A mild dose of aspirin promotes hippocampal neurogenesis and working memory in experimental ageing mice

A mild dose of aspirin promotes hippocampal neurogenesis and working memory in experimental ageing mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A mild dose of aspirin promotes hippocampal neurogenesis and working memory in experimental ageing mice Jemi Feiona Vergil Andrews, Divya Bharathi Selvaraj, Akshay Kumar, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2789201/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aspirin treatment is the most widely used preventive measure against cardiovascular diseases. Aspirin is also expected to provide beneficial effects on the brain. However, the association between aspirin treatment and neurocognitive functions is a subject of debate. Ample reports strongly advocate that a mild dose of aspirin positively modulates hippocampal plasticity responsible for memory. Aspirin is a selective cyclooxygenase (COX)-2 inhibitor but the underlying mechanism through which aspirin modulates neuroplasticity remains unclear. Adult neurogenesis in the hippocampus has been established as an underlying basis of learning and memory. Therefore, aspirin treatment might be linked to the regulation of hippocampal neurogenesis. Thus, this study revisited the effect of low-dose aspirin on learning and memory in correlation with the regulation of hippocampal neurogenesis in the brains of ageing experimental mice. Results from the novel object recognition (NOR) test, Morris water maze (MWM), and cued radial arm maze (cued RAM) revealed that aspirin treatment enhances working memory in experimental ageing mice. Further, the co-immunohistochemical assessments on the brain sections indicated an increased number of doublecortin (DCX) positive immature neurons and bromodeoxyuridine (BrdU)/neuronal nuclei (NeuN) double-positive newly generated neurons in the hippocampi of mice in aspirin-treated group compared to the control group. Recently, enhanced activity of acetylcholinesterase (AChE) in circulation has been identified as an indicative biomarker of dementia. The biochemical assessment in the blood of aspirin-treated mice showed decreased activity of AChE than that of the control group. This study supports the procognitive effects of aspirin which can be translated to treat dementia. Aspirin memory hippocampus doublecortin adult neurogenesis Morris water maze Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aspirin is one of the most widely used generic non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment regime of pain and fever [ 1 , 2 ]. Regular intake of aspirin appears to provide preventive measures against various aging-associated diseases including cardiovascular illness, cerebral stroke, thrombosis, and cancer [ 3 ]. The blockade of cyclooxygenase (COX)-2 enzyme responsible for the synthesis of inflammatory prostaglandins is a well-recognized mode of action for aspirin [ 4 ]. Besides, aspirin has also been identified to modulate the molecular pathways related to cholesterol rafts, proton pumps, capsaicin receptors, N-methyl-D-aspartate (NMDA) receptors, voltage-gated calcium channels, and peroxisome proliferator-activated receptor alpha (PPAR-α) [ 5 – 10 ]. An elevated level of COX-2 has been linked to neuroinflammation and oxidative stress in the brain during ageing, infections, and upon exposure to neurotoxic agents [ 11 ]. While many brain disorders have been characterized by disruption of neuroplasticity and apoptotic events in the brain due to overexpression of expression of COX-2, pharmacological blockade of COX-2 has been considered as a therapeutic intervention to treat various mood disorders, neurocognitive deficits, and psychiatric illnesses [ 11 , 12 ]. Among various COX-2 inhibitors, aspirin has been speculated to enhance memory in ageing population and mitigate neuropathogenesis in various brain diseases. While some correlative studies indicated that the association between aspirin treatment and memory is uncertain, ample scientific evidence strongly implies that aspirin prevents memory decline in elderly subjects [ 13 , 14 ]. Eventually, recent reports demonstrated that aspirin treatment considerably improves the synaptic plasticity in the cognitive centres of the experimental brains [ 15 ]. Recently, the implementation of aspirin has been considered for the treatment regime of memory loss in aging and neurocognitive disorders including Alzheimer's disease (AD) [ 16 , 17 ]. However, aspirin-mediated possible cellular mechanisms by which regulation of neuroplasticity responsible for cognitive function remains to be established. Adult neurogenesis is an inimitable and dynamic neuro-regenerative process of the brain in which new functional neurons are continuously generated from neural stem cells (NSCs) in the dentate gyrus (DG) of the hippocampus and subventricular zone (SVZ) of the brain [ 18 – 20 ]. Notably, hippocampal neurogenesis has been regarded as an underlying cellular basis of pattern separation, mood, spatial learning, and memory in physiological conditions [ 21 , 22 ]. Whereas decreased hippocampal neurogenesis has been linked to memory loss upon aging [ 23 ]. Moreover, neurodegenerative disorders have been characterized by impaired hippocampal neurogenesis accounting for neurological deficits and dementia [ 18 , 19 , 24 ]. Therefore, targeting a centralized molecular pathway that interlinks various pathogenic mechanisms and suppresses the hippocampal neuroregenerative plasticity during ageing and pathogenic conditions with dementia is crucial. It can be speculated that aspirin treatment might be involved in the regulation of hippocampal neurogenesis. However, the reports on the effects of aspirin in the neuroregenerative potential of the brain are highly limited. A progress in the understanding of the neuropharmacological effects of aspirin on the modulation of neuroplasticity and neuroregenerative measures responsible for memory functions is an important scientific perusal. Therefore, this study revisited the effect of aspirin on the regulation of hippocampal neurogenesis and neurocognitive behaviours in experimental aging mice. Materials And Methods Experimental animals Male Bagg albino laboratory-bred (BALB)/c mice were procured from Liveon Biolabs private limited, Karnataka India. A set of seven to eight months old experimental mice (N = 12) were divided into 2 groups namely, Control (N = 6) and Aspirin (Disprin, Reckitt Benckiser Healthcare India PVT Ltd) treated (ASP) (N = 6) groups. Animals were maintained under standard laboratory conditions of a 12-hour light/ dark cycle at 22–25°C in the animal house facility of Bharathidasan University with free access to feed and water. All animal experiments were conducted in accordance with the approval of the institutional animal ethical committee (IAEC), Bharathidasan University under the regulations of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), India (Ref No: BDU/IAEC/2017/NE/41/Dt.21.03.2017). The Treatment Of Aspirin And Bromodeoxyuridine Each mouse in the treatment group was given a daily dose of aspirin (60 milligram (mg) /kilogram (kg) body weight (BW)) in drinking water for 40 days, while mice in the control group received normal drinking water. During the treatment period of aspirin from day 9 to day 13, the labelling of dividing NSCs was performed using the thymidine analog 5-Bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich, USA). BrdU was dissolved in a warm sterile solution of 0.9% NaCl (Sisco Research Laboratories (SRL), India) and thoroughly mixed. A single dose of BrdU solution at 50 mg/Kg BW was intraperitoneally injected into all the animals in both the control and the aspirin group for 5 consecutive days. During the treatment period of aspirin from day 16 to day 40, the animals were subjected to various behavioural tests such as novel object recognition (NOR), cued radial arm maze (cued RAM), and Morris water maze (MWM). A camera was placed above the test fields and connected to a computer equipped with SMART 3.0 video tracking software (Pan lab, Harvard Apparatus, Spain). The entire activities of each animal were tracked and the behavioural parameters were assessed using SMART 3.0 software. After 30 days from the last injection of BrdU coupled with behavioural experiments, mice were perfused and the brain tissues were taken for the analysis of neurogenesis. Novel Object Recognition Test To assess the effect of aspirin on the non-spatial recognition memory and discrimination index, the experimental animals were subjected to a standard NOR test as previously described by Yesudhas and colleagues 2020 [ 25 ]. During the habituation period, each animal was allowed to explore an empty quadrilateral arena (30 × 15 x 30 cm). The test arena was equally digitally divided into two squares and identical cylindrical shaped yellow-colored wooden objects namely A1 and A2 were placed in the middle of each square. Using SMART 3.0 software, two circular zones were digitally introduced around each object, such as zone 3 (sky blue circle) and zone 4 (pink circle). In the trial phase, each animal was released in the middle of the arena and allowed to explore and familiarize itself with identical objects for 5 minutes (mins) with 3 consecutive trials. After the task, the animal was returned to the home cage. Next, the test phase was performed after 2 hours in which object A2 was replaced by a novel green-coloured rectangular-shaped wooden object B but familiarized object A1 was retained at the same place. During the test phase, each mouse was released again into the arena to re-explore the objects for 5 mins. The overall activities of each animal were captured using SMART 3.0. In particular, the time spent by experimental animals in the respective zones and the tendency to explore the novel objects were estimated. The discrimination index was calculated using the formula discrimination index = (Exploration time in novel object − exploration time in familiar object) / (total exploration time in both objects) × 100 [ 25 ]. Morris Water Maze Task A black circular MWM tank with a diameter of 150 cm and a depth of 50 cm was used to investigate the aspirin-mediated effect on spatial learning and memory in experimental