Based on correlative human neuropathology studies, antioxidants would be predicted to be associated with healthy aging, might serve to reduce the risk of developing AD, and may improve cognitive function in AD patients. Studies in humans have shown either a positive effect of antioxidant use on cognition and risk reduction for developing AD [17, 42, 51], or no significant effects [18, 30, 38, 41]. There have been few systematic and controlled clinical trials evaluating the effects of antioxidants on cognition in aged individuals or patients with AD. Vitamin E delays institutionalization in AD patients, suggesting some beneficial effects [65]. However, vitamin E alone did not improve cognition in patients with mild cognitive impairment, which is thought to be a precursor to AD [57]. Furthermore, in nondemented elderly women, vitamin E treatment was associated with minimal improvements in cognition [30].

In addition to studying the effects of cellular antioxidants on cognition and risk of AD, there are several studies of the effects of targeted cofactors that improve mitochondrial function, including acetylcarnitine (ALCAR) and lipoic acid (LA) [34]. ALCAR and LA may improve mitochondrial function and reduce the production of ROS, thus also reducing oxidative damage to proteins, lipids, and DNA/RNA [35]. In studies where ALCAR was administered to patients with moderate to severe AD, cognition either improved and/or slower deterioration was observed [5, 58, 62, 70]. In early-onset AD patients (less than 65 years of age), only small cognitive improvements were noted [75] but in other studies of younger patients with AD (less than 61 years) there was evidence for slowed disease progression [7, 76]. When the results of all these studies were combined in a meta-analysis, there were clear benefits of ALCAR administration in patients with AD, particularly with respect to slowing cognitive decline [48]. Moreover, combining ALCAR with acetylcholinesterase therapy in AD may provide additional benefits [3]. Similar evidence of maintenance of function was observed in a study of nine patients with AD or related dementias receiving 600 mg/day of LA for an average of 337 days [21]. In a follow-up study of 48 patients for a longer 48-month treatment period, maintenance of function similar to the smaller study was observed [20].

When taken together, however, studies of dietary or supplemental antioxidant intake in humans reveal variable results and appear far less robustly associated with positive functional outcomes than those reported in aging rodents [4, 28, 29]. Variability in outcomes of human antioxidant clinical trials may reflect inconsistencies in amounts of supplements administered, their form and source, compliance, as well as assessment and documentation of exact background of dietary intake of antioxidants. Interestingly, combinations of antioxidants may be superior to single compound supplementation [80] and dietary intake of antioxidants has been shown to be superior to supplements in human studies on cognition and risk of developing AD [2, 50]. In elderly women, supplementation with a combination of vitamins E and C can lead to improved memory [11]. Thus, single antioxidant administration (e.g., vitamin E alone) may prove to be more efficacious if administered in combination with other antioxidants (e.g., vitamin C, which helps to recycle vitamin E) and administered through diet rather than a tablet supplement. As described in later sections, the combination of antioxidants administered by way of a fortified food proved to be a potent intervention for improvement of cognition and reducing brain pathology when tested in a canine model of human brain aging.

Advanced age in dogs is frequently associated with severe behavioral and cognitive deficits [52]. Age-dependent cognitive deficits in canines can be observed on many different measures of learning and memory. Deficits in complex learning tasks such as oddity discrimination learning [15, 47], size discrimination learning [22, 74], and spatial learning [12] occurs with age in dogs. Tasks sensitive to prefrontal cortex function, including reversal learning and visuospatial working memory, also deteriorate with age [22, 72]. In addition, egocentric spatial learning and reversal, measuring the ability of animals to select a correct object based on their own body orientation is age-sensitive [12]. Interestingly, on simple learning tasks and procedural learning measures, aged dogs performed equally as well as younger animals [45], suggesting that a subset of cognitive functions remains intact with age as it does in aging humans.

