April 1999
Abstract Male C57BL/6NIA mice were provided one of six different antioxidant diets: vitamin E, glutathione, vitamin E plus glutathione, melatonin, strawberry extract, or control, beginning at 18 months of age. A battery of motor tests—rod walk, wire hang, plank walk, and inclined screen—was administered either: 1) before dietary treatment and then 6 months later at 24 months of age; or 2) only after 6 months of dietary treatment at age 24 months. An untreated group of 4-month-old mice served as young controls. Psychomotor performance was lower in 18-month-old mice compared with 4-month-old mice in the rod walk, wire hang, and inclined screen tests; however, no further decline was seen from 18 to 24 months on any measure. Chronic dietary antioxidant treatments were not effective in reversing age-related deficits in psychomotor behavior, except for the glutathione diet on inclined screen performance. It seems that motor performance deteriorates profoundly with age, because deficits at 18 months of age were as severe as they were at 24 months, and these age-associated motor deficits may be difficult to reverse, even with antioxidant treatment. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Oxidative stress; Motor behavior; Aging; Chronic dietary treatment; Vitamin E; Glutathione; Melatonin; Strawberry
1. Introduction Oxidative stress (OS) is thought to be a contributing factor to the decrements in motor performance seen in aging. The “free radical hypothesis of aging” maintains that, with age, oxidative damage increases and endogenous antioxidant defense mechanisms become insufficient to detoxify the generation of reactive oxygen species (ROS), such as super* Corresponding author. Tel.: ⫹001-617-556-3118; fax: ⫹1-617-556-3222. E-mail address: [email protected] (B. Shukitt–Hale) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 3 9 - X
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oxide and hydroxyl radicals (Harman, 1992; Ames et al., 1993; Mo et al., 1995). The subsequent accumulation of these oxygen free radicals results in oxidative damage to critical biological molecules, particularly in the brain (Olanow, 1992), and contributes to the detrimental effects of aging (Harman, 1992; Ames et al., 1993; Carney et al., 1994). Manipulations shown to increase the presence of ROS in the brain increase oxidized protein and worsen performance deficits, whereas interventions designed to quench ROS decrease oxidized protein levels and normalize associated behavioral deficits (Carney et al., 1994). Therefore, if the generation and accumulation of ROS are important factors in causing age-related psychomotor decrements, chronic enhancement of antioxidant defenses could slow, or possibly reverse, this process by reducing OS, resulting in improved motor performance (Socci et al., 1995). It has been postulated that accumulation of oxidized proteins or lipid peroxidation in aging may be involved in the age-related functional neuronal loss contributing to deficits in motor performance (Joseph, 1992; Forster et al., 1996). Specifically, these alterations in motor function may include decreases in balance, muscle strength, and coordination. Both aged rats and mice show decrements in performance on several tasks requiring coordinated control of motor and reflexive responses, such as suspension time on a horizontal wire or inclined wire mesh screen and the length of time it takes for the animal to traverse a wooden rod or plank (Dean et al., 1981; Joseph et al., 1983a; Joseph and Lippa, 1986; Ingram et al., 1994b, Shukitt–Hale et al., 1998b). Age-related deficits in motor performance are thought to be the result of alterations in the striatal dopamine (DA) system; the striatum, a brain area which mediates several aspects of motor behavior (Joseph and Roth, 1988), shows marked neurodegenerative changes with age (Joseph, 1992). Antioxidant nutrients added to the diet are one defense strategy to prevent, intercept, or repair age-induced OS (Harman, 1992; Sies, 1993; Joseph et al., 1998). Vitamin E (vit E), glutathione (GSH), melatonin, and strawberry extract have all been found to have antioxidant properties. Vit E is a radical chain-breaking antioxidant (Sies, 1993) and is the most important free radical scavenger within membranes and