animals, as previously described [ 25 ]. Four extra maze visual cues were placed on the walls of the behavioural room with an appropriate lighting setup. Using SMART 3.0, the whole MWM was designated as an arena, and four subdivisions namely zone 1, zone 2, zone 3, and zone 4 were digitally introduced. For habituation, all animals were randomly exposed to the MWM for 2 days without the platform. Then, an escape hidden platform was placed in the middle of zone 4 and submerged 1 cm below the surface of the water. The temperature of water in the MWM pool was maintained at around 20–22°C. During the training session, each mouse was gently released into the water maze from four different directions in a systematic manner and trained to find the hidden platform within 1 min. At the end of each trial, the animal was allowed to stay on the platform for 30 seconds and then returned to the home cage. The learning session was carried out for 14 consecutive days at 4 trials for 1 min each per day with a minimum of 30 mins of intervals. The tracking SMART 3.0 module was calibrated and programmed to automatically stop when the mouse reaches the platform before 1 min. For each trial, the swimming path, distance moved, speed, and time spent in each quadrant were automatically recorded during the trial and analyzed at the end of each day. The learning curve was established by the time taken to find the platform and represented as escape latency. One day after the learning session, the probe test was conducted, in which the platform was removed from zone 4, each mouse was released into the MWM pool and the performance of the animal was recorded for 1 min. The time spent in the platform zone was considered to assess the retention memory of the animals. On the following day, the platform was placed in the opposite quadrant (zone 2) for the reversal training. Similar to the learning phase, each animal was released from all four sides systematically and the time taken to find the submerged hidden platform in a new zone was measured. The reversal learning was carried out for 3 consecutive days consisting of 4 trials each for 1 min. The short-term learning curve for the reversal training was established to determine spatial working memory [ 25 ]. Cued Radial Arm Maze Paradigm To validate the effect of aspirin on working memory, experimental animals were further challenged with cued RAM. 24 hours prior to the cued RAM experiment, the feed was removed from the animal cage. A standard 8-arm radial maze with eight horizontal equidistantly spaced arms (44 cm length x 14 cm breadth x 12 cm height) radiating from a circular central middle zone (32 cm in diameter). Using SMART 3.0, each arm was digitally divided into 8 arms (arm 1 to arm 8). In the learning phase, a visible wooden proximal maze cue was placed on the parapet end of arm 5 and choco flakes were placed inside arm 5. Each mouse was released in the middle of the cued RAM and allowed to explore freely all the arms. The uninterrupted movement of the mouse was recorded using SMART 3.0 for 5 mins. At the end of each training, the mouse was placed in the arm for 30 seconds and choco flakes were given as a reward. Then the mouse was gently removed from the cued RAM and returned to the home cage. In the learning phase, 5 mins x 3 trials per day were conducted for consecutive 3 days. The next day, the test phase was performed, in which the cue was shifted to arm 1 and the choco flakes were removed from arm 5. Each animal was released in the middle and the uninterrupted moments of the animal were recorded using SMART 3.0. The time taken to enter, the number of entries, and the time spent in the newly cued arm were calculated as the measure of working explicit memory [ 26 ]. Animal Perfusion And Cryosection Of The Brains After the behavioural experiments paralleled by 30 days from the last injection of BrdU, the experimental mice were deeply anesthetized. Blood samples were collected from cardiac puncture for biochemical assay and animals were transcardially perfused with sterile 0.9% NaCl (SRL, India) followed by 4% paraformaldehyde (PFA) (Himedia, India), as previously described [ 25 , 27 ]. The whole brains were dissected and soaked in 4% PFA overnight at 4°C. The next day, brains were transferred to fresh tubes containing 30% sucrose (SRL, India) and stored for a week at 4°C. Then the brain tissues were further processed for cryosectioning using a sliding microtome (Weswox, India) with a custom-built specimen block holder surrendered by the platform for dry ice. The poly freeze tissue freezing medium (Sigma Aldrich, USA) was added to the specimen block holder and was allowed to freeze using dry ice. Each brain was carefully separated into two hemispheres and embedded in poly freeze tissue freezing medium and kept on the specimen block holder. The brain tissue was optimally frozen using dry ice and subjected to the sagittal sections of 30 µM thickness and serially distributed in 12 tubes containing the cryoprotectant solution in a 1:1:2 ratio of glycerol (Merck, Germany), ethylene glycol (SRL, India) in 0.1M sodium phosphate buffer (NaH 2 PO 4 and K 2 HPO 4 (SRL, India), pH 7.5) and stored at -20°C for further immunohistochemical analysis as per the previously described [ 27 , 28 ]. Epifluorescence Immunohistochemical Analysis Of Neurogenesis 1 out of 12 free-floating brain sections from the left hemisphere of the brain with the 360 µM distance apart was taken in 12 well plates (Tarson, India) filled with 1× Tris Buffered Saline (TBS) (Tris HCl (Himedia, India) and NaCl (SRL, India), pH 7.4) and washed thrice using a shaker (Tarson, India) for 10 mins each at room temperature. Next, in the antigen retrieval step, the brain sections were placed in sodium citrate buffer (10 millimolar (Mm) Sodium citrate dihydrate (Thermo Fisher Scientific, USA) and 0.05% Triton X (Himedia, India), pH 6.0 for two hours at 65°C in a water bath (Kemi, India). Then the brain sections were washed thrice in 1×TBS for 10 mins each at room temperature. After washing, the sections were incubated in a blocking solution of 3% Bovine Serum Albumin (BSA) (Himedia, India) in 0.1% TBST (1×TBS and 0.1% Triton X) for 1 hour on a shaker at room temperature. After blocking, the primary antibodies were reconstituted in the blocking solution and added to the sections, and incubated for 48 hours at 4°C. To label the doublecortin (DCX) positive immature neurons, brain sections were incubated with rabbit anti-DCX antibody (Cell Signalling Technology (CST), USA; 1:250 dilution) for 48 hours at 4°C. After 48 hours the solution containing primary antibodies was removed and washed thrice in 1×TBS for 10 mins each at room temperature. Then, the brain sections were incubated with a fluorescent conjugated secondary antibody namely, goat anti-rabbit Dylight 594 (Novus Biologicals, USA; 1:500 dilution) for 24 hours at 4°C. After 24 hours, the secondary antibodies were removed and the sections were washed twice in 1×TBS for 10 mins each at room temperature. The cell nuclei were labelled with 0.1 mg/mL of 4',6-diamidino-2-phenylindole (DAPI) (Himedia, India) in TBST for 10 mins. Further, the brain sections were washed in 1×TBS for 10 mins and placed on the double frosted slides (Borosil, India), and allowed to dry overnight in the dark. Upon complete drying, the sections were mounted using the Prolong Glass Antifade Mountant (Thermo Fisher Scientific, USA) and dried properly in dark. Then the brain sections were visualized under a fluorescence microscope (Leica Microsystems, Germany). Slides were blind coded and the DG of the hippocampus was traced. DCX-positive cells were counted in the entire hippocampal DG using the ImageJ software cell counter plugin and the total number of DCX-positive cells per hippocampal DG was estimated as per previous reports [ 27 , 29 ]. The length of the dendrites of DCX cells was also measured using ImageJ software. Next, to assess the neuronal differentiation and survival, an additional set of brain sections were taken and washed in 1×TBS washed thrice for 10 mins each at room temperature. Next, the antigen retrieval step was performed in which the brain sections were placed in sodium citrate buffer for two hours at 65°C. After antigen retrieval, the brain sections were placed in 2 normal (N) HCl (Finar, India) for 10 mins at 37°C on a shaker. Then the brain sections were treated with 0.1 Molar borate buffer (Boric acid (Himedia, India), pH 8.5 for 10 mins. Then the brain sections were washed thrice in 1×TBS for 10 mins each at room temperature. After washing, the sections were incubated in 3% BSA for 1 hour on a shaker at room temperature. After blocking, two primary antibodies namely, mouse anti-BrdU (Novus Biologicals, USA; 1:100 dilution) and rabbit anti-Neuronal nuclear protein (NeuN) (Novus Biologicals, USA); 1:100 the dilution) at 4°C for 48 hours. Then, the primary antibodies were removed and the sections were washed thrice in 1×TBS buffer for 10 mins. The brain sections were incubated together with two different secondary antibodies such as sheep anti-mouse Dylight 488 (Novus Biologicals, USA; 1: 500 dilution) and goat anti-rabbit Dylight 594 (Novus Biologicals, USA) antibodies at 4°C for 24 hours. After 24 hours, the secondary antibodies were removed and the sections were washed thrice in 1×TBS for 10 mins each. Finally, the brain sections were placed on the double frosted slides (Borosil, India) and allowed to dry overnight in the dark. After complete drying, the sections were mounted using the Prolong Glass Antifade Mountant (Thermo Fisher Scientific, USA) and dried properly in dark. A laser-scanning confocal microscope (SM 710, Laser Scanning Confocal Microscope, Carl Zeiss, Germany) was used from the central instrumentation facility of Bharathidasan University to assess the immunofluorescence assessment. The number of BrdU-positive cells and BrdU co-localization with NeuN was analyzed from confocal z-stacks throughout the hippocampal DG. For the cell survival assessment, the number of BrdU-positive cells was counted in the entire non-overlapping confocal z-stack images of hippocampi. For the frequency of neuronal differentiation, all BrdU-positive cells in hippocampal DG from each animal were assessed, and BrdU-positive cells (green) that