Memory also declines with age in dogs both for information about objects and location in space (spatial) [10, 24, 45]. Furthermore, studies of the time course of the development of cognitive decline demonstrate that deterioration in spatial ability occurs early in the aging process in canines, between 6 and 7 years of age [72] and provides researchers with guidelines for ages at which to start a treatment study. The neurobiological basis for cognitive decline in aging dogs may depend in part upon the progressive accumulation of oxidative damage to proteins, lipids, and DNA/RNA.

In dog brain, the accumulation of carbonyl groups, which is a measure of oxidative damage to proteins, increases with age [23, 68] and is associated with reduced endogenous antioxidant enzyme activity or protein levels such as in glutamine synthetase and superoxide dismutase (SOD) [23, 27, 31, 55]. In several studies, a relation between age and increased oxidative damage has been inferred by measuring the amount of endproducts of lipid peroxidation (oxidative damage to lipids) including the extent of 4-hydroxynonenal (4HNE) [63, 64], or malondialdehyde [23]. Also, evidence of increased oxidative damage to DNA or RNA (8OHdG) in aged dog brain has been reported [14, 64]. If oxidative damage leads to progressive age-associated neuropathology and cognitive decline, then one could hypothesize that dietary antioxidants may prove beneficial.

A variety of antioxidant or antioxidant defense-associated molecules are derived from food sources. Vitamin E is found in high concentrations in nuts and oils, vitamin C is found in high concentrations in fruits, and beta-carotene is found in certain vegetables. In addition trace minerals such as selenium, copper, zinc, and manganese, which are important to enzymes that specifically detoxify free radicals (Cu/Zn SOD) or help recycle antioxidants that detoxify free radicals (glutathione peroxidase), may be acquired from different food sources.

Recent research has shown that some molecules classified as mitochondrial cofactors (lipoic acid, l-carnitine) may enhance function of aged mitochondrion such that fewer ROS are produced during aerobic respiration. Chronic oxidative damage to enzymes and cell membranes may reduce the capability to bind mitochondrial enzyme cofactors thus reducing metabolic capacity [35]. Supplementation of foods with these mitochondrial cofactors increases the concentration within cells and restores binding to the enzymes that require them, which restores mitochondrial efficiency [35] and reduces oxidative damage to RNA [34].

If brain aging in dogs is attributable to progressive accumulation of oxidative damage, which results in cognitive dysfunction, then reduction of oxidative damage via dietary fortification of antioxidants appears as a viable intervention option. A longitudinal investigation of the effects of dietary fortification of antioxidants on cognitive function of beagle dogs was thus completed, which included 48 aged (10–13 years of age) and 17 young beagles (3–5 years old). Each animal was assigned into one of two food groups using a counterbalanced design based on extensive baseline cognitive testing. No differences existed between cognitive ability of groups prior to dietary intervention.

An antioxidant-enriched food for maintenance of adult dogs was formulated to include a broad spectrum of antioxidants and two mitochondrial cofactors. The control and test foods had the following differences in formulation on an as-fed basis, respectively: dl-alpha-tocopherol acetate (120 vs. 1050 ppm), ascorbic acid as Stay-C (30 vs. 80 ppm), l-carnitine (20 vs. 260 ppm), and dl-alpha-lipoic acid (20 vs. 128 ppm). Based on an average weight of 10 kg per animal, the daily doses for each compound were 800 IU or 210 mg/day (21 mg/kg/day) of vitamin E, 16 mg/day (1.6 mg/kg/day) of vitamin C, 52 mg/day (5.2 mg/kg/day) of carnitine, and 26 mg/day (2.6 mg/kg/day) of lipoic acid. Fruits and vegetables were also incorporated at a 1-to-1 exchange ratio for corn, resulting in 1% inclusions of each of the following: spinach flakes, tomato pomace, grape pomace, carrot granules, and citrus pulp. This was equivalent to raising fruit and vegetable servings from 3 to 5–6/day. Serum vitamin E was increased ∼75% by the antioxidant food in treated dogs [44].

