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. Total number 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. CF1 mouse species is ideal for general multipurpose model, safety and efficacy testing and infectious disease model, and also have been used in various neuroscience research studies worldwide. 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 ⁰C, 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.

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 (tail vein) intravenous injections for 5 days in a 30-day period and EE group of mice were exposed to enriched environment for 30 days.

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-cm ² culture flasks to incubate with 5% CO ₂ 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.

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.

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. 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.

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.

The present study shows that normal aging in mice is associated with impairment in cognition, learning and memory capabilities. Comparison of potential strategies to attenuate age-associated cognitive and memory impairments during aging in this study has shown interesting findings. In the radial arm maze task during the 3 ^rd and 4 ^th trial days of acquisition phase of the test, it was observed that aged mice supplemented with Ch-DHA performed significantly better in spatial learning with reduced numbers of working and reference memory errors and higher ability to correctly choose the baited arms in comparison to same in age-matched normal aging mice. Comparatively, HEK-CM treated aged mice also showed similar findings except that on the 4 ^th trial day of acquisition phase they performed significantly better compared to the same in age-matched Ch-DHA supplemented aged mice as well as aged mice exposed to EE. Whereas aged mice exposed to EE performed significantly better compared to same in age-matched NAC mice only on the 4 ^th trial day of acquisition phase. Additionally, it was observed that during the retention phase of the radial arm maze test too, aged mice supplemented with Ch-DHA and those treated with HEK-CM performed significantly better than the same in age-matched NAC mice, indicating better memory of the learnt correct choices. These findings indicate that all three strategies help to some extent in attenuating spatial memory deficit 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. ^{30 – 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 Many studies have also shown the beneficial effects of cell therapies 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 could potentiate megakaryopoiesis due to the presence of erythropoietin and other cytokines. ⁴³ Erythropoietin is a known neuroprotectant. ^{44 , 45} In kainic acid induced hippocampal damaged mice, HEK-CM treatment showed significant increase in neuro-protection and improvement of hippocampal cognitive function. ⁴⁶ Further, previous studies in aged animals also showed that environmental enrichment prevents age-associated impairments in spatial learning and cognition. ^{26 , 47}

In the present study, behavioral testing for novel object recognition showed that, aging mice treated with HEK-CM had significantly higher preference for familiar object as well as novel object when compared to the same in age-matched NAC as well as HIHEK-CM mice groups. Additionally, aging mice supplemented Ch and DHA although had higher visits to familiar object, they were not significant as compared to the same in age-matched NAC, whereas visits to novel object were significantly higher when compared to the same in age-matched NAC mice. Whereas aging mice exposed to EE had significantly higher preference for familiar object but not for novel object when compared to the same in age-matched NAC mice.

Studies show that kainic acid (KA) lesioned mice that received HEK-CM showed 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