were co-labelled with NeuN (red) as considered double-positive cells (yellow) and calculated the percentage of newly differentiated neurons per hippocampal DG [ 27 , 30 ]. Estimation Of Ache Activity In The Blood The whole blood was collected in a container containing an anticoagulant, 3.2% buffered trisodium citrate (Thermofischer Scientific, USA). The blood was centrifuged at 800 rpm for 10 mins. The plasma was transferred into another sterile tube and then centrifuged at a speed of 2500 rpm for 15 mins to obtain platelet concentration. The lower 1/3rd is platelet-rich plasma (PRP) and the upper 2/3rd is platelet-poor plasma (PPP). PPP was removed and the tube was shaken to suspend the platelet pellets present at the bottom of the tube. The microplate AChE activity assay was carried out as described by Ellman et al in 1961 as soon as the PRP was obtained [ 31 ]. The samples were aliquoted into the wells of a microtiter plate and 0.1mM acetylthiocholine iodide (SRL, India) and 0.5 mM 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) (Himedia, India) was added. AChE present in the PRP catalyzes the hydrolysis of acetylthiocholine into acetate and thiocholine. The enzyme activity of PRP was measured by the yellow colour from thiocholine when it reacted with DTNB. The plate was shaken for a few seconds and the absorbance at 412 nm was taken using UV-Vis Spectrophotometer (Synergy™ HTX Multi-Mode Microplate Reader, Biotek, Winooski, USA). Statistical Analyses The values have been represented as mean ± standard deviation (SD). Student t-test was applied to measure the statistical significance for the number of entries, duration and discrimination index in NOR test, probe test in MWM, latency and duration in cued RAM, number of DCX positive cells, length of the dendrites, percentage of BrdU/NeuN double-positive cells and AChE activity between the control and Aspirin treated groups. The escape latency to find the platform and reversal learning in the MWM test, the learning curve in cued RAM, was assessed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. All the statistical analyses were made using Graph Pad Prism. The significance level was assumed at P < 0.05 unless otherwise indicated. Results Aspirin treatment improved novel object recognition in experimental mice In experimental animals, the time taken to explore a novel object has been considered a reflection of non-spatial memory function [ 32 ]. NOR test was conducted to assess the effect of aspirin mediated on the non-spatial object memory based on the ability of the animals to distinguish the difference between a known object and a new object in an experimental task. The experimental animals that show a preference to explore a new object compared to a familiar object have been considered to have improved memory. During the training session, experimental mice were exposed to a test arena and freely allowed to explore two identical objects. In the test phase, experimental mice were exposed to the replacement of a familiar object with a novel object. Though mice in both the control and the aspirin-treated group were able to recognize the novel object, experimental mice that are treated with aspirin showed an enhanced tendency to visit the novel object zone and explore the new object more than the familiarized object compared to the mice in the control group (Control = 3.5 ± 1.6 vs Aspirin = 6.5 ± 0.83). As a result, time spent by the aspirin-treated animals in the novel object zone was significantly increased than the control group (Control = 35 ± 10 vs Aspirin = 54 ± 13). Eventually, the percentage of the discrimination index was significantly enhanced in the aspirin-treated group compared to the control group (Control = 24.8 ± 9 vs Aspirin = 38 ± 9) thereby, indicating that aspirin treatment improved non-spatial recognition memory in experimental ageing mice (Fig. 1 ). Aspirin Treatment Enhanced Spatial Learning And Working Memory In The Morris Water Maze Task The MWM is an effective behavioural test of spatial learning and retention of memory in experimental rodents [ 33 ]. In the MWM task, the animals were trained to navigate from a releasing point to locate a submerged escape platform in an open water pool with the use of extra maze cues or spatial signs in the behavioural room. The spatial learning performance of experimental animals was evaluated as latency to find the hidden platform across repeated trials and memory retention was assessed by time spent by the animals in the target zone during the probe trial without the platform. In the MWM- assessment, the learning performance, and preference of experimental mice to find the platform in identifying the hidden platform were gradually improved during the training period of 14 days in both control and aspirin-treated groups. Valuation of data obtained from the learning curve indicated that the escape latency was decreased in the aspirin-treated group compared to the control group noticeably from the 5th day of training. Next, the experimental animals were challenged for the degree of memory retention during the probe test. During the probe test, the platform was removed from the MWM, and each animal was released to the pool time spent in the platform zone was measured for 1 min per single trial. The duration of search in the target quadrant by the aspirin-treated animals was found to be significantly increased than that of control animals (Control = 14 ± 4 vs Aspirin = 27 ± 8). Next, to assess the reversal learning and working memory, the platform was placed in the opposite quadrant and reversal learning was conducted for the next three days. Eventually, the time taken to find the platform in the opposite quadrant was reduced by experimental mice in the aspirin-treated group than the control group. Taken together, the MWM test indicates that the aspirin treatment prevents memory decline in experimental mice (Fig. 2 ). Aspirin Treatment Improved Working Memory In Cued Ram The use of cued RAM is highly instrumental in assessing the working memory of experimental rodents. Notably, the eight-arm radial maze with a proximal cue at the target arm enables the animals to locate flavoured feed during the training session. In the test phase, the ability of experimental animals to explore the shifted proximal cue in a new location as a strategic attempt in search of feed has been considered the degree of working memory [ 34 ]. In the trial phase, the time spent by the experimental mice in both the control and aspirin-treated group in the cued arms were nearly similar. However, during the test phase, while the latency to enter the cue shifted arm was reduced (Control = 207 ± 70 vs Aspirin = 232 ± 37), time spent in the newly cued arm was significantly increased in the aspirin-treated group than that of the control (Control = 22 ± 10 vs Aspirin = 38 ± 3). Taken together, the cued RAM experiment validates that the aspirin treatment facilities working and declarative memory in experimental mice (Fig. 3 ). Aspirin Treatment Increased The Hippocampal Neurogenesis In the stem cell niches of the mammalian brain, the number of DCX-positive immature neurons has been considered to reflect ongoing neurogenesis [ 35 ]. The immuno labelling study of the DCX-positive cells in the hippocampal DG revealed that the number of immature neurons was significantly increased in the aspirin-treated group than that of the control group. Further, in the morphological assessment of DCX-positive cells (Control = 3742 ± 1029 vs Aspirin = 6087 ± 1316) and the length of the dendrites (Control = 90 ± 23 vs Aspirin = 172 ± 42) was found to be increased in hippocampal DG region of the brains of the experimental mice in the aspirin-treated group than the control group. BrdU labelling of newly dividing NSCs followed by the co-expression with a mature neural mark has been a critical step in assessing the neuronal fate in the brain [ 27 ]. In the confocal microscope-based co-immunolabelling study, the percentage of BrdU/NueN double positive cells was significantly increased in the granule cell layer of hippocampal DG regions of the brains of the experimental mice in the aspirin-treated group than the control group (Control = 76 ± 8 vs Aspirin = 91 ± 3). Considering the fact aspirin treatment appears to promote neuronal differentiation of NCS and facilitate hippocampal neurogenesis in experimental mice (Fig. 4 ). Aspirin-treated Animals Exhibited Reduced Activity Of Ache In The Blood Recently, the activity of AChE in the blood has been considered a diagnostic measure to correlate memory [ 36 ]. The colorimetric determination of AChE activity was performed to measure the increase in the intensity of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. Results revealed that the absorbance values of AChE activity in blood samples of aspirin-treated animals were significantly reduced when compared to that of control animals (Control = 1.8 ± 0.2 vs Aspirin = 1.3 ± 0.18) (Fig. 4 ). Discussion Progressive memory loss is one of the increasing clinical concerns worldwide that pose major challenges to the ageing population, health care providers, and biomedical research sectors [ 19 , 37 , 38 ]. The investigation into underlying mechanisms and therapeutic targets is an ongoing quest and the identification of drugs that exert precognitive action has become an unmet need [ 19 , 39 ]. Aspirin is a widely used painkiller that blocks the enzymatic action of COX-2 thereby, mitigating inflammatory processes in various diseases [ 40 , 41 ]. Numerous experimental studies suggest that aspirin treatment modulates brain functions positively at behavioural, biochemical, and cellular levels [ 42 ]. Based on the animal behavioural and immunobiological assessments, the present study demonstrates that aspirin treatment improves hippocampal neurogenesis thereby ageing experimental mice enables. In response to ageing-mediated biological changes and neurodegenerative events, proinflammatory molecules are discharged from activated immune cells in the brain [ 18 , 27 ]. The abnormally elevated levels of proinflammatory have been known to impair the neurogenic process at the level of proliferation, and differentiation survival of NSCs leading to progressive memory impairments [ 18 , 43 ]. Notably, aspirin has