Stress-induced cognitive impairment is related to the suppression of hippocampal neurogenesis that results from an increase of oxidative stress. Therefore, the aim of this study was to investigate the effects of administration of a blueberry drink, having a high antioxidant power, on the cognitive performance of adult rats exposed to chronic mild stress.
Twelve-week-old male Sprague-Dawley rats (n = 48) were randomly divided into four groups: control (CO), stress (ST), control + 5% blueberry drink (CO + B), and stress + 5% blueberry drink (ST + B). After eight weeks, the cognitive performance was assessed using a multiple T-maze water test. Levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and ascorbic acid were measured in the brain, and catecholamine concentrations were measured in plasma.
The brain weights of the rats from the ST and ST + B groups were significantly lower than those of the rats from the CO and CO + B groups. The cognitive performance of the ST group was impaired when compared to that of the CO group. This impairment was significantly improved by the blueberry drink supplementation (
These findings suggest that the blueberry drink may protect against the cognitive impairment induced by chronic mild stress.
Stress is defined as any situation capable of perturbing physiological or psychological homeostasis [
Oxidative stress, reflecting the accumulation of oxygencontaining free radicals, increases with aging and may play a critical role in age-related functional deficits of the brain [
Recently, the majority of studies have focused on the cognitive effects of flavonoid-rich foods in aged animals [
The aim of this study was to investigate the effects of eight-week administration of a blueberry drink on the cognitive performance in 12-week-old rats exposed to chronic mild stress. Loss of cognitive abilities during aging is a complex process that starts to become evident during middle age in rats (12-24-month-old), even in the absence of a specific neurodegenerative disease [
A freeze-dried 100% wild blueberry powder was purchased from Bactolac Pharmaceutical, Inc. (Hauppauge, NY, USA). A blueberry drink was prepared by adding the blueberry powder to fresh tap water (PurePlus, Inc., Incheon, Korea). Assay kits for catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The standard of ascorbic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA), and all reagents used in ascorbic acid analysis were high-performance liquid chromatography (HPLC) grade. Catecholamines were measured using the 3-CAT plasma enzyme-linked immunosorbent assay (ELISA) kit from Labor Diagnostika (Nord, Nordhorn, Germany).
Twelve-week-old male Sprague-Dawley rats (Hanlim Experimental Animal Laboratory, Seoul, Korea) weighing 470 ± 10 g were fed a laboratory diet. The animals were kept individually and housed in a temperature-controlled room (24 ± 1℃) with a relative humidity of 50-60% and a 12/12 h light/dark cycle (lights on at 6:00 a.m.). The rats had
After acclimation to laboratory conditions for one week, the rats were randomly divided into four groups (n = 12 per group): a control group (CO, no chronic mild stress/tap water), a blueberry drink only group (CO + B, no chronic mild stress/5% blueberry drink), a stress only group (ST, chronic mild tress/tap water), and a stress + blueberry drink group (ST + B, chronic mild stress/5% blueberry drink). To prepare a 5% blueberry drink, 5 g of the blueberry powder was mixed daily with 100 mL of tap water and then stirred with a magnetic stir bar under protection from light until completely dissolved. The blueberry drink was provided to the CO + B and ST + B groups instead of the regular drinking water, and the water bottles for the groups were wrapped in aluminum foil to protect from light in order to prevent the destruction of bioactive compounds.
Feed and water consumption was recorded daily, and the rats were weighed weekly. A feed efficiency ratio (FER) was also calculated. All animals were maintained under the appropriate experimental conditions for eight weeks before performing the multiple T-maze water test.
The chronic mild stress model was slightly modified from those described previously for rats by Willner et al. [
The multiple T-maze water test was based on the method of Ishizaki [
After completing the multiple T-maze water test, the rats were anesthetized with diethyl ether, and blood was collected from the heart and immediately placed on ice. The whole intact brain was then carefully removed and placed in an ice-chilled Petri dish. The brain was weighed and washed with isotonic saline. The blood was collected into ethylenediaminetetraacetic acidcoated tubes and centrifuged at 3,000 rpm for 15 min at 4℃. The plasma was transferred into 1.5-mL microtubes for catecholamine analysis. All samples were stored at -80℃ until analysis.
