Exploring potential strategies to enhance memory and cognition in aging mice

Animals

Approval for the study was obtained from Institutional Animal Care and Use Committee [No. IAEC/KMC/107/2014], MAHE, Manipal. All experiments were carried out in accordance with guidelines provided by the IAEC and CPCSEA, Government of India. A total of 66 middle-aged (12 to 15-months-old) CF1 male mice housed in Central animal house research facility, MAHE, Manipal, were used for the study. Experiment was conducted in 44 days including 30 days of intervention period and 14 days of behaviour analysis. During 44 days of experimental period [09/03/2019 to 22/04/2019], 4 to 6 mice were housed and bedded in standard polypropylene cages containing paddy husk as bedding material that was replaced every two days. Standard lab conditions, with temperatures ranging between 23±2°C, humidity (50±5%), and 12 hrs light-dark cycle were maintained for all the mice. Pellet feed with Ch content of 1 mg/kg, procured from VRK Nutritional Solutions [VRK’s “Scientist’s Choice” Laboratory Animal Diets, Pune] and water ad libitum were freely accessible to these mice. As expected, 10 to 15% of mortality was observed in the mice due to ageing.

Experimental groups

Middle-aged male CF1 mice were randomly grouped [n=6/group] into normal aging control (NAC), saline vehicle control (SVC), Ch-DHA, HEK-CM, heat inactivated HEK-CM (HIHEK-CM) and EE groups. The NAC mice group remained undisturbed in the home cage throughout the 30 days interventional period of the experiment. The SVC group mice (included only for eight arm radial maze test) were fed equi-volume of saline for 30 days, Ch-DHA group mice were fed 45mg/kg body weight of Ch and 300mg/kg body weight of DHA for 30 days, HEK-CM group of mice were injected 100 μL of HEK-CM and HIHEK-CM group mice were injected 100 μL of heat inactivated HEK-CM through intravenous (tail vein) injections for 5 days, specifically on the 1^st, 6^th, 11^th, 16^th and 21^st day of the experimental period and EE group of mice were exposed to enriched environment for 30 days.

HEK-CM derivation

Cryopreserved 293T cells, which are derived from human embryonic kidney (HEK), were rapidly thawed at 37°C. HEK cells from a cryovial which contained dimethyl sulfoxide were transferred to a centrifuge tube (15 mL) and 5 mL of HEK media (Dulbecco’s modified Eagle’s medium-high glucose) was added along with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids and 1% of 200 mmol/L L-glutamate and centrifuged at 1800 rpm for 5 min. After discarding the supernatant, the pellets were re-suspended in 3 mL of HEK media and the cell suspension was then transferred into 25-cm² culture flasks to incubate with 5% CO₂ at 37°C for 24 hrs. The HEK cells were grown until they attained a confluency of 70 to 80%. HEK-CM, the conditioned media in which the HEK cells were grown was stored at -80°C for further use. HIHEK-CM was used as the vehicle control for HEK-CM and was prepared by subjecting HEK-CM to heat inactivation at 60°C in a water bath for 30 minutes, followed by cooling to room temperature. This process was employed to ensure that HIHEK-CM has no cognitive effects, as the heat treatment inactivates all the protein constituents of HEK-CM.

Environmental enrichment

For the enriched condition large wire-mesh cages (70 × 70 × 45 cm) were provided with various objects (toys), which were changed daily. These consisted of small mazes, ladders, running wheels, swings, plastic and metal cubes, spheres, cylinders, and trays of small objects, that could be carried about by the mice.

Eight-arm radial maze test

The eight-arm radial maze is an elevated plexiform maze placed 80 cm above the floor. It consists of an octagonal central platform from which equally spaced eight arms (each arm is 42 cm long, 11.4 cm high, and 11.4 cm wide) radiate and has a video monitor attached to a computer. After 30 days of treatment, mice were semi-starved for 2 days to reduce their body weight up to 85% and then subjected to eight arm radial maze test for 10 days, which consisted of 2 days of habituation phase followed by 4 days each of acquisition and retention phases.

During the habituation/orientation phase, all the eight arms were baited with food pellets and mice were allowed to orient and get habituated to the maze during two trials per day carried out for 2 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. Prior to each trial and each session, the maze was wiped with 50% ethanol to avoid any olfactory cues. The mice were placed in the centre of the maze and allowed to explore the maze freely. The mice were trained to take the food from baited arm without making a re-entry into the already visited arm. The trial was ended when the mice have taken the food from all four baited arms or after 5 min if mice did not visit the baited arms. During the trial, the animal’s performance was monitored and the number of entries into the arms were noted. Each mouse was given two trials per day for 4 days. The performance of the animal was scored by calculating the percentage of correct responses (a correct entry is the animal’s number of first visits to the baited arms) divided by the total number of entries made by the animal. Re-entries into previously visited baited arms were counted as working memory errors and entries into the unbaited arms were taken as reference memory errors.