been reported to induce the production of endogenous lipoxins, a potent anti-inflammatory molecule, which diminishes the pathogenic activation of microglia thereby reducing the load of various inflammatory molecules including C-reactive protein tumour necrosis factor (TNF)-α and interleukin − 6 in experimental animals [ 44 – 46 ]. For the past few decades, the beneficial effects of aspirin treatment against cardiovascular and cerebrovascular diseases have unequivocally been established [ 47 – 49 ]. In recent years, the neuroprotective role of aspirin in preventing cognitive decline has also increasingly been recognized. In contrast, few randomized controlled trials and meta-analyses have stated no association between aspirin and cognitive functions. Thus, there exists an ongoing considerable ambiguity about the beneficial effects of aspirin against dementia and neurocognitive impairment [ 50 ]. However, convincing experimental evidence supports the procognitive effect of aspirin as it appears to positively modulate hippocampal plasticity. In the experimental models challenged with glutamate excitotoxicity and hypoxia, aspirin treatment has been reported to provide neuroprotection in the brain [ 51 ]. Notably, the neuroprotective role of aspirin has been linked to the inhibition of NF-kB signaling in the brain [ 52 ]. Depeng Feng et al reported that aspirin facilitates neuroprotection in experimental animal models treated with lipopolysaccharide (LPS), and cerebral ischemia-reperfusion (CIRP) injury [ 53 ]. Eventually, cohort and population-based studies revealed an inverse relationship between long-term intake of low-dose aspirin and the severity of dementia [ 54 , 55 ]. Chandra et al 2018, reported that a low dose of aspirin decreases amyloid load through the activation of peroxisome proliferator-activated receptor alpha (PPAR)-α in the hippocampus of a 5×FAD mouse model of memory loss [ 56 ]. Saima Rizwan et al reported that a low-dose aspirin treatment improves memory in the AlCl3-induced mouse model of amnesia in association with an alteration of the opioid system [ 57 ]. While the circulating platelets have been recognized to contribute to amyloid pathology in the brain upon ageing, the platelet-reducing effect of aspirin has been considered to minimize the neuropathogenic events in the brain of subjects with neurodegenerative disorders [ 50 , 58 , 59 ]. In corroboration with the previous reports, in the present study, the aspirin-treated animals showed a better performance in NOR, MWM, and cued RAM compared to the control group. Moreover, the behaviour paradigms clearly indicated precognitive properties of aspirin were highly evident as the mice in the treatment group exhibited enhanced working memory. Aspirin-mediated memory enchantment could arise from both COX-dependent or indented pathways that positively contribute to the regulation of hippocampal plasticity. Considering the COX inhibition nature, the neuroprotective effect of aspirin treatment has been reported to be mediated via the inhibition of neuroinflammation, and mitochondrial oxidative phosphorylation [ 51 , 60 ]. The neuroprotective property of aspirin could play a direct role in the prevention of memory decline. Moreover, aspirin has been reported to mitigate free radical production thereby mimicking the antioxidant potential in different organs including the brain [ 42 , 61 ]. Patel et al demonstrated that aspirin interacts with PPARα and concedes memory functions through cAMP response element-binding protein (CREB) mediated hippocampal plasticity [ 10 , 15 ]. In addition, aspirin treatment has been reported to be involved in the epigenetic modification upregulation of the expression of BDNF responsible for preserving the memory function in Benzo[a]pyrene (BaP) treated experimental mice [ 62 ]. Notably, recent experimental data indicated that aspirin may be involved in the regenerative plasticity of the hippocampus [ 63 ]. In this study, the immunohistochemical assessments of DCX-positive cells and confocal-based BrdU/NeuN revealed a concomitant increase in the neurogenic process at the level of neuronal differentiation of NSCs in the hippocampus of ageing experimental mice. Notably, dendritic arborization of DCX-positive cells was found to be increased in the hippocampal DG of aspirin-treated animals. The augmented dendritic arborization and length signifies the state of neural differentiation and integration of DCX-positive cells in the hippocampal circuit [ 64 , 65 ]. Recently, Giacomo Pozzoli et al reported that aspirin promotes the differentiation of SK-N‐SH (N) human neuroblastoma cells through the modulation of the expression of cell cycle checkpoint markers p21 Waf1 and Rb1 independent of the COX pathway [ 66 ]. Considering the facts, it can be presumed that the procognitive effect of aspirin might be resulting from the neuronal differentiation of NSCs augmenting the new neurons in the hippocampus [ 67 ]. Notably, regulation of neurogenesis in the hippocampus has been linked to levels of some neurotransmitters that are crucial for learning and memory. Acetylcholine (ACh) is one of the key neurotransmitters responsible for cognitive function [ 25 , 68 ]. Abnormal levels of ACh in the circulation and brain has been identified as a cause of neurocognitive impairments [ 69 ]. As age increases, a decline in the level of ACh is evident in the circulation and the brain [ 25 , 70 ]. Activation of certain enzymes responsible for biochemical pathways of ACh synthesis appears to result in abnormal levels of ACh. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the rate-limiting enzymes that breakdowns ACh into choline and acetate [ 25 , 71 , 72 ]. However, AChE appears to break down ACh more than BuChE to a greater extent as BuChE has a lesser affinity for ACh [ 26 ]. Remarkably, increased activities of AChE leading to reduced levels of ACh have been identified as an underlying pathological determinant of cognitive impairments and memory loss in various neurocognitive diseases including AD [ 73 , 74 ]. Eventually, increased levels of AChE in the blood have been considered a biomarker of dementia [ 36 , 75 ]. Thus, the implementation of AChE inhibitors has been considered to mitigate memory loss in aging and AD [ 76 ]. Recently, few COX inhibitors have been known to diminish the activities of AChE [ 77 ]. Notably, bioinformatics-based docking studies revealed that aspirin binds to the active sites of AChE and acts as a potential inhibitor of its activities [ 78 , 79 ]. Ample reports suggest that inhibition of AChE facilitates hippocampal neuroregenerative plasticity in the adult brain [ 80 ]. Considering the aforementioned factors, it can be hypothesized that aspirin treatment might play a role in the regulation of hippocampal regenerative plasticity responsible for memory enhancement in association with the inactivation of AChE. Therefore, this study supports the procognitive and proneurogenic effects of aspirin which can be considered to translate for the treatment regimens to boost neuroregenerative plasticity noticed during various disease conditions with neurocognitive impairments. Conclusion Consumption of aspirin, a potent COX inhibitor has been reported to yield diversified results with reference to cognitive functions. The present study demonstrates that aspirin treatment enhances working memory in ageing experimental mice. This study revealed that aspirin promotes neuronal differentiation in the hippocampus in correlation with a reduction in AChE activities in circulation. Considering the facts, better pattern recognition, spatial working memory, and declarative memory functions noticed in the animal in the aspirin treatment group could be largely due to enhanced neuroregenerative plasticity in the hippocampus despite its neuroprotective roles. This study supports the memory-enhancing capacity of aspirin and necessitates further studies to reveal the mechanisms reasonable for pro-neurogenic effects in the brain Declarations Data availability The data will be made available from the corresponding author upon reasonable request. Acknowledgment This work was supported by Early Career Research Award (SERB-ECR/2016/000741) from the Science and Engineering Research Board (SERB) under the Department of Science and Technology (DST), Government of India. The authors also thank the research grant (SERB-EEQ/2016/000639) from the Science and Engineering Research Board (SERB) under the Department of Science and Technology (DST), Government of India for the financial support. Dr. Mahesh Kandasamy has been supported by University Grants Commission–Faculty Recharge Programme (UGC-FRP), New Delhi, India Dr. Anusuyadevi Muthuswamy would like to thank ICMR, New Delhi, India (2019-2605/CMB/Adhoc/BMS) and DST, New Delhi, India (DST/CSRI/2018/343(G)) for the financial support. Authors also thank RUSA 2.0, Biological Sciences, Bharathidasan University for their financial support (TN RUSA: 311/RUSA (2.0) / 2018 dt. 02/12/2020). Divya Bharathi is the recipient of the RUSA 2.0 project fellowship (Ref. No. BDU/RUSA 2.0/TRP/BS/Date 22/04/2021). Jemi Feiona was supported as a project assistant from the project grant SERB EEQ/2016/000639. The authors would like to acknowledge UGC-SAP and DST-FIST for the infrastructure of the Department of Animal Science and Biochemistry, Bharathidasan University. Authors extend acknowledgment to the University Science Instrumentation Centre (USIC)-BDU & DST-PURSE (Phase 1 & 2) for the confocal analysis. The authors would like to thank Dr. Muthu Kumar, USIC-BDU for the excellent technical support in the confocal microscopy. We express sincere thanks to Dr. R. Thirumurugan, Professor and Head, the Department of Animal Science for providing the facility to utilize the Multimode Plate Reader. Ethics declarations Conflict of interest The authors declare no conflict of interest. Authors contributions MK conceived and designed the experiment. JFAV, DBS, AK, and MK conducted the experiments and analyzed the data. MK JFAV, DBS, AK, and MA wrote, reviewed, and edited the manuscript. All authors read and approved the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2789201","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":190140773,"identity":"f92f6820-77d0-4e75-ba7a-eb31f34d4dfd","order_by":0,"name":"Jemi