lipoproteins (Halliwell, 1994). Vit E inhibits lipid peroxidation by scavenging peroxyl radicals (Halliwell, 1994) and severe deprivation of vit E in humans produces neurological damage (Muller and Goss–Sampson, 1990). GSH is an endogenous protective agent that plays a critical role in intracellular antioxidant defense. GSH acts to destroy ROS and is capable of neutralizing free radicals generated by OS (Pileblad and Magnusson, 1990); brain GSH is decreased in aging (Chen et al., 1989). Melatonin, the primary hormone of the pineal gland, has been shown to be a free radical scavenger and a general antioxidant (Reiter, 1995). Melatonin preserves macromolecules including DNA, protein, and lipids from oxidative damage and, compared with other antioxidants, melatonin seems to have a greater efficacy in protecting against cellular OS (Reiter, 1995). Fruits and vegetables are the main source of antioxidants in the diet (Ames et al., 1993). Strawberries have been shown to have a high total antioxidant capacity (Wang et al., 1996), possibly because of flavonoids, specifically the anthocyanins (Wang et al., 1996; Cao et al., 1997; Wang et al., 1997). Fruit and vegetable intake is associated with a lowered risk of degenerative disease, whereas the lack of adequate consumption of fruits and vegetables is linked to cancer incidence (Ames et al., 1993). Recent research from our laboratory demonstrated that strawberry, spinach, and vit E diets were effective in retarding age-associated deficits in muscarinic receptor sensitivity, as assessed via oxotremorine enhancement of striatal dopamine release; isoproteronal facilitation of
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Table 1 Mouse survival at 24 months by dietary group and behavioral testing group
Dietary Group
Control Vitamin E GSH E ⫹ GSH Melatonin Strawberry Overall survival
Behavioral Group Pre & Post
Post only
Survival per group
% Survived
9/19 14/20 14/20 14/19 12/19 12/20 75/117
13/20 12/20 17/19 13/19 15/19 14/18 84/115
22/39 26/40 31/39 27/38 27/38 26/38 159/232
56% 65% 79% 71% 71% 68% 69%
GABAergic inhibition of cerebellar Purkinje neurons; calcium regulation; and cognitive performance as measured by the Morris water maze (Joseph et al., 1998). The objective of the present study was to determine the efficacy of dietary supplementation with antioxidants in reversing/restoring age-related declines in motor performance in mice. Treatment of mice with one of six diets (control, vit E, GSH, E⫹GSH, melatonin, or strawberry) was begun at 18 months of age and carried through to 24 months of age. Motor performance was assessed on a battery of age-sensitive tests, both before and after treatment with these antioxidants in the diets. This investigation was part of a larger study designed to investigate the effects of dietary antioxidants in aged animals on oxidative stress, immune response, age-associated pathogenesis (Lipman et al., 1998), and survival; results from these other studies are forthcoming.
2. Materials and methods 2.1. Animals Male C57BL/6NIA mice (bred and raised at the HNRCA under barrier conditions, in four monthly onset groups) were used in this study (see Table 1). The 232 ad libitum chow-fed mice were 18 months of age before being started on a 6-month dietary treatment regimen consisting of a control diet (Meydani et al., 1987) or control diet supplemented with one of five antioxidants. From the original group, 159 mice (69%) survived until age 24 months. An untreated group (n ⫽ 11) of 4-month-old mice served as young, chow-fed, controls. Throughout the 6 months, the mice were individually housed in autoclaved, filtered, polycarbonate cages with wood shavings as bedding and free access to food and water, and maintained on a 12-h light/dark cycle. An ongoing health surveillance ensured that the animals remained viral antibody free. These animals were used in compliance with all applicable laws and regulations as well as principles expressed in the National Institutes of Health, United States Public Health Service Guide for the Care and Use of Laboratory Animals. This study was reviewed and approved by the Animal Care and Use Committee at the U.S. Department of Agriculture, Human Nutrition Research Center on Aging at Tufts University (an AAALAC accredited facility).
