Prevention of cognitive deficits and brain oxidative stress with superoxide dismutase/catalase mimetics in aged mice

Abstract

Continuous decline in cognitive performance accompanies the natural aging process in humans, and multiple studies in both humans and animal models have indicated that this decrease in cognitive function is associated with an age-related increase in oxidative stress. Treating aging mammals with exogenous free radical scavengers has generally been shown to attenuate age-related cognitive decline and oxidative stress. The present study assessed the effectiveness of the superoxide dismutase/catalase mimetics EUK-189 and EUK-207 on age-related decline in cognitive function and increase in oxidative stress. C57/BL6 mice received continuous treatment via osmotic minipumps with either EUK-189 or EUK-207 for 6 months starting at 17 months of age. At the end of treatment, markers for oxidative stress were evaluated by analyzing levels of free radicals, lipid peroxidation and oxidized nucleic acids in brain tissue. In addition, cognitive performance was assessed after 3 and 6 months of treatment with fear conditioning. Both EUK-189 and EUK-207 treatments resulted in significantly decreased lipid peroxidation, nucleic acid oxidation, and reactive oxygen species (ROS) levels. In addition, the treatments also significantly improved age-related decline in performance in the fear-conditioning task. Our results thus confirm a critical role for oxidative stress in age-related decline in learning and memory and strongly suggest a potential usefulness for salen-manganese complexes in reversing age-related declines in cognitive function and oxidative load.

Figure 1. Effects of 3-month chronic treatment with EUK-189 or EUK-207 on context fear conditioning

17 month-old mice were treated for 3 months with a low (0.15 mM) or a high (1.5 mM) concentration of EUK-189 or EUK-207 and then trained in a contextual fear conditioning paradigm and tested 24 h after training. Results were calculated as percent time the mouse expressed freezing behavior during the 8-min observation period for context minus the percent time the mouse expressed freezing behavior prior to training. Shown are means ± SEM of 9-17 mice. One-way ANOVA indicated that the effect of age was significant (*p <0.05 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-207 (**p <0.05 vs. vehicle control).

Figure 2. Effects of 6-month chronic treatment with EUK-189 or EUK-207 on context fear conditioning

After 6 months of treatment, 23 month-old mice were retrained in a contextual fear conditioning paradigm, but in a new context, and tested 24 h after training. Results were calculated as percent time the mouse expressed freezing behavior during the 8-min observation period for context minus the percent time the mouse expressed freezing behavior prior to training. Shown are means ± SEM of 9-14 mice. One-way ANOVA indicated that the effect of age was significant (*p <0.001 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-207 (**p <0.05 vs. vehicle control).

Figure 3. Effects of chronic treatment with EUK-189 or EUK-207 on lipid peroxidation in brain homogenates

At the end of the 6 month treatment, mice were decapitated and their brains (minus cerebellum and pons) were removed and homogenized. Lipid peroxidation was then quantified by the thiobarbituric acid-reactive substances (TBARS) assay. Lipid peroxidation was also determined in brain homogenates from 16 month-old control mice. Levels of lipid peroxidation were expressed as nmol malondialdehyde equivalent per mg of protein. Shown are means ± SEM of 8-10 mice. One-way ANOVA indicated that the effect of age was highly significant (*p <0.001 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-207 (**p <0.001 vs. vehicle control).

Figure 4. Effects of chronic treatment with EUK-189 or EUK-207 on reactive oxygen species (ROS) levels in brain homogenates

At the end of the 6-month treatment, brain ROS content was quantified by incubating brain homogenates with 2′-7-dichlorofluorescein diacetate (DCFH-DA). ROS content was also determined in brain homogenates from 16 month-old control mice. Results were expressed as percentage of 16 month value. Shown are means ± SEM of 8-10 mice. One-way ANOVA indicated that the effect of age was highly significant (*p <0.001 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-207 (**p <0.001 vs. vehicle control).

Figure 5. Effects of chronic treatment with EUK-189 or EUK-207 on oxidized nucleic acids in brain homogenates

At the end of the 6-month treatment, oxidized guanine was quantified by extracting DNA from brain homogenates and then blotting onto a membrane, which was then probed with an anti-oxo8dG/oxoG antibody. Oxidized nucleic acid content was also determined in brain homogenates from 16 month-old control mice. (A) Representative 8oxoG dot blot. (B) Results were expressed as percentage of 16 month value. Shown are means ± SEM of 8-10 mice. One-way ANOVA indicated that the effect of age was significant (*p <0.05 vs. 16 month-old mice), as was the effect of EUK-189 and EUK-207 (**p <0.05 vs. vehicle control, ***p <0.01 vs. vehicle control).

Figure 6. Correlation between performance in the contextual fear conditioning task and brain levels of free radicals and lipid peroxidation

Individual data for contextual fear conditioning and brain free radical content (A) or lipid peroxidation (B) were plotted for 16-month-old control mice, 23-month-old vehicle control mice, and 23-month-old EUK-189 and EUK-207 treated mice. Free radical content was expressed as percentage of 16-month-old control value (A) and levels of lipid peroxidation were expressed as nmol malondialdehyde equivalent per mg of protein (B). Contextual fear conditioning performance was calculated as percent time the mouse expressed freezing behavior during the 8-min observation period for context minus the percent time the mouse expressed freezing behavior prior to training (A & B). Regression lines were plotted for the control animals and analysis indicated a significant negative correlation between contextual fear conditioning performance and brain free radical content (Pearson’s correlation coefficient ρ= −0.68, p = 0.0054, n =15) (A) and lipid peroxidation levels (Pearson’s correlation coefficient, ρ= −0.76, p = 0.0011, n =15) (B). No significant correlation between performance in the contextual fear conditioning task and brain levels of free radicals or lipid peroxidation was observed in 23-month-old EUK-189 (n =17) and EUK-207 (n =19) treated mice (A & B).