SOD, CAT, and GPx activities in the brain were measured using their respective assay kits. The total ascorbic acid in the brain was measured using an HPLC method [
Plasma norepinephrine, epinephrine, and dopamine concentrations were quantified using the 3-CAT plasma ELISA kit.
Statistical analysis was performed using the SPSS for Windows 19.0 software (SPSS, Inc., Chicago, IL, USA). The effects of the chronic mild stress, blueberry drink, and their interaction were tested using two-way analysis of variance (ANOVA), followed by Fisher's least significant difference (LSD) test. Results were considered statistically significant at
There were no significant differences in the final body and brain weights at the end of the experiment between the CO and CO + B groups (
The numbers of errors that the rats made when they entered the blind alley of the multiple T-maze are shown in
The effects of the chronic mild stress and blueberry drink on the brain SOD, CAT, GPx, and ascorbic acid levels are shown in
The plasma norepinephrine levels were affected by the blueberry drink. The concentration in the ST group (167.28 ± 68.71 pg/mL) was significantly increased by the blueberry drink supplementation (425.09 ± 64.78 pg/mL in the ST + B group) (
Recently, there has been an increase in diseases related to psychological stress, and thus, more attention has been given to the prevention of stress-induced injuries. Stress is associated with the activation of the HPA axis. The hippocampus is critical for the learning, consolidation, and retrieval of declarative memories. It is also important for the formation of spatial memory [
Exposure to stress situations can influence the feeding behavior, which results in changes of body weight of rats [
Various studies have used the multiple T-maze test for analyzing spatial memory in mice and rats [
Studies have suggested that polyphenolic compounds may be involved in protecting cognitive functioning through their antioxidant activities [
The metalloproteins SOD, CAT, and GPx provide the first line of antioxidant defense against reactive oxygen species through enzyme-catalyzed dismutation of O2- to H2O2, which is further reduced to oxygen and water [
It is well known that the sympathetic nervous system is closely involved in the regulation of the stress response. Psychological stress activates the sympathetic nervous system, and, in turn, catecholamines are released from the sympathetic nerve terminal and the adrenal medulla. Catecholamines include norepinephrine, epinephrine, and dopamine, which are involved in the modulation of the body's cognitive and emotional state and other psychoactivities. Norepinephrine and dopamine levels decrease upon experiencing psychological stress [
Based on the compositional analysis, 1 g of the blueberry powder in this study contained 19.96 mg of total polyphenols in gallic acid equivalents (data not shown). The approximately 5.5 g/kg of body weight of the blueberry powder consumed daily by the rats equated to the amount of polyphenols in 53 g of fresh whole blueberries, based on the polyphenol content [
In conclusion, our findings suggest that blueberry supplementation may protect against the cognitive impairment induced by chronic psychological stress. These results may be due to the antioxidant and neuroprotective effects of a blueberry drink, resulting from the high concentrations of polyphenols and flavonoids found in blueberry. Based on these findings, blueberry appears to have potential benefits in terms of prevention of a cognitive decline during stress, and these effects may extend to the cognitive decline associated with other neuropathic diseases.
The authors declare no potential conflicts of interests.
(A) mean time to reach the goal on the first day of the multiple T-maze water test and (B) mean time to reach the goal on the second day of the multiple T-maze water test. The data are expressed as the mean ± SE of 12 animals per group. Significant differences between the stress (ST) and blueberry drink (B) or interaction between these factors (ST × B) were tested by two-way ANOVA and expressed as
The data are expressed as the mean ± SE of 12 animals per group. Significant differences between the stress (ST) and blueberry drink (B) or interaction between these factors (ST × B) were tested by two-way ANOVA and expressed as
Data are expressed as the mean ± SE of 12 animals per group.
1) Significant differences between the chronic mild stress (ST) and blueberry drink (B) or interaction between these factors (ST × B) were tested by two-way ANOVA and expressed as
2) Mean values with different superscript letters are significantly different (
Data are expressed as the mean ± SE of 12 animals per group.
1) Significant differences between the stress (ST) and blueberry drink (B) or interaction between these factors (ST × B) were tested by two-way ANOVA and expressed as
2) Mean values with different superscript letters are significantly different (
Data are expressed as the mean ± SE of 12 animals per group.
1) Significant differences between the stress (ST) and blueberry drink (B) or interaction between these factors (ST × B) were tested by two-way ANOVA and expressed as
2) Mean values with different superscript letters are significantly different (