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2.2. Diets Initially, mice were weight matched and randomly assigned to one of six diet groups (38 – 40 mice in each group, see Table 1): control (a modified AIN76 diet with 30 ppm vit E), vit E (dl-␣-tocopheryl acetate, 500 ppm vit E), GSH (0.5% GSH), E⫹GSH (500 ppm vit E ⫹ 0.5% GSH), melatonin (11 ppm melatonin), or freeze-dried aqueous strawberry extract (1%); all doses were based on previous investigations [vit E: (Meydani et al., 1986); GSH: (Furukawa et al., 1987; Favilli et al., 1997); melatonin: (Pierpaoli and Regelson, 1994); strawberry: (Wang et al., 1996; Joseph et al., 1998)] that have shown increased levels in plasma and other tissues following feeding. The basal diet contained casein (18%), corn starch (33.55%), sucrose (33.4%), cellulose (5%), DL-methionine (0.3%), choline bitartrate (0.25%), AIN76A salt (3.5%) and vitamin mix (1%), plus 5% soybean oil (Meydani et al., 1987). Because melatonin is light-sensitive, the melatonin diet was provided in ceramic (lead-free) jars. For a more complete description of the diets, see Lipman et al. (1998). 2.3. Behavioral Testing Mice were randomly assigned to one of two behavioral groups: pre & post (motor testing before and after 6 months of dietary treatment) and post only (motor testing following 6 months of dietary treatment) (see Table 1). A young control group was tested one time, at age 4 months. For each test session, a battery of psychomotor behavioral tests was performed that consisted of complex movement tasks which have been shown to deteriorate with age (Joseph et al., 1983; Joseph, 1992; Ingram et al., 1994b; Shukitt–Hale et al., 1998b), including in C57BL/6J mice (Dean et al., 1981; Ingram, 1983; Ingram et al., 1993). These tests were administered one time at each test session in the following order, with a short break between them. Briefly, the tests included: 1) Rod walking, which measures psychomotor coordination and the integrity of the vestibular system by requiring the animal to balance on a stationary, horizontal rod (120 cm long, 12 mm in diameter, positioned 35 cm above the table surface); 2) Wire suspension/wire hanging, which measures muscle strength and the prehensile reflex, an animal’s ability to grasp a taut horizontal wire (2 mm in diameter, 62 cm above the table top) with its forepaws and to remain suspended; 3) Plank walking, which measures balance and coordination by exposing the mice to three different sizes of horizontal planks (wide ⫽ 20 mm; medium ⫽ 13 mm; narrow ⫽ 6 mm, each 100 cm long, placed 33 cm above the floor); and 4) Inclined screen, which measures muscle tone, strength, stamina, and balance by placing the mouse on a wire mesh screen that was tilted 60o or 85o to the horizontal plane of the floor. For a more complete description of the tests, see Shukitt–Hale et al. (1998b). 2.4. Statistics For each behavioral measure, between-subjects analyses of variance (ANOVA) models comparing the different diet groups were performed by using Biomedical Data Package (BMDP) to test for statistical significance at the p ⱕ 0.05 level; post hoc comparisons were performed by using Duncan’s multiple range test. Additionally, ANOVAs were run to compare differences between the age groups, by using data from the 4, 18 (pre & post), and 24 (post only) month control groups only; these groups were comparable because they were all naive at the time of testing.
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Fig. 1. Latency to fall (mean ⫹ SEM: s) in the rod walk, wire hang, and inclined screen (85o tilt) tests for two or three different age groups: 4-month (n ⫽ 11), 18-month (n ⫽ 19), and 24-month (n ⫽ 8). Asterisks indicate significant differences from 4 months (** p ⬍ 0.01).
