Previous in vitro studies have shown that walnut extract can inhibit amyloid-beta (Aβ) fibrillization and solubilize its fibrils. The in vitro research also shows that walnut extract has a protective effect against Aβ -induced oxidative stress and cellular death. The researchers in this study utilized mice to see if there was an association between dose of walnut intake and cognitive function in a transgenic mouse model of Alzheimer’s disease.1
Supplementing their diet with walnuts reversed the cognitive deficits of ‘Alzheimer’s mice’ (transgenic mice with Alzheimer’s-like features, known as AD-tg mice). Normally these mice perform more poorly than their normal (‘wild-type’) counterparts in cognitive tests including various skill tests, but walnut supplementation reversed these differences. The researchers concluded that dietary supplementation with walnuts may have a beneficial effect in reducing the risk of, or slowing the progressing of, Alzheimer’s disease.
Researchers purchased 11-week old transgenic female mice with Swedish double mutations K670N and M671L of amyloid precursor protein (APP), who would mimic an Alzheimer’s patient, and wild type mice, all from the same facility. The wild type mice were littermate controls that were produced in the same litters as the transgenic Alzheimer’s mice (AD-tg), but did not carry the manipulated gene of interest. The study included 37 mice, which were each housed in a separate cage, and their body weight was measured. They were all given the same diet for five weeks during a period of acclimatization. Then, at the age of four months, the AD-tg mice were randomly divided into three study groups and fed either a control diet without walnuts (n=9) or a custom-mixed diet enriched with either 6% (n=11) or 9% (n=10) walnuts. The wild type mice (n=7) were also fed the control diet.
All mice in the study were given behavioral tests at the age of 13-14 months to test for motor coordination (rotarod), emotionality (elevated plus maze), perseveration (Tmaze), and cognitive capacity (Morris water maze).
The rotarod test for motor coordination consisted of a 3cm diameter rod with a rubber surface and four lanes of spindle, each 5cm wide. The mice were each given two tries on the rod without rotation for pre-training. During training, the mice were placed on the rod and it gradually increased in rotational speed from 5RPM to 40RPM over 10 minutes. The trial was stopped when the mouse fell off onto a plastic surface 15cm below the rod. The training involved four sessions, administered on two consecutive days.
The elevated plus maze (EPM) test involved a wooden maze that was painted black and placed 1 m from the floor. It consisted of two open arms (5 × 30 cm) and two closed arms (5 × 30 × 15 cm), which extended at a right angle from a center arena (5 × 5 cm). It was used to evaluate the animals’ emotionality by challenging the mice to exhibit two conflicting responses. One response was to seek safety in a closed arm and the other was to explore a salient arm. The mice were placed one at a time in the center of the EPM, facing the closed arm. The researchers video-recorded the exploratory behavior of each mouse for 5 minutes, and they analyzed the frequency and duration of entry into the center, closed arm, and open arm.
The Tmaze was constructed of 5mm clear Plexiglas, with a 20 x 6 x 24cm start box affixed at a right angle to two 18 x 6 x 24cm goal arms, submerged in opaque water that was 10cm in depth. There was an escape platform at the end of each arm that was not visible from the choice point. The mice were allowed to swim through the arm to the platform and rest on the platform for 10 seconds before being removed and dried. The mice then were involved in discrimination training of two blocks of 10 massed trials a day with the platform placed in the opposite goal box of the preference arm the mouse chose the initial time. If the mouse was unable to reach the platform in 60 seconds it was rescued. The criterion for learning the researchers chose was 18 errorless trials (90%) in a day, and once learning was complete the reversal training was given with the platform placed in the opposite arm of the one originally trained.