Feiona Vergil Andrews","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jemi","middleName":"Feiona Vergil","lastName":"Andrews","suffix":""},{"id":190140774,"identity":"8aafbbe2-a76f-45d9-996d-3d26b7d6eeb6","order_by":1,"name":"Divya Bharathi Selvaraj","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Divya","middleName":"Bharathi","lastName":"Selvaraj","suffix":""},{"id":190140775,"identity":"9b5c7698-93ca-4820-a92b-08443c679dc7","order_by":2,"name":"Akshay Kumar","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Akshay","middleName":"","lastName":"Kumar","suffix":""},{"id":190140776,"identity":"7471bde8-bcd1-4074-9348-bcdccdd54d19","order_by":3,"name":"Syed Aasish Roshan","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Syed","middleName":"Aasish","lastName":"Roshan","suffix":""},{"id":190140777,"identity":"17d1b274-5612-459a-9d3d-4c3c64f031f3","order_by":4,"name":"Muthuswamy Anusuyadevi","email":"","orcid":"","institution":"Bharathidasan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muthuswamy","middleName":"","lastName":"Anusuyadevi","suffix":""},{"id":190140778,"identity":"a03004b0-633b-437f-b058-bd4ca8b0cb8a","order_by":5,"name":"Mahesh Kandasamy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIiWNgGAWjYFACHgYGxgYGAyDLAMznl4Ay2IjWIjmDZC0GN6AMXEC3/ezBhz93MBjzz27e+PFHzbbEzbebt0kw1Ngx8Ek3YNVidiYv2Zj3DIOZxJ1jxdI8x24nbrtzrEyC4VgyA5vMAexaDuSYSTO2Mdgw3MgxkGZgA2q5kWMmwcB2gIFNIgG7lvNvzH/+BGqRv5Fj/PPHv9uJm2eAtPzDowVoJgNvG4OZAchw3rbbiRskgAzGNnxa3hhL87ZJGBveSCuz5u27bTwD6CmLxL5kHtwOyzH8+LPNxnDejeTNN398uy3bDwy6Gx++2cnJz8CuBQok4CzHBhCZAI4vIoE90SpHwSgYBaNgxAAAarFf0ikFewsAAAAASUVORK5CYII=","orcid":"","institution":"Bharathidasan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mahesh","middleName":"","lastName":"Kandasamy","suffix":""}],"badges":[],"createdAt":"2023-04-07 09:14:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2789201/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2789201/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":35603175,"identity":"d46aab70-b616-442a-8357-eab0d187909f","added_by":"auto","created_at":"2023-04-11 18:28:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":357363,"visible":true,"origin":"","legend":"\u003cp\u003eNovel object recognition (NOR) test. (A) Representative tracking image of NOR test of control and aspirin-treated mice. The sky-blue colored circle indicates the familiar object and the pink-colored circle indicates the novel object. (B) The bar graph represents the number of entries into the novel object zone. (C) The bar graph represents the time spent in the novel object zone. (D) The bar graph depicts the percentage of the discrimination index.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2789201/v1/fa16759df050babadfd2187a.png"},{"id":35603176,"identity":"0daa96e9-51f8-4d6c-8203-04a5054837b6","added_by":"auto","created_at":"2023-04-11 18:28:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398814,"visible":true,"origin":"","legend":"\u003cp\u003eSpatial learning and memory of animals in Morris Water Maze (MWM) test. (A) Representative tracking image of MWM during the initial and late stages of the learning phase. (B) Representative tracking image of MWM during the probe test. (C) Representative tracking image of MWM during platform switch. (D) Result of the learning curve with escape latency to find the hidden platform. (E) The bar graph describes the time spent by the animals in the platform zone during the probe test. (F) The learning curve with escape latency to find the hidden platform in the opposite quadrant during the platform switch.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2789201/v1/830c4d4d28e92415db2eab95.png"},{"id":35604296,"identity":"e31db673-23f9-430a-a74a-3d761d351d83","added_by":"auto","created_at":"2023-04-11 18:36:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":360321,"visible":true,"origin":"","legend":"\u003cp\u003eCued Radial Arm Maze test. (A) Representative tracking image of cued RAM at the learning phase. The sun symbol represents the proximal cue in arm 5 in which rewards were placed in the learning phase. (B) Representative tracking image of cued RAM at the test phase and the sun symbol represents the proximal cue in which the cue was replaced in arm 1 and the food was removed in the test phase. (C) Result of the learning curve with latency to reach the arm in which cue and food was placed. . (D) The bar graph represents the time taken to reach the cue shifted arm by the animals in the test phase. (E) The bar graph describes the time spent in the cue-shifted arm by the animals in the test phase.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2789201/v1/3aa431c2c0f0ff027109ac6b.png"},{"id":35603177,"identity":"4d707e2d-1c2a-4f5d-84de-d9c7fbf59040","added_by":"auto","created_at":"2023-04-11 18:28:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1276708,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical assessment of immature neurons. (A) Representative image of DCX-positive cells in the dentate gyrus (DG) of the experimental animals.\u003cstrong\u003e \u003c/strong\u003eThe image illustrates the DCX positive cells, DAPI, and overlay of the same in the DG of the hippocampus. The scale bar =25 µm (B) The enlarged image illustrates the length of the dendrites. (C) The bar graph represents the number of DCX-positive cells in the dentate gyrus (DG) of the experimental animals. (D) The bar graph describes the length of the dendrites in the dentate gyrus (DG) of the experimental animals.\u003cstrong\u003e \u003c/strong\u003e(E)\u003cstrong\u003e \u003c/strong\u003eRepresentative confocal image of BrdU/NeuN double-positive cells in the DG of the hippocampus. (F) The bar graph represents the percentage of BrdU/NeuN double-positive cells. The scale bar =25 µm (G) The bar graph represents the enzymatic activity of acetylcholine esterase in the PRP of the experimental animals.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2789201/v1/5dadb69c88f2265175643d9b.png"},{"id":35604305,"identity":"48f10d0a-7934-4855-bbc7-893d43ae5843","added_by":"auto","created_at":"2023-04-11 18:36:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2479192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2789201/v1/88e989dd-5aaf-4dac-bf9e-36901123c764.pdf"},{"id":35604304,"identity":"87a37808-51ec-40ba-ac4c-783c7e2f9732","added_by":"auto","created_at":"2023-04-11 18:36:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2479192,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2789201/v1/7d515e66-7736-44a7-b2a9-9328eca99713.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A mild dose of aspirin promotes hippocampal neurogenesis and working memory in experimental ageing mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAspirin is one of the most widely used generic non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment regime of pain and fever [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Regular intake of aspirin appears to provide preventive measures against various aging-associated diseases including cardiovascular illness, cerebral stroke, thrombosis, and cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The blockade of cyclooxygenase (COX)-2 enzyme responsible for the synthesis of inflammatory prostaglandins is a well-recognized mode of action for aspirin [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Besides, aspirin has also been identified to modulate the molecular pathways related to cholesterol rafts, proton pumps, capsaicin receptors, N-methyl-D-aspartate (NMDA) receptors, voltage-gated calcium channels, and peroxisome proliferator-activated receptor alpha (PPAR-α) [\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. An elevated level of COX-2 has been linked to neuroinflammation and oxidative stress in the brain during ageing, infections, and upon exposure to neurotoxic agents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While many brain disorders have been characterized by disruption of neuroplasticity and apoptotic events in the brain due to overexpression of expression of COX-2, pharmacological blockade of COX-2 has been considered as a therapeutic intervention to treat various mood disorders, neurocognitive deficits, and psychiatric illnesses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among various COX-2 inhibitors, aspirin has been speculated to enhance memory in ageing population and mitigate neuropathogenesis in various brain diseases. While some correlative studies indicated that the association between aspirin treatment and memory is uncertain, ample scientific evidence strongly implies that aspirin prevents memory decline in elderly subjects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Eventually, recent reports demonstrated that aspirin treatment considerably improves the synaptic plasticity in the cognitive centres of the experimental brains [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recently, the implementation of aspirin has been considered for the treatment regime of memory loss in aging and neurocognitive disorders including Alzheimer's disease (AD) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, aspirin-mediated possible cellular mechanisms by which regulation of neuroplasticity responsible for cognitive function remains to be established. Adult neurogenesis is an inimitable and dynamic neuro-regenerative process of the brain in which new functional neurons are continuously generated from neural stem cells (NSCs) in the dentate gyrus (DG) of the hippocampus and subventricular zone (SVZ) of the brain [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, hippocampal neurogenesis has been regarded as an underlying cellular basis of pattern separation, mood, spatial learning, and memory in physiological conditions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Whereas decreased hippocampal neurogenesis has been linked to memory loss upon aging [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, neurodegenerative disorders have been characterized by impaired hippocampal neurogenesis accounting for neurological deficits and dementia [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Therefore, targeting a centralized molecular pathway that interlinks various pathogenic mechanisms and suppresses the hippocampal neuroregenerative plasticity during ageing and pathogenic conditions with dementia is crucial. It can be speculated that aspirin treatment might be involved in the regulation of hippocampal neurogenesis. However, the reports on the effects of aspirin in the neuroregenerative potential of the brain are highly limited. A progress in the understanding of the neuropharmacological effects of aspirin on the modulation of neuroplasticity and neuroregenerative measures responsible for memory functions is an important scientific perusal. Therefore, this study revisited the effect of aspirin on the regulation of hippocampal neurogenesis and neurocognitive behaviours in experimental aging mice.