3. Results Initially, ANOVAs were run to compare motor performance between ages to determine if there were significant differences between 4, 18, and 24 months (controls only). Several tests showed deficits from 4 to 18 months; however, no tests showed additional performance decrements from 18 to 24 months. Specifically, in the rod walking test (Fig. 1), ANOVA showed a significant age difference [F(2, 35) ⫽ 8.11, p ⬍ 0.01]; post-hoc testing
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showed that latency to fall was longer for the young group than for the 18-month (p ⬍ 0.01) or the 24-month group (p ⬍ 0.01). There was also a significant age effect in the wire-hang test (Fig. 1) [F(2,35) ⫽ 12.77, p ⬍ 0.001]; again, latency to fall was longer for the 4-month group when compared with the 18-month (p ⬍ 0.01) or 24-month group (p ⬍ 0.01). Only data from the 4- and 24-month groups were comparable for the inclined screen, as both of these groups were tested at the 85o angle (the pre & post group animals were tested at the 60o angle, but this angle proved too easy for the mice as they all achieved the 600-s max time, regardless of age or diet). There was a significant age effect on the inclined screen (Fig. 1) [F(1, 17) ⫽ 115.93, p ⬍ 0.001], with the 24-month-old animals having a shorter latency to fall than the 4-month-group. There were no age differences for plank walk latency, distance, or turns; again, this test proved too easy for the mice, as almost all of them achieved the 60-s max score for latency, regardless of age. In summary, although some deficits were seen from 4 to 18 months, motor performance for the 18-month group on all measures was never different from the 24-month group. Additionally, there was a significant effect of age on body weight [F(2, 35) ⫽ 130.74, p ⬍ 0.001] for these control groups. The 4-month group weighed, on average, 26.1 ⫾ 0.26 g (mean ⫾ SEM), the 18 month group weighed 40.6 ⫾ 0.77 g, and the 24 month group weighed 51.1 ⫾ 1.88 g. Body weight increased with age, with both the 18- and 24-month groups weighing more than the 4-month group (p ⬍ 0.01) and the 24 month group weighing more than the 18-month group (p ⬍ 0.01). However, Ingram et al. (1983, 1994b) found that even though body weight in mice is correlated with motor performance, the age-related decline in motor function is still intact after adjusting for this factor, i.e., weight is not the main factor contributing to age-related performance impairments. Because motor performance was not decreased from 18 to 24 months in the control group, and because initial analysis of the data from the group tested at 18 and 24 months showed no difference between the diet groups on any test of motor performance (because of variation and practice effects), we primarily looked at the effect for the antioxidant diets in the 24-month post only group. This group, tested for the first time at 24 months of age after 6 months on the various diets, was naive to the tests; prior practice was therefore not a factor (as it was in the pre & post group). An initial ANOVA revealed a difference among the diet groups with respect to weight [F(5, 78) ⫽ 2.67, p ⬍ 0.05] and inclined screen performance [F(5, 78) ⫽ 2.96, p ⬍ 0.05]; the GSH group weighed significantly (p ⬍ 0.05) less than the control, vit E, and melatonin groups and performed significantly (p ⬍ 0.05) better than these groups on the inclined screen. Therefore, to control for this difference in body weight and prevent its influence on the behavioral tests, subsequent ANOVAs were run on a subset of the animals by discarding mice with a weight of greater or less than one standard deviation away from the mean weight of all the mice. This weight-matching strategy eliminated 32 mice from the sample: 5 in the control group, 3 in the vit E group, 8 in the GSH group, 7 in the E⫹GSH group, 5 in the melatonin group, and 4 in the strawberry group; the group effect for weight by using this subset of animals was now nonsignificant (p ⫽ 0.26). A diet effect was seen for only one parameter (when analyzing the weight-matched dataset), inclined screen latency [F(5, 46) ⫽ 5.00, p ⬍ 0.001]. The GSH group had a longer latency to fall and performed significantly (p ⬍ 0.05) better than all the other groups on this measure (Fig. 2). Interestingly, this parameter was the only one that showed a diet effect before the groups were weight-matched, although using the weight-matched dataset revealed that the performance of the GSH group was improved relative to all other groups, not just the ones in which the weights differed. It was also found that the GSH group had a significantly higher survivability rate (79% vs. 56%) than the control group
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Fig. 2. Latency to fall (mean ⫹ SEM: s) in the inclined screen test (85o tilt) for six different diet groups: control (n ⫽ 8), vit E (n ⫽ 9), GSH (n ⫽ 9), melatonin (n ⫽ 10), E⫹GSH (n ⫽ 6), and strawberry (n ⫽ 10). The GSH group is significantly (p ⬍ 0.05) different from all other groups.
(Mann–Whitney U(1) ⫽ 585.00, p ⬍ 0.05; there were no other significant differences between groups in survivability). No effect of diet was seen for rod latency, wire latency, plank walk latency, distance, or turns (Fig. 3).
