Background

Aging population is rapidly expanding worldwide, and age-related cognitive impairments proves detrimental for achieving a better productive and quality of life. Lack of effective therapies for age-related cognitive impairment focuses attention on developing preventive strategies, such as nutritional interventions, cell therapies and environmental manipulations. The objective of present study was to explore the comparative benefits of potential memory-enhancing strategies like supplementation of choline and docosahexaenoic acid (DHA) or administration of conditioned media derived from human embryonic kidney stem cells (HEK-CM) or exposure to environmental enrichment (EE), that attenuates cognitive impairments in aging mice.

Results

Spatial memory and cognition were decreased in normal aging mice. Aged mice exposed to dietary Ch-DHA or HEK-CM showed significant enhancement in spatial learning tasks, memory and cognition compared to the same in age-matched NAC mice. Ch-DHA and HEK-CM treated mice committed significantly lesser reference memory errors and attained a higher percentage of correct choices in spatial learning and memory tasks. Moreover, on testing for cognition in NORT, significantly higher number of visits to the novel object was observed in Ch-DHA supplemented and HEK-CM administered aging mice whereas HEK-CM and EE mice groups showed significantly greater number of visits to familiar object, when compared to same in age-matched NAC and HI-HEKCM groups respectively.

Conclusion

Supplementation of Ch-DHA and HEK CM treatment strategies have a higher potential [~ 20-30%] for enhancing spatial learning, memory and cognition in normal aged mice, whereas exposure to enriched environment seems to enhance only their short term memory.

Eight-arm radial maze test

The eight-arm radial maze is an elevated plexiform maze placed 80 cm above the floor. It consists of eight equally spaced arms [each arm is 42 cm long, 11.4 cm high, and 11.4 cm wide] radiating from an octagonal central platform and has a video monitor attached to a computer.

Mice were allowed to familiarize themselves with the radial maze during the orientation /habituation phase. Before the test, mice were semi-starved for 48 hrs to reduce their body weight to 85% of the original body weight. During the orientation phase all the eight arms of the radial maze were baited with food pellets. Mice were allowed to orient and get habituated to the maze during two trials per day carried out for two days.

Acquisition phase of a spatial task is conducted following habituation in the radial maze. During this phase, bait of food pellets was placed only in four arms of the radial maze. Before each trial and prior to each session, the maze was wiped with 50% alcohol to avoid any olfactory cues. The mice were placed at the centre of the maze and allowed to freely explore the maze. The mice were trained to take food pellets from the baited arm without making a re-entry into the arm already visited. The trial was terminated when the animal had taken the food reward from all four baited arms or after 5 min if all the four baited arms were not visited. During the trial, the animal’s performance was monitored from a marked location in the room and the number of entries into the arms and the total time required to visit all the baited arms were video recorded. Each animal was given two trials per day for four days. The performance of the animal was scored by calculating the percentage of correct responses (a correct entry is when the animal has not previously entered that arm) divided by the total number of entries made by the animal. The average criteria for acquisition are attaining 7.5/8 correct choices. Re-entry into an already visited baited arm was considered as working memory error and entry into the never baited arm was considered as reference memory error.

Retention phase of spatial task in the radial maze: Subsequent to learning /acquisition phase of the eight-arm radial task, mice were retained in their respective cages for 4 days without training. In order to assess whether they retained the learned / acquired task, the performance of mice in the eight-arm radial maze were again assessed for a single trial on 4^th day and the scores were noted. The experimental protocol remained same as that for the acquisition test.

Novel object recognition test (NORT)

The cognitive functions of mice were assessed by using NORT. It is based on the innate tendency of rodents to visit novel objects more frequently, as compared with familiar objects. Number of visits to a novel object provides a behavioural measure of memory retention and discriminating ability between familiar and novel objects, thereby revealing their cognitive abilities and hippocampal function. The test was done as a new one-trial NORT method. In the habituation phase mice were allowed to orient and habituate in an opaque open field arena for 30 min. Mice were retained in their home-cage for 5 minutes after the thirty-minute habituation phase. Subsequently during acquisition phase mice were exposed to 2 identical objects placed in the open field arena. Mice were allowed to explore and get familiarized with the objects for 5 min and then placed back in their home cage. During the test phase [one day after the acquisition phase], these mice were re-exposed to the open-field arena with one familiar object and a novel object, for 5 min. The entire experiment was videotaped, and scoring was carried out offline. During the 5-min test phase, the number of visits by the mice to the familiar or the novel object was scored. The number of visits was considered as when the mouse’s mouth/nose/paws were in contact with the objects. Accidental contact of the mouse with the object was not considered for scoring. The test arena and the objects were cleaned with 70% alcohol before exposing a subsequent mouse for the test, to remove any olfactory clues. 

DATA SET FILE DETAILS

File 1. Percentage of correct choices made by mice during learning phase of eight arm radial maze test 

File 2. Number of working memory errors made by mice during trial phase of eight arm radial maze test

File 3. Number of reference memory errors made by mice during trial phase of eight arm radial maze test

File 4. Percentage of correct choices made by mice during retention phase of eight arm radial maze test 

File 5. Number of visits to familiar object made by mice during the test phase of novel object recognition test

File 6. Number of visits to novel object made by mice during the test phase of novel object recognition test