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals\u003c/h2\u003e \u003cp\u003eMale Bagg albino laboratory-bred (BALB)/c mice were procured from Liveon Biolabs private limited, Karnataka India. A set of seven to eight months old experimental mice (N\u0026thinsp;=\u0026thinsp;12) were divided into 2 groups namely, Control (N\u0026thinsp;=\u0026thinsp;6) and Aspirin (Disprin, Reckitt Benckiser Healthcare India PVT Ltd) treated (ASP) (N\u0026thinsp;=\u0026thinsp;6) groups. Animals were maintained under standard laboratory conditions of a 12-hour light/ dark cycle at 22\u0026ndash;25\u0026deg;C in the animal house facility of Bharathidasan University with free access to feed and water. All animal experiments were conducted in accordance with the approval of the institutional animal ethical committee (IAEC), Bharathidasan University under the regulations of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), India (Ref No: BDU/IAEC/2017/NE/41/Dt.21.03.2017).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe Treatment Of Aspirin And Bromodeoxyuridine\u003c/h3\u003e\n\u003cp\u003eEach mouse in the treatment group was given a daily dose of aspirin (60 milligram (mg) /kilogram (kg) body weight (BW)) in drinking water for 40 days, while mice in the control group received normal drinking water. During the treatment period of aspirin from day 9 to day 13, the labelling of dividing NSCs was performed using the thymidine analog 5-Bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich, USA). BrdU was dissolved in a warm sterile solution of 0.9% NaCl (Sisco Research Laboratories (SRL), India) and thoroughly mixed. A single dose of BrdU solution at 50 mg/Kg BW was intraperitoneally injected into all the animals in both the control and the aspirin group for 5 consecutive days. During the treatment period of aspirin from day 16 to day 40, the animals were subjected to various behavioural tests such as novel object recognition (NOR), cued radial arm maze (cued RAM), and Morris water maze (MWM). A camera was placed above the test fields and connected to a computer equipped with SMART 3.0 video tracking software (Pan lab, Harvard Apparatus, Spain). The entire activities of each animal were tracked and the behavioural parameters were assessed using SMART 3.0 software. After 30 days from the last injection of BrdU coupled with behavioural experiments, mice were perfused and the brain tissues were taken for the analysis of neurogenesis.\u003c/p\u003e\n\u003ch3\u003eNovel Object Recognition Test\u003c/h3\u003e\n\u003cp\u003eTo assess the effect of aspirin on the non-spatial recognition memory and discrimination index, the experimental animals were subjected to a standard NOR test as previously described by Yesudhas and colleagues 2020 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. During the habituation period, each animal was allowed to explore an empty quadrilateral arena (30 \u0026times; 15 x 30 cm). The test arena was equally digitally divided into two squares and identical cylindrical shaped yellow-colored wooden objects namely A1 and A2 were placed in the middle of each square. Using SMART 3.0 software, two circular zones were digitally introduced around each object, such as zone 3 (sky blue circle) and zone 4 (pink circle). In the trial phase, each animal was released in the middle of the arena and allowed to explore and familiarize itself with identical objects for 5 minutes (mins) with 3 consecutive trials. After the task, the animal was returned to the home cage. Next, the test phase was performed after 2 hours in which object A2 was replaced by a novel green-coloured rectangular-shaped wooden object B but familiarized object A1 was retained at the same place. During the test phase, each mouse was released again into the arena to re-explore the objects for 5 mins. The overall activities of each animal were captured using SMART 3.0. In particular, the time spent by experimental animals in the respective zones and the tendency to explore the novel objects were estimated. The discrimination index was calculated using the formula discrimination index = (Exploration time in novel object\u0026thinsp;\u0026minus;\u0026thinsp;exploration time in familiar object) / (total exploration time in both objects) \u0026times; 100 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eMorris Water Maze Task\u003c/h3\u003e\n\u003cp\u003eA black circular MWM tank with a diameter of 150 cm and a depth of 50 cm was used to investigate the aspirin-mediated effect on spatial learning and memory in experimental animals, as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Four extra maze visual cues were placed on the walls of the behavioural room with an appropriate lighting setup. Using SMART 3.0, the whole MWM was designated as an arena, and four subdivisions namely zone 1, zone 2, zone 3, and zone 4 were digitally introduced. For habituation, all animals were randomly exposed to the MWM for 2 days without the platform. Then, an escape hidden platform was placed in the middle of zone 4 and submerged 1 cm below the surface of the water. The temperature of water in the MWM pool was maintained at around 20\u0026ndash;22\u0026deg;C. During the training session, each mouse was gently released into the water maze from four different directions in a systematic manner and trained to find the hidden platform within 1 min. At the end of each trial, the animal was allowed to stay on the platform for 30 seconds and then returned to the home cage. The learning session was carried out for 14 consecutive days at 4 trials for 1 min each per day with a minimum of 30 mins of intervals. The tracking SMART 3.0 module was calibrated and programmed to automatically stop when the mouse reaches the platform before 1 min. For each trial, the swimming path, distance moved, speed, and time spent in each quadrant were automatically recorded during the trial and analyzed at the end of each day. The learning curve was established by the time taken to find the platform and represented as escape latency. One day after the learning session, the probe test was conducted, in which the platform was removed from zone 4, each mouse was released into the MWM pool and the performance of the animal was recorded for 1 min. The time spent in the platform zone was considered to assess the retention memory of the animals. On the following day, the platform was placed in the opposite quadrant (zone 2) for the reversal training. Similar to the learning phase, each animal was released from all four sides systematically and the time taken to find the submerged hidden platform in a new zone was measured. The reversal learning was carried out for 3 consecutive days consisting of 4 trials each for 1 min. The short-term learning curve for the reversal training was established to determine spatial working memory [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eCued Radial Arm Maze Paradigm\u003c/h3\u003e\n\u003cp\u003eTo validate the effect of aspirin on working memory, experimental animals were further challenged with cued RAM. 24 hours prior to the cued RAM experiment, the feed was removed from the animal cage. A standard 8-arm radial maze with eight horizontal equidistantly spaced arms (44 cm length x 14 cm breadth x 12 cm height) radiating from a circular central middle zone (32 cm in diameter). Using SMART 3.0, each arm was digitally divided into 8 arms (arm 1 to arm 8). In the learning phase, a visible wooden proximal maze cue was placed on the parapet end of arm 5 and choco flakes were placed inside arm 5. Each mouse was released in the middle of the cued RAM and allowed to explore freely all the arms. The uninterrupted movement of the mouse was recorded using SMART 3.0 for 5 mins. At the end of each training, the mouse was placed in the arm for 30 seconds and choco flakes were given as a reward. Then the mouse was gently removed from the cued RAM and returned to the home cage. In the learning phase, 5 mins x 3 trials per day were conducted for consecutive 3 days. The next day, the test phase was performed, in which the cue was shifted to arm 1 and the choco flakes were removed from arm 5. Each animal was released in the middle and the uninterrupted moments of the animal were recorded using SMART 3.0. The time taken to enter, the number of entries, and the time spent in the newly cued arm were calculated as the measure of working explicit memory [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eAnimal Perfusion And Cryosection Of The Brains\u003c/h3\u003e\n\u003cp\u003eAfter the behavioural experiments paralleled by 30 days from the last injection of BrdU, the experimental mice were deeply anesthetized. Blood samples were collected from cardiac puncture for biochemical assay and animals were transcardially perfused with sterile 0.9% NaCl (SRL, India) followed by 4% paraformaldehyde (PFA) (Himedia, India), as previously described [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The whole brains were dissected and soaked in 4% PFA overnight at 4\u0026deg;C. The next day, brains were transferred to fresh tubes containing 30% sucrose (SRL, India) and stored for a week at 4\u0026deg;C. Then the brain tissues were further processed for cryosectioning using a sliding microtome (Weswox, India) with a custom-built specimen block holder surrendered by the platform for dry ice. The poly freeze tissue freezing medium (Sigma Aldrich, USA) was added to the specimen block holder and was allowed to freeze using dry ice. Each brain was carefully separated into two hemispheres and