4. Discussion This study showed that motor performance was lower in 18-month-old animals compared with 4-month-old animals, however, no further decline was seen from 18 to 24 months. Dietary antioxidant supplementation was not effective in reversing age-related deficits in psychomotor behavior, except for the GSH diet on inclined screen performance. One possible reason why the diets were not efficacious could be related to the fact that motor performance deteriorates rapidly and profoundly with age (Shukitt–Hale et al., 1998b); this hypothesis is supported by the result that no decline in motor performance was seen past 18 months in the control group. Behaviors requiring a greater degree of motor coordination, balance, or strength have been shown to decline systematically and quickly with aging, all dropping sharply beginning in mid-life (Wallace et al., 1980; Shukitt–Hale et al., 1998b). Consistent with another investigation (Ingram et al., 1981), C57BL/6J mice show motor behavioral decrements from 4 to 18 months on the rod walk and wire-hang tests, but no further decrement at 24 months. These age-associated motor deficits may be difficult to reverse, even with antioxidant treatment. It could be that it is much more difficult to reverse the effects of aging than to try to prevent or protect against them, which could be accomplished by beginning dietary treatment at an earlier age. These results agree with those of other studies which showed no beneficial effect of various chronic treatments on motor performance in older animals. One previous study (Ingram et al., 1993) found no beneficial effects of chronic L-deprenyl treatment (beginning at 18 months of age) on motor performance in mice, with one exception—performance on the rotodrum in mice treated for 9 months. L-Deprenyl is a potent, irreversible monoamine oxidase Type B (MAO-B) inhibitor thought to have a general effect on reducing impairment caused by ROS by decreasing MAO-B activity in the brain. A
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Fig. 3. Latency to fall (mean ⫹ SEM: s) in the rod walk, wire hang, and plank walk, as well as distance traveled and number of turns in the plank walk test for six different diet groups: control (n ⫽ 8), vit E (n ⫽ 9), GSH (n ⫽ 9), melatonin (n ⫽ 10), E⫹GSH (n ⫽ 6), and strawberry (n ⫽ 10).
second study (Ingram et al., 1994a) found that chronic nimodipine (a calcium channel blocker) had no significant effects on motor performance (inclined screen, rotorod, and rod hang) when treatment was initiated at 24 months of age. Another study found that four months of antioxidant treatment (phenyl-␣-tert-butyl nitrone, ␣-tocopherol, and ascorbate) did not improve motor activity, but did improve cognitive performance, of aged rats (Socci et al., 1995). Finally, one study (Pitsikas et al., 1990) tested psychomotor perfor-
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mance of rats who had been kept on a life-long hypocaloric diet from the age of 3 weeks, because dietary restriction has been shown to have a sparing effect on age-related deficits. No differences were found between aged (24 month) animals who had been fed a standard versus a hypocaloric diet on motor tests (plank walking and rotarod test), yet beneficial dietary effects were found in adult (12 month) rats. Old rats performed very poorly, independently of their diet, particularly on tests calling for balance, suggesting that mechanisms controlling body balance are those that deteriorate most in old age (Pitsikas et al., 1990). Some studies have shown reduction of motor behavioral deficits in old animals. Treatments that increased striatal DA receptor density, e.g., haloperidol (Joseph et al., 1983a) and prolactin (Joseph and Lippa, 1986), or retarded their loss, e.g., dietary restriction (Joseph et al., 1983b), improved motor performance in senescent animals, suggesting the importance of the functional integrity of the striatum in these behaviors (Joseph and Roth, 1988). Further, another study (Ingram et al., 1987) found that the age-related declines in rotorod performance were attenuated in 31- to 35-month-old mice when dietary restriction began at weaning. It is interesting that only the GSH diet in the present study improved motor behavior, specifically inclined