Retention phase of spatial task in the radial maze: Subsequent to learning/acquisition phase, mice were retained in their individual cages for 4 days without any training. In order to assess the retention of the learned/acquired task, the performance of mice in the radial maze was again assessed for a single trial on the 4^th day. 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. Rodents have an innate tendency to visit the novel objects repeatedly than to the familiar objects. Two round plastic container boxes filled with sand were used as familiar objects, and a wooden cube box with different shape and color was used as novel object. The objects were positioned in the center of the open field, with equal distance separating them from one another and from the sides of the field. Number of visits to a novel object gives a behavioral measure of retention of memory and discriminating ability between familiar and novel objects, thus revealing their cognitive and hippocampal function. The test was done as a new one-trial NORT method. In the habituation phase, animals were allowed to orient and habituate in an open field for 30 min. Mice were retained in their home-cage for 5 min after the 30 min habituation phase. Subsequently, during the acquisition phase, mice were exposed to two identical objects and allowed to explore and get familiarized with the objects for 5 min. Subsequently, mice were placed back in their respective cages. During the test phase [one day after the acquisition phase], mice were again exposed to the open-field arena with one familiar object and a novel object, for 5 min. The test phase was video monitored, and the number of visits by each animal to the familiar and novel objects were marked manually from the monitor and then counted. Only the active contact of animal with its nose, mouth or paws to the objects was considered as the number of visits. The accidental touch, such as if animal was backing into the object or bumping the object accidentally as it passed was not included for scoring. To remove any olfactory clues, the test arena and the objects were cleaned with 70% alcohol before placing a successive mouse for the test.

Statistics

Data were presented as mean ±SEM and one-way analysis of variance [ANOVA] with Bonferroni's post-hoc test were used to compare the treatment effects between the groups, and a value of p< 0.05 was considered as statistically significant. SPSS (RRID: SCR_002865) was used for statistical analysis.

Spatial learning and memory

The present study results reveal that normal aging in mice is associated with cognitive and memory impairments, particularly in spatial learning. Aged mice supplemented with Ch-DHA demonstrated improved spatial learning, reduced memory errors, and better performance in choosing baited arms during the radial arm maze task. HEK-CM treatment also yielded positive outcomes, surpassing Ch-DHA supplementation on the 4^th trial day. EE exposure demonstrated benefits, particularly on the 4^th trial day, outperforming NAC-treated mice. Overall, Ch-DHA, HEK-CM, and EE interventions exhibited potential in mitigating spatial memory deficits in aging mice. High consumption of Ch during the perinatal period showed neuroprotective effect in animal models including during age related neuronal dysfunction. Perinatal Ch supplemented rats showed improvement in spatial memory performance and showed less errors relative to the untreated group when subjected to a 12-arm radial maze task.^31,32 Rats which were supplemented with Ch as infants were able to retain a larger number of items in working memory during their adulthood.³³ Studies also report that, dietary DHA supplementation enhances spatial memory in DHA deficient rats.³⁴ Recent studies observed that, combined supplementation of both Ch and DHA was more efficacious in enhancing hippocampal neurodevelopment. Prenatal supplementation of Ch and DHA showed significant improvement in spatial learning and other cognitive functions during adolescence.^35,36 Supplementation of Ch and DHA reduced memory errors in obese rats when subjected to eight arm radial maze test.³⁷ The hippocampus plays a major role in memory function. Hippocampal damage disturbs the memory consolidation.³⁸ Cell therapies are beneficial in restoring spatial learning in animal models of hippocampal degeneration.^39,40 Conditioned media which are derived from cell cultures are known to have excellent sources of neurotrophic factors and cytokines.^41,42 Furthermore, HEK-CM contain a neurotropic factor erythropoietin and other cytokines.^43,44 In kainic acid induced hippocampal damaged mice, HEK-CM treatment showed significant increase in neuro-protection and improvement of hippocampal cognitive function.^45,46 Further, previous studies in aged animals also showed that EE prevents age-associated impairments in spatial learning and cognition.^26,47

Cognition

In the present study, HEK-CM-treated aging mice showed increased preference for both familiar and novel objects, surpassing NAC and HIHEK-CM groups. Ch-DHA supplementation led to higher visits to novel objects, while EE-exposed mice exhibited a significant preference for familiar objects. These results highlight the varied effects of interventions on novel object recognition in aging mice.

Studies show that kainic acid (KA) lesioned mice that received HEK-CM showed a significantly greater number of visits towards the novel object as compared with KA alone treated mice. Normal mice that received HEK-CM also showed higher preference for novel object as compared with familiar objects.⁴⁶ Combined supplementation of nutrients like Ch and DHA after perinatal brain injury in mice showed a substantial preference for the novel object when subjected to NORT, indicating that recognition memory after brain injury was improved by the supplementation of DHA and Ch.⁴⁸ Aged mice, exposed to standard environment rather than EE, were able to discriminate between familiar and novel objects. Animals exposed to EE were able to distinguish stationary from displaced objects with different preferences independent of age. It was observed that young mice spent more time with displaced objects and aged mice with stationary objects.⁴⁹

Previous studies have detailed a few of the molecular mechanisms underlying the relationship between these strategies and their role in learning, memory, and cognition. Choline is known for its ability to enhance cholinergic function, while DHA, as an essential nutrient, facilitates neuronal development and processes such as neurogenesis, synaptogenesis, and synaptic plasticity in the hippocampus, ultimately enhancing cognitive capabilities.⁴⁸ Additionally, the HEK-CM is an excellent source of neurotrophic factors and contains known neuroprotectants such as erythropoietin and other cytokines that enhance cognition.^44–46 Exposure to EE has been found to stimulate neurogenesis, enhance the granule cell layer, and increase the number of dentate gyrus granule cells in the hippocampus. Moreover, EE exposure elevates the cAMP response element-binding protein (CREB) level in the hippocampus of aged animals.^27,47 Hence, it is evident that these strategies employ various underlying mechanisms to regulate both the structure and function of the hippocampus and related areas involved in cognitive processing. This variability could potentially account for the differences in behavioral outcomes observed in the present study in mice following exposure to these distinct age-associated cognitive-enhancing strategies. The current study might not capture the long-term effects of the interventions and sustained cognitive improvements. Although it assesses the comparative benefits of various interventions, further research is essential to unravel the molecular or cellular mechanisms, thereby enhancing the therapeutic approach.