¹ Division of Physiology,, DBMS, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India
² Department of Regenerative Medicine, Manipal Institute of Regenerative Medicine, Yelahanka, Bengaluru, Karnataka, 560065, India

Shreevatsa Bhat M
Roles: Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Resources, Software, Validation, Writing – Original Draft Preparation

Ramesh Babu M G
Roles: Data Curation, Formal Analysis, Software, Validation, Writing – Review & Editing

Anandh Dhanushkodi
Roles: Conceptualization, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

Prof Kiranmai S Rai
Roles: Conceptualization, Data Curation, Formal Analysis, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

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 36 middle-aged (12 to 15-months-old) CF1 male mice housed in Central animal house research facility, MAHE, Manipal, were used for the study. Male mice were selected as a highly sensitive experimental model for choline-containing supplementation because they lack the estrogen driven endogenous choline synthesis mediated by the phosphatidylethanolamine N-methyltransferase pathway in females, making them significantly more responsive to dietary choline alterations.⁵² 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 70% 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. The presentation of the entire experimental protocol is shown in Figure 1.

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.

Figure 1. Diagrammatic representation of experimental protocol.

Table 1. Spatial memory performance of mice groups during learning phase in the eight-arm radial maze task.

Table 2. Spatial working memory errors by mice groups during learning phase in the eight-arm radial maze task.

Table 3. Spatial reference memory errors by mice groups during learning phase in the eight-arm radial maze task.

Table 4. Correct choices made by mice groups during spatial memory retention phase in the eight-arm radial maze task.

Table 5. Cognitive performance of mice groups showing visits to familiar object in the NORT.

Table 6. Cognitive performance of mice groups showing visits to novel object in the NORT.

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 Previous studies have demonstrated neuroprotective potential of HEK-CM in animal models and in vitro neurodegenerative conditions attributing to endogenous upregulations of erythropoietin, BDNF and anti-apoptotic factors.;^46,53 Further, previous studies in aged animals also showed that EE prevents age-associated impairments in spatial learning and memory.^26,47

Cognition

The NORT was used to assess recognition memory in aging mice following the different interventions. The present findings suggest that Ch-DHA supplementation, HEK-CM treatment, and EE may differentially influence recognition memory during aging. In particular, HEK-CM and Ch-DHA treatments were associated with greater exploration of novel objects, suggesting improved recognition memory, whereas EE produced a different pattern of object preference with greater exploration of familiar object. These observations indicate that nutritional, cell-derived, and environmental interventions may influence memory-related behaviors through distinct mechanisms.

The present findings are generally consistent with previous reports demonstrating beneficial effects of HEK-CM and Ch-DHA on recognition memory. Kainic acid-lesioned mice treated with HEK-CM exhibited a significantly greater preference for novel objects than untreated animals, and normal mice receiving HEK-CM also showed enhanced exploration of novel objects.⁴⁶Similarly, combined choline and DHA supplementation following perinatal brain injury improved performance in the novel object recognition test, indicating enhanced recognition memory.⁴⁸ AThese observations support the increased novel object preference observed in the HEK-CM and Ch-DHA treated groups in the present study. EE has also been reported to influence object recognition behavior, although the nature of this effect may vary with age. A previous study showed that animals exposed to EE displayed age-dependent object preferences. It was observed that young mice spent more time exploring novel objects, whereas aged mice showed a greater preference for familiar objects.⁴⁹ Such age-related variability may explain the distinct pattern of object preference observed in the EE group in the present study.

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. In addition, the present study has several limitations, including the absence of molecular and histological analyses to elucidate underlying mechanisms, the inclusion of only male mice which may limit the generalizability of the findings to females. Future studies incorporating both sexes, mechanistic assessments, characterization of conditioned media, longer follow-up periods, and adequately powered experimental designs are warranted.

Data availability

Acknowledgments

The authors would like to thank Manipal Academy of Higher Education for providing all infrastructure and support needed for this study.

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Published: 25 Jun 2026, 12:141

Published: 29 Jan 2024, 12:141

Published: 07 Jun 2023, 12:141

Published: 07 Feb 2023, 12:141

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VERSION 4

PUBLISHED 25 Jun 2026

Revised

Reviewer Report 02 Jul 2026

David V C Brito, ABC-RI, Algarve Biomedical Center Research Institute, Faro, Portugal;  Faculty of Medicine and Biomedical Sciences (FMCB), University of Algarve, Portugal, Portugal;  ABC Collaborative Laboratory: Integrated Ageing and Rejuvenation Solutions (ABC CoLAB – Ageing Better), Portugal, Portugal 

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Reviewer Report 05 Jul 2024

Laibaik Park, Weill Cornell Medical College, New York, NY, USA 

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Reviewer Report 21 Mar 2024

Sareesh Narayanan Naduvil, Department of Physiology, School of Medicine and Dentistry, AUC-UK Track, University of Central Lancashire, Preston, England, UK 

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VERSION 2

PUBLISHED 07 Jun 2023

Revised

Reviewer Report 12 Jun 2023

Sareesh Narayanan Naduvil, Department of Physiology, School of Medicine and Dentistry, AUC-UK Track, University of Central Lancashire, Preston, England, UK 

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Reviewer Report 09 Mar 2023

Kumar M.R. Bhat, Department of Anatomy, Ras Al Khaimah College of Medical Sciences, RAK Medical and Health Sciences University, Ras Al Khaimah, United Arab Emirates 

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