screen performance; the GSH group also had the highest survival rate—79%—which was significantly higher than the control group. Brain GSH is decreased in aging (Chen et al., 1989) and the GSH/GSSG (oxidized GSH) ratio falls with age in C57BJ mice (Vina et al., 1992); these age-related changes can be prevented with oral administration of GSH (Vina et al., 1992). Additionally, administration of GSH also improved the performance of these mice, in that they did not lose their ability to pass a motor coordination test at 18 months of age (Vina et al., 1992). In the same manner, GSH supplementation in this study reversed the age-related effect on inclined screen performance, possibly by destroying ROS and neutralizing free radicals generated by OS through increased brain levels of GSH. This improvement in the GSH group at 24 months of age was not due to differences in body weight, as the changes were still evident after adjusting for body weight. In this study, the 44% mortality rate in the control group, while not unusual for C57BL/6 mice, may account for the lack of a difference in motor performance between 18 and 24 months (i.e., mice that may have been impaired at 24 months died before they could be tested). This attrition is a problem with aging studies in that only animals that survive can be tested. However, additional analyses run on the 117 original mice in the Pre & Post behavioral group showed that there were no differences on any measure of performance at 18 months between survivors (those mice who lived until 24 months, n ⫽ 75) and casualties (those who died, n ⫽ 42), showing that those animals who die earlier do not initially have worse motor performance. This study showed that age-related deficits in motor behavior appear early in life and are fairly resistant to change. Further research should continue to explore the protective effects of antioxidants implemented at an earlier age so as to prevent OS effects. Our laboratory has shown beneficial effects of strawberry, spinach, and vit E diets on agerelated neuronal signal-transduction and cognitive behavioral deficits in animals fed supplemented diets from age 6 months to 15 months (Joseph et al., 1998), and we are conducting additional studies along these lines. Preliminary results show that a blueberry diet (similar to the strawberry diet in the present study) reversed deficits on two measures of motor performance (rod walk and accelerating rotarod) and reversed age-related changes in neuronal signal transduction (Bickford et al., 1998; Shukitt–Hale et al., 1998a).
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Age-related deficits in motor performance are thought to be the result of OS-induced alterations in the striatum, which shows marked neurodegenerative changes beginning at an early age (Joseph, 1992). A recent study that justifies this antioxidant approach found that the age-related loss of motor coordination in mice (aged 22 months) was correlated with OS damage within the cerebellum (Forster et al., 1996). OS damage within the hippocampus and striatum was not predictive of functional impairment; however the authors believe that the relatively high level of damage in these areas may indicate a more advanced stage of aging in which oxidative damage is no longer a reliable indication of functional status (Forster et al., 1996). Therefore, these results (Forster et al., 1996) support the view that OS is a causal factor in brain senescence and possibly age-related behavioral changes, justifying the continued exploration of the potential for prevention or reversal by antioxidant treatments as assessed in this study.
Acknowledgments The authors thank Simin Nikbin Meydani for providing the mice used in this study. We also thank George Mouzakis for graphical assistance and Ippolita Cantuti–Castelvetri for assisting with data collection. Additionally, we thank Sung Nim Han and Dayong Wu for preparing the diets, Ronald L. Prior and Guohua Cao for supplying the Strawberry extract, and the animal carestaff (Department of Comparative Biology and Medicine) of the U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University for the feeding and maintenance of the mice used in this study. We also thank Kyowa Hakko Kogyo, Tokyo, Japan, for providing the glutathione.