The Morris maze consisted of a steel, circular pool with a 90cm diameter that was 36cm in height. It was painted white and filled to a depth of 20cm with opaque, milk water. The Morris maze had a10 x 10 x 9cm clear Plexiglas escape platform, which was placed 24 cm from the pool wall. A video camera on the ceiling above the pool monitored and recorded the time it took for each mouse to reach the platform, as well as the distance it swam. The training involved five spaced trials for five days, totaling 25 trials. The platform was placed in a fixed position, deemed northeast) and the start position randomly alternated between north, south, and west. Once they reached the platform, the each mouse was allowed to rest for 10 seconds on the platform before it was removed and, dried with a towel, and placed in a dry cage under a heat lamp until the next trial (about a 10 minute interval). If the mouse did not find the platform within 3 minutes, it was gently guided to the platform. Also, a 60-second probe trial was conducted, where the platform was removed from the maze, and the amount of time the mouse swam in each of the four quadrants of the pool was recorded. Finally, a reversal test (five trials a day for three days) was given after a two-day resting period, in which the platform location was moved to the southeast location in the pool (180◦ from the original position). The measure of performance for the probe trial was the percent distance travelled in the trained quadrant, and the measure of performance for the other trials was the time each mouse took to reach the escape platform.
At the ages of 4 and 9 months, there was no significant difference in body weight among any of the groups of mice. However, at 13 months of age, the body weights of AD-tg mice on the control diet and the AD-tg mice eating 6% walnuts were significantly less (p < 0.05) than those of the wild type mice. The body weights of the AD-tg mice eating 9% walnuts were similar to those of the wild type mice, and they were significantly higher (p < 0.05) than the weights of AD-tg mice fed the control diet and the AD-tg mice fed 6% walnuts.
For the rotarod testing, a significant interaction between treatments indicated differential rates of motor learning during the two-day sessions. For trial 1, the durations of the four groups were not significantly different from each other. However, on the last trial the duration of the AD-tg mice fed the control diet was significantly shorter than that of the wild type mice, while the durations of the AD-tg mice fed 6% and 9% walnuts were indistinguishable from the duration of the wild type mice fed the control diet. Also, the wild type mice fed the control diet, the AD-tg mice fed 6% walnuts, and the AD-tg mice fed 9% walnuts recorded a significantly longer time in trial 4 than trial 1, whereas AD-tg mice fed the control diet showed no improvement. There was no significant difference in duration between the AD-tg mice fed 6% and 9% walnuts.
The elevated plus maze (EPM) evaluated responses to anxiety-producing environmental cues. There was a significant effect of diet with walnuts on total open arm activity. A significant interaction between genotype/treatment and exposure days indicates that there were changes in responses during the two days of exposure. All three AD-tg groups (control diet, 6% walnuts, and 9% walnuts) had a significantly higher level of activity than wild type animals on day 1. On day 2, however, AD-tg mice on a diet with 6% or 9% walnuts significantly reduced their open arm activity to the level of wild type mice, whereas activity remained elevated for AD-tg mice on the control diet, as compared to all three other groups. The activity levels of the two groups of AD-tg mice on a diet with either 6% or 9% walnuts were not different from each other on either day.
During the water Tmaze, the AD-tg mice fed a 6% or 9% walnut diet, while not different from the wild type mice, made significantly fewer errors than the AD-tg mice fed the control diet for acquisition. For reversal, although the AD-tg mice fed the control diet showed higher error scores than others, no group differences reached a statistical difference. Analyses of the escape data yielded similar results. For the Morris water maze, all groups reduced their escape time over the training period, but only the AD-tg mice fed 6% and 9% walnuts showed a significant reduction by the last day of learning, whereas the AD-tg mice fed a control diet failed to show any significant change which indicated that they were deficient in learning to locate the hidden platform. The AD-tg mice fed 6% and 9% walnuts spent more time in the area where the escape platform had been placed than the AD-tg mice fed the control diet. AD-tg mice fed 6% and 9% walnuts and wild type mice had higher activity than the AD-tg mice fed the control diet in the trained quadrant after acquisition and reversal trials.
Overall, the AD-tg mice fed a diet comprised of either 6% or 9% walnuts performed better on the skills assessed than the AD-tg mice fed the control diet. The researchers concluded that results from their study suggests that dietary supplementation with walnuts may have a beneficial effect in reducing the risk, delaying the onset, or slowing the progression of, or preventing AD.
- Muthaiyah B., Essa MM., Lee M., Chauhan V., Kaur K, Chauhan K. Dietary Supplementation of Walnuts Improves Memory Deficits and Learning Skills in Transgenic Mouse Model of Alzheimer’s Disease. Journal of Alzheimer’s Disease. 2014 ;42(4):1397-1405.