Treatment with the antioxidant fortified food led to improvements in spatial attention (landmark task) learning as early as within 2 weeks of beginning the food intervention [44]. Subsequent testing of animals with a more difficult complex learning task, oddity discrimination, also revealed benefits of the fortified food [15]. Improved learning ability was maintained over time with the antioxidant treatment whereas untreated animals showed a progressive decline [46]. Interestingly, cognitive improvements were initially limited to aged animals as young dogs treated with the antioxidant fortified food were not different from control-fed dogs [67]. However, when initiated at a young age (2–4 years of age) it was found that prolonged administration of antioxidant fortified food resulted in significant improvement on a visual discrimination task compared to age-matched controls fed a nonfortified food over the same time period [16]. These results suggest that a relatively short administration period of a food fortified with antioxidants in aged dogs resulted in a slowing of the rate of decline in cognitive abilities, possibly attributable to either repair of age-associated oxidative damage or improved cellular function through improved mitochondrial function [25]. As a corollary to this finding the results in young dogs might suggest that early administration of an antioxidant fortified food acts in a way to delay onset of cognitive decline by either prevention of oxidative damage or continuance of optimal cellular function.

The improved cognitive outcomes were hypothesized to be attributable to enhanced mitochondrial function resulting in decreased oxidative damage in brain tissue from aged dogs administered antioxidant fortified food. Mitochondrial function was measured in aged dogs and revealed that antioxidant fortified food reduced age-associated mitochondrial dysfunction by reducing ROS production [25]. These results suggest that one mechanism by which the antioxidant fortified food improved cognition was by maintaining mitochondrial homeostasis by either the antioxidant fortification or the mitochondrial nutrient (l-carnitine, lipoic acid) targeted fortification. Oxidative damage to proteins from the parietal cortex was measured by derivatization with 2,4-dinitrophenylhydrazine (DNPH) which revealed reduced damage in dogs fed antioxidant fortified food [55]. These results suggest that oxidative damage to proteins was reduced in the parietal cortex, thought to be involved with landmark discrimination learning and other visual learning tasks that were improved in the cognitive assessments.

It is important to note that reduced oxidative damage to proteins and increased endogenous antioxidant activity were associated with improved cognition in the aging dogs on antioxidant fortified foods. Higher levels of one measure of protein oxidation (3-nitrotyrosine) and lower levels of antioxidant activity (glutathione-S-transferase) were subsequently correlated with higher error scores on a reversal learning problem and on a visuospatial task (i.e., impaired function) [55]. These results strongly suggest that oxidative damage, particularly to vulnerable proteins involved with energy metabolism, neuronal integrity, and antioxidant systems are key contributors to cognitive decline associated with aging in dogs. The most important aspect of this work is the discovery that cognitive performance may be improved relatively quickly in aged canines by dietary manipulation as well as slowing the onset of cognitive decline in younger dogs administered the fortified food at an early age for a prolonged period. Antioxidants may potentially act, therefore, to mitigate development of age-associated behavioral changes, and possibly even neuropathology, by counteracting oxidative stress.

Oxidative damage is a consistent feature of brain aging in all species studied. Decline in cognitive functions that accompanies aging may have a biological basis, and many of the disorders associated with aging may be preventable through dietary modifications that incorporate specific nutrients. Based on previous research and results of both laboratory and clinical studies in the canine model of human aging and disease, antioxidants may be one class of nutrient that may be beneficial. Brains of aged dogs accumulate oxidative damage to proteins and lipids, as well as mitochondrial dysfunction that may lead to dysfunction of neuronal cells. The production of free radicals and lack of increase in compensatory antioxidant enzymes may lead to detrimental modifications to important macromolecules within neurons. Reducing oxidative damage and mitochondrial dysfunction through food ingredients rich in a broad spectrum of antioxidants and mitochondrial cofactors significantly improves, or slows the decline of, learning and memory in aged dogs as well as delaying the onset of decline in younger dogs. Furthermore, there are clear links between the reduction of brain oxidative damage and mitochondrial impairments and improved or maintained cognitive function. However, determining which compounds, which combinations and dosage range, when to initiate intervention, and long-term effects constitute critical gaps in knowledge.