References Ames, B. N., Shigenaga, M. K., & Hagen, T. M. (1993). Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA, 90, 7915–7922. Bickford, P. C., Shukitt–Hale, B., Gould, T. J., Briedrick, L., Denisova, N., Bielinski, D., & Joseph, J. A. (1998). Reversal of age-related declines in CNS neurotransmission with diets supplemented with fruit or vegetable extracts. Soc Neurosci Abst, 24, 2157. Cao, G., Sofic, E., & Prior, R. L. (1997). Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships. Free Radic Biol Med, 22, 749 –760. Carney, J. M., Smith, C. D., Carney, A. M., & Butterfield, D. A. (1994). Aging- and oxygen-induced modifications in brain biochemistry and behavior. Ann N Y Acad Sci, 738, 44 –53. Chen, T. S., Richie, J. P., & Lang, C. A. (1989). The effect of aging on glutathione and cysteine levels in different regions of the mouse brain. Proc Soc Exp Biol Med, 190, 399 – 402. Dean, R. L., Scozzafava, J., Goas, J. A., Regan, B., Beer, B., & Bartus, R. T. (1981). Age-related differences in behavior across the life span of the C57BL/6J mouse. Exp Aging Res, 7, 427– 451. Favilli, F., Marraccini, P., Iantomasi, T., & Vincenzizi, M. T. (1997). Effect of orally administered glutathione on glutathione levels in some organs of rats: role of specific transporters. Br J Nutr, 78, 293–300. Forster, M. J., Dubey, A., Dawson, K. M., Stutts, W. A., Lal, H., & Sohal, R. S. (1996). Age-related losses of cognitive function and motor skills in mice are associated with oxidative protein damage in the brain. Proc Natl Acad Sci, USA 93, 4765– 4769. Furukawa, T., Meydani, S. N., & Blumberg, J. B. (1987). Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech Ageing Dev, 38, 107–117. Halliwell, B. (1994). Free radicals and antioxidants: a personal view. Nutr Rev, 52, 253–265. Harman, D. (1992). Role of free radicals in aging and disease. Ann NY Acad Sci, 673, 126 –141. Ingram, D. K. (1983). Toward the behavioral assessment of biological aging in the laboratory mouse: concepts, terminology, and objectives. Exp Aging Res, 9, 225–238.
B. Shukitt–Hale et al. / Experimental Gerontology 34 (1999) 797– 808
807
Ingram, D. K., Joseph, J. A., Spangler, E. L., Roberts, D., Hengemihle, J., & Fanelli, R. J. (1994a). Chronic nimodipine treatment in aged rats: analysis of motor and cognitive effects and muscarinic-induced striatal dopamine release. Neurobiol Aging, 15, 55– 61. Ingram, D. K., Jucker, M., & Spangler, E. L. (1994b). Behavioral manifestations of aging. In: U. Mohr, D.L. Cungworth, C.C. Capen (Eds.), Pathobiology of the Aging Rat, vol. 2 (pp. 149 –170). Washington, DC: ILSI Press. Ingram, D. K., London, E. D., Reynolds, M. A., Waller, S. B., & Goodrick, C. L. (1981). Differential effects of age on motor performance in two mouse strains. Neurobiol Aging, 2, 221–227. Ingram, D. K., Weindruch, R., Spangler, E. L., Freeman, J. R., & Walford, R. L. (1987). Dietary restriction benefits learning and motor performance of aged mice. J Gerontol, 42, 78 – 81. Ingram, D. K., Wiener, H. L., Chachich, M. E., Long, J. M., Hengemihle, J., & Gupta, M. (1993). Chronic treatment of aged mice with L-deprenyl produces marked striatal MAO-B inhibition but no beneficial effects on survival, motor performance, or nigral lipofuscin accumulation. Neurobiol Aging, 14, 431– 440. Joseph, J. A. (1992). The putative role of free radicals in the loss of neuronal functioning in senescence. Integ Physiol Behav Sci, 27, 216 –227. Joseph, J. A., Bartus, R. T., Clody, D., Morgan, D., Finch, C., Beer, B., & Sesack, S. (1983a). Psychomotor performance in the senescent rodent: reduction of deficits via striatal dopamine receptor up-regulation. Neurobiol Aging, 4, 313–319. Joseph, J. A. & Lippa, A. S. (1986). Reduction of motor behavioral deficits in senescent animals via chronic prolactin administration - II. Non-stereotypic behaviors. Neurobiol Aging, 7, 37– 