embedded in poly freeze tissue freezing medium and kept on the specimen block holder. The brain tissue was optimally frozen using dry ice and subjected to the sagittal sections of 30 \u0026micro;M thickness and serially distributed in 12 tubes containing the cryoprotectant solution in a 1:1:2 ratio of glycerol (Merck, Germany), ethylene glycol (SRL, India) in 0.1M sodium phosphate buffer (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (SRL, India), pH 7.5) and stored at -20\u0026deg;C for further immunohistochemical analysis as per the previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEpifluorescence Immunohistochemical Analysis Of Neurogenesis\u003c/h3\u003e\n\u003cp\u003e1 out of 12 free-floating brain sections from the left hemisphere of the brain with the 360 \u0026micro;M distance apart was taken in 12 well plates (Tarson, India) filled with 1\u0026times; Tris Buffered Saline (TBS) (Tris HCl (Himedia, India) and NaCl (SRL, India), pH 7.4) and washed thrice using a shaker (Tarson, India) for 10 mins each at room temperature. Next, in the antigen retrieval step, the brain sections were placed in sodium citrate buffer (10 millimolar (Mm) Sodium citrate dihydrate (Thermo Fisher Scientific, USA) and 0.05% Triton X (Himedia, India), pH 6.0 for two hours at 65\u0026deg;C in a water bath (Kemi, India). Then the brain sections were washed thrice in 1\u0026times;TBS for 10 mins each at room temperature. After washing, the sections were incubated in a blocking solution of 3% Bovine Serum Albumin (BSA) (Himedia, India) in 0.1% TBST (1\u0026times;TBS and 0.1% Triton X) for 1 hour on a shaker at room temperature. After blocking, the primary antibodies were reconstituted in the blocking solution and added to the sections, and incubated for 48 hours at 4\u0026deg;C. To label the doublecortin (DCX) positive immature neurons, brain sections were incubated with rabbit anti-DCX antibody (Cell Signalling Technology (CST), USA; 1:250 dilution) for 48 hours at 4\u0026deg;C. After 48 hours the solution containing primary antibodies was removed and washed thrice in 1\u0026times;TBS for 10 mins each at room temperature. Then, the brain sections were incubated with a fluorescent conjugated secondary antibody namely, goat anti-rabbit Dylight 594 (Novus Biologicals, USA; 1:500 dilution) for 24 hours at 4\u0026deg;C. After 24 hours, the secondary antibodies were removed and the sections were washed twice in 1\u0026times;TBS for 10 mins each at room temperature. The cell nuclei were labelled with 0.1 mg/mL of 4',6-diamidino-2-phenylindole (DAPI) (Himedia, India) in TBST for 10 mins. Further, the brain sections were washed in 1\u0026times;TBS for 10 mins and placed on the double frosted slides (Borosil, India), and allowed to dry overnight in the dark. Upon complete drying, the sections were mounted using the Prolong Glass Antifade Mountant (Thermo Fisher Scientific, USA) and dried properly in dark. Then the brain sections were visualized under a fluorescence microscope (Leica Microsystems, Germany). Slides were blind coded and the DG of the hippocampus was traced. DCX-positive cells were counted in the entire hippocampal DG using the ImageJ software cell counter plugin and the total number of DCX-positive cells per hippocampal DG was estimated as per previous reports [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The length of the dendrites of DCX cells was also measured using ImageJ software.\u003c/p\u003e \u003cp\u003eNext, to assess the neuronal differentiation and survival, an additional set of brain sections were taken and washed in 1\u0026times;TBS washed thrice for 10 mins each at room temperature. Next, the antigen retrieval step was performed in which the brain sections were placed in sodium citrate buffer for two hours at 65\u0026deg;C. After antigen retrieval, the brain sections were placed in 2 normal (N) HCl (Finar, India) for 10 mins at 37\u0026deg;C on a shaker. Then the brain sections were treated with 0.1 Molar borate buffer (Boric acid (Himedia, India), pH 8.5 for 10 mins. Then the brain sections were washed thrice in 1\u0026times;TBS for 10 mins each at room temperature. After washing, the sections were incubated in 3% BSA for 1 hour on a shaker at room temperature. After blocking, two primary antibodies namely, mouse anti-BrdU (Novus Biologicals, USA; 1:100 dilution) and rabbit anti-Neuronal nuclear protein (NeuN) (Novus Biologicals, USA); 1:100 the dilution) at 4\u0026deg;C for 48 hours. Then, the primary antibodies were removed and the sections were washed thrice in 1\u0026times;TBS buffer for 10 mins. The brain sections were incubated together with two different secondary antibodies such as sheep anti-mouse Dylight 488 (Novus Biologicals, USA; 1: 500 dilution) and goat anti-rabbit Dylight 594 (Novus Biologicals, USA) antibodies at 4\u0026deg;C for 24 hours. After 24 hours, the secondary antibodies were removed and the sections were washed thrice in 1\u0026times;TBS for 10 mins each. Finally, the brain sections were placed on the double frosted slides (Borosil, India) and allowed to dry overnight in the dark. After complete drying, the sections were mounted using the Prolong Glass Antifade Mountant (Thermo Fisher Scientific, USA) and dried properly in dark. A laser-scanning confocal microscope (SM 710, Laser Scanning Confocal Microscope, Carl Zeiss, Germany) was used from the central instrumentation facility of Bharathidasan University to assess the immunofluorescence assessment. The number of BrdU-positive cells and BrdU co-localization with NeuN was analyzed from confocal z-stacks throughout the hippocampal DG. For the cell survival assessment, the number of BrdU-positive cells was counted in the entire non-overlapping confocal z-stack images of hippocampi. For the frequency of neuronal differentiation, all BrdU-positive cells in hippocampal DG from each animal were assessed, and BrdU-positive cells (green) that were co-labelled with NeuN (red) as considered double-positive cells (yellow) and calculated the percentage of newly differentiated neurons per hippocampal DG [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eEstimation Of Ache Activity In The Blood\u003c/h3\u003e\n\u003cp\u003eThe whole blood was collected in a container containing an anticoagulant, 3.2% buffered trisodium citrate (Thermofischer Scientific, USA). The blood was centrifuged at 800 rpm for 10 mins. The plasma was transferred into another sterile tube and then centrifuged at a speed of 2500 rpm for 15 mins to obtain platelet concentration. The lower 1/3rd is platelet-rich plasma (PRP) and the upper 2/3rd is platelet-poor plasma (PPP). PPP was removed and the tube was shaken to suspend the platelet pellets present at the bottom of the tube. The microplate AChE activity assay was carried out as described by Ellman et al in 1961 as soon as the PRP was obtained [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The samples were aliquoted into the wells of a microtiter plate and 0.1mM acetylthiocholine iodide (SRL, India) and 0.5 mM 5,5\u0026rsquo;-dithiobis-2-nitrobenzoic acid (DTNB) (Himedia, India) was added. AChE present in the PRP catalyzes the hydrolysis of acetylthiocholine into acetate and thiocholine. The enzyme activity of PRP was measured by the yellow colour from thiocholine when it reacted with DTNB. The plate was shaken for a few seconds and the absorbance at 412 nm was taken using UV-Vis Spectrophotometer (Synergy\u0026trade; HTX Multi-Mode Microplate Reader, Biotek, Winooski, USA).\u003c/p\u003e\n\u003ch3\u003eStatistical Analyses\u003c/h3\u003e\n\u003cp\u003eThe values have been represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Student t-test was applied to measure the statistical significance for the number of entries, duration and discrimination index in NOR test, probe test in MWM, latency and duration in cued RAM, number of DCX positive cells, length of the dendrites, percentage of BrdU/NeuN double-positive cells and AChE activity between the control and Aspirin treated groups. The escape latency to find the platform and reversal learning in the MWM test, the learning curve in cued RAM, was assessed by one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test for multiple comparisons. All the statistical analyses were made using Graph Pad Prism. The significance level was assumed at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 unless otherwise indicated.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAspirin treatment improved novel object recognition in experimental mice\u003c/h2\u003e \u003cp\u003eIn experimental animals, the time taken to explore a novel object has been considered a reflection of non-spatial memory function [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. NOR test was conducted to assess the effect of aspirin mediated on the non-spatial object memory based on the ability of the animals to distinguish the difference between a known object and a new object in an experimental task. The experimental animals that show a preference to explore a new object compared to a familiar object have been considered to have improved memory. During the training session, experimental mice were exposed to a test arena and freely allowed to explore two identical objects. In the test phase, experimental mice were exposed to the replacement of a familiar object with a novel object. Though mice in both the control and the aspirin-treated group were able to recognize the novel object, experimental mice that are treated with aspirin showed an enhanced tendency to visit the novel object zone and explore the new object more than the familiarized object compared to the mice in the control group (Control\u0026thinsp;=\u0026thinsp;3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6 vs Aspirin\u0026thinsp;=\u0026thinsp;6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83). As a result, time spent by the aspirin-treated animals in the novel object zone was significantly increased than the control group (Control\u0026thinsp;=\u0026thinsp;35\u0026thinsp;\u0026plusmn;\u0026thinsp;10 vs Aspirin\u0026thinsp;=\u0026thinsp;54\u0026thinsp;\u0026plusmn;\u0026thinsp;13). Eventually, the percentage of the discrimination index was significantly enhanced in the aspirin-treated group compared to the control group (Control\u0026thinsp;=\u0026thinsp;24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;9 vs Aspirin\u0026thinsp;=\u0026thinsp;38\u0026thinsp;\u0026plusmn;\u0026thinsp;9) thereby, indicating that aspirin treatment improved non-spatial recognition memory in experimental ageing mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAspirin Treatment Enhanced Spatial Learning And Working Memory In The Morris Water Maze Task\u003c/h3\u003e\n\u003cp\u003eThe MWM is an effective behavioural test of spatial learning and retention of memory in experimental rodents [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In the MWM task, the animals were trained to navigate from a releasing point to locate a submerged escape platform in an open water pool with the use of extra maze cues or spatial signs in the behavioural room. The spatial learning performance of experimental animals was evaluated as latency to find the hidden platform across repeated trials and memory retention was assessed by time spent by the animals in the target zone during the probe trial without the platform. In the MWM- assessment, the learning performance, and preference of experimental mice to find the platform in identifying the hidden platform were gradually improved during the training period of 14 days in both control and aspirin-treated groups. Valuation of data obtained from the learning curve indicated that the escape latency was decreased in the aspirin-treated group compared to the control group noticeably from the 5th day of training. Next, the experimental animals were challenged for the degree of memory retention during the probe test. During the probe test, the platform was removed from the MWM, and each animal was released to the pool time spent in the platform zone was measured for 1 min per single trial. The duration of search in the target quadrant by the aspirin-treated animals was found to be significantly increased than that of control animals (Control\u0026thinsp;=\u0026thinsp;14\u0026thinsp;\u0026plusmn;\u0026thinsp;4 vs Aspirin\u0026thinsp;=\u0026thinsp;27\u0026thinsp;\u0026plusmn;\u0026thinsp;8). Next, to assess the reversal learning and working memory, the platform was placed in the opposite quadrant and reversal learning was conducted for the next three days. Eventually, the time taken to find the platform in the opposite quadrant was reduced by experimental mice in the aspirin-treated group than the control group. Taken together, the MWM test indicates that the aspirin treatment prevents memory decline in experimental mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAspirin Treatment Improved Working Memory In Cued Ram\u003c/h3\u003e\n\u003cp\u003eThe use of cued RAM is highly instrumental in assessing the working memory of experimental rodents. Notably, the eight-arm radial maze with a proximal cue at the target arm enables the animals to locate flavoured feed during the training session. In the test phase, the ability of experimental animals to explore the shifted proximal cue in a new location as a strategic attempt in search of feed has been considered the degree of working memory [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the trial phase, the time spent by the experimental mice in both the control and aspirin-treated group in the cued arms were nearly similar. However, during the test phase, while the latency to enter the cue shifted arm was reduced (Control\u0026thinsp;=\u0026thinsp;207\u0026thinsp;\u0026plusmn;\u0026thinsp;70 vs Aspirin\u0026thinsp;=\u0026thinsp;232\u0026thinsp;\u0026plusmn;\u0026thinsp;37), time spent in the newly cued arm was significantly increased in the aspirin-treated group than that of the control (Control\u0026thinsp;=\u0026thinsp;22\u0026thinsp;\u0026plusmn;\u0026thinsp;10 vs Aspirin\u0026thinsp;=\u0026thinsp;38\u0026thinsp;\u0026plusmn;\u0026thinsp;3). Taken together, the cued RAM experiment validates that the aspirin treatment facilities working and declarative memory in experimental mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAspirin Treatment Increased The Hippocampal Neurogenesis\u003c/h3\u003e\n\u003cp\u003eIn the stem cell niches of the mammalian brain, the number of DCX-positive immature neurons has been considered to reflect ongoing neurogenesis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The immuno labelling study of the DCX-positive cells in the hippocampal DG revealed that the number of immature neurons was significantly increased in the aspirin-treated group than that of the control group. Further, in the morphological assessment of DCX-positive cells (Control\u0026thinsp;=\u0026thinsp;3742\u0026thinsp;\u0026plusmn;\u0026thinsp;1029 vs Aspirin\u0026thinsp;=\u0026thinsp;6087\u0026thinsp;\u0026plusmn;\u0026thinsp;1316) and the length of the dendrites (Control\u0026thinsp;=\u0026thinsp;90\u0026thinsp;\u0026plusmn;\u0026thinsp;23 vs Aspirin\u0026thinsp;=\u0026thinsp;172\u0026thinsp;\u0026plusmn;\u0026thinsp;42) was found to be increased in hippocampal DG region of the brains of the experimental mice in the aspirin-treated group than the control group. BrdU labelling of newly dividing NSCs followed by the co-expression with a mature neural mark has been a critical step in assessing the neuronal fate in the brain [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the confocal microscope-based co-immunolabelling study, the percentage of BrdU/NueN double positive cells was significantly increased in the granule cell layer of hippocampal DG regions of the brains of the experimental mice in the aspirin-treated group than the control group (Control\u0026thinsp;=\u0026thinsp;76\u0026thinsp;\u0026plusmn;\u0026thinsp;8 vs Aspirin\u0026thinsp;=\u0026thinsp;91\u0026thinsp;\u0026plusmn;\u0026thinsp;3). Considering the fact aspirin treatment appears to promote neuronal differentiation of NCS and facilitate hippocampal neurogenesis in experimental mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAspirin-treated Animals Exhibited Reduced Activity Of Ache In The Blood\u003c/h3\u003e\n\u003cp\u003eRecently, the activity of AChE in the blood has been considered a diagnostic measure to correlate memory [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The colorimetric determination of AChE activity was performed to measure the increase in the intensity of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. Results revealed that the absorbance values of AChE activity in blood samples of aspirin-treated animals were significantly reduced when compared to that of control animals (Control\u0026thinsp;=\u0026thinsp;1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 vs Aspirin\u0026thinsp;=\u0026thinsp;1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eProgressive memory loss is one of the increasing clinical concerns worldwide that pose major challenges to the ageing population, health care providers, and biomedical research sectors [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The investigation into underlying mechanisms and therapeutic targets is an ongoing quest and the identification of drugs that exert precognitive action has become an unmet need [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Aspirin is a widely used painkiller that blocks the enzymatic action of COX-2 thereby, mitigating inflammatory processes in various diseases [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Numerous experimental studies suggest that aspirin treatment modulates brain functions positively at behavioural, biochemical, and cellular levels [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Based on the animal behavioural and immunobiological assessments, the present study demonstrates that aspirin treatment improves hippocampal neurogenesis thereby ageing experimental mice enables. In response to ageing-mediated biological changes and neurodegenerative events, proinflammatory molecules are discharged from activated immune cells in the brain [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The abnormally elevated levels of proinflammatory have been known to impair the neurogenic process at the level of proliferation, and differentiation survival of NSCs leading to progressive memory impairments [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Notably, aspirin has been reported to induce the production of endogenous lipoxins, a potent anti-inflammatory molecule, which diminishes the pathogenic activation of microglia thereby reducing the load of various inflammatory molecules including C-reactive protein tumour necrosis factor (TNF)-α and interleukin \u0026minus;\u0026thinsp;6 in experimental animals [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the past few decades, the beneficial effects of aspirin treatment against cardiovascular and cerebrovascular diseases have unequivocally been established [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In recent years, the neuroprotective role of aspirin in preventing cognitive decline has also increasingly been recognized. In contrast, few randomized controlled trials and meta-analyses have stated no association between aspirin and cognitive functions. Thus, there exists an ongoing considerable ambiguity about the beneficial effects of aspirin against dementia and neurocognitive impairment [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, convincing experimental evidence supports the procognitive effect of aspirin as it appears to positively modulate hippocampal plasticity. In the experimental models challenged with glutamate excitotoxicity and hypoxia, aspirin treatment has been reported to provide neuroprotection in the brain [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Notably, the neuroprotective role of aspirin has been linked to the inhibition of NF-kB signaling in the brain [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Depeng Feng et al reported that aspirin facilitates neuroprotection in experimental animal models treated with lipopolysaccharide (LPS), and cerebral ischemia-reperfusion (CIRP) injury [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Eventually, cohort and population-based studies revealed an inverse relationship between long-term intake of low-dose aspirin and the severity of dementia [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Chandra et al 2018, reported that a low