40. Joseph, J. A. & Roth, G. S. (1988). Altered striatal dopaminergic and cholinergic reciprocal inhibitory control and motor behavioral decrements in senescence. Ann NY Acad Sci, 521, 110 –122. Joseph, J. A., Shukitt–Hale, B., Denisova, N. A., Prior, R. L., Cao, G., Martin, A., Taglialatela, G., & Bickford, P. C. (1998). Long-term dietary strawberry, spinach, or vitamin E supplementation retards the onset of age-related neuronal signal-transduction and cognitive behavioral deficits. J Neurosci, 18, 8047– 8055. Joseph, J. A., Whitaker, J., Roth, G. S., & Ingram, D. K. (1983b). Life-long dietary restriction affects striatally-mediated behavioral responses in aged rats. Neurobiol Aging, 4, 191–196. Lipman, R. D., Bronson, R. T., Wu, D., Smith, D. E., Prior, R., Cao, G., Han, S. N., Martin, K. R., Meydani, S. N., & Meydani, M. (1998). Disease incidence and longevity are unaltered by dietary antioxidant supplementation initiated during middle age in C57BL/6 mice. Mech Ageing Dev, 103, 269 –284. Meydani, S. N., Meydani, M., Verdon, C. P., Shapiro, A. A., Blumberg, J. B., & Hayes, K. C. (1986). Vitamin E supplementation suppresses prostaglandin E2 synthesis and enhances the immune response of aged mice. Mech Ageing Dev, 34, 191–201. Meydani, S. N., Shapiro, A. C., Meydani, M., Macauley, J. B., & Blumberg, J. B. (1987). Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BL/6Nia mice. Lipids, 22, 345–350. Mo, J. Q., Hom, D. G., & Andersen, J. K. (1995). Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain. Mech Ageing Dev, 81, 73– 82. Muller, D. P. R. & Goss–Sampson, M. A. (1990). Neurochemical, neurophysiological, and neuropathological studies in Vitamin E deficiency. Crit Rev Neurobiol, 5, 239 –263. Olanow, C. W. (1992). An introduction to the free radical hypothesis in Parkinson’s disease. Ann Neurol, 32, S2–S9. Pierpaoli, W. & Regelson, W. (1994). Pineal control of aging: Effect of melatonin and pineal grafting on aging mice. Proc Natl Acad Sci USA, 91, 787–791. Pileblad, E. & Magnusson, T. (1990). Effective depletion of glutathione in rat striatum and substantia nigra by L-buthionine sulfoximine in combination with 2-Cyclohexene-1-one. Life Sci, 47, 2333–2342. Pitsikas, N., Carli, M., Fidecka, S., & Algeri, S. (1990). Effect of life-long hypocaloric diet on age-related changes in motor and cognitive behavior in a rat population. Neurobiol Aging, 11, 417– 423. Reiter, R. J. (1995). The role of the neurohormone melatonin as a buffer against macromolecular oxidative damage. Neurochem Int, 27, 453– 460. Shukitt–Hale, B., Bickford, P. C., McEwen, J. J., Cao, G., Prior, R. L., & Joseph, J. A. (1998a). Reversal of age-related motor and cognitive behavioral deficits with diets supplemented with fruit or vegetable extracts. Soc Neurosci Abst, 24, 2157. Shukitt–Hale, B., Mouzakis, G., & Joseph, J. A. (1998b). Psychomotor and spatial memory performance in aging male Fischer 344 rats. Exp Geron, 33, 615– 624. Sies, H. (1993). Strategies of antioxidant defense. Eur J Biochem, 215, 213–219. Socci, D. J., Crandall, B. M., & Arendash, G. W. (1995). Chronic antioxidant treatment improves the cognitive performance of aged rats. Brain Res, 693, 88 –94.
808
B. Shukitt–Hale et al. / Experimental Gerontology 34 (1999) 797– 808
Vina, J., Sastre, J., Anton, V., Bruseghini, L., Esteras, A., & Asensi, M. (1992). Effect of aging on glutathione metabolism: Protection by antioxidants. In: Emerit, I., Chance, B. (Eds.), Free Radicals and Aging (pp. 136 –144). Basel, Switzerland: Birkhauser Verlag. Wallace, J. E., Krauter, E. E., & Campbell, B. A. (1980). Motor and reflexive behavior in the aging rat. J Gerontol, 35, 364 –370. Wang, H., Cao, G., & Prior, R. L. (1996). Total antioxidant capacity of fruits. J Agric Food Chem, 44, 701–705. Wang, H., Cao, G., & Prior, R. L. (1997). Oxygen radical absorbing capacity of anthocyanins. J Agric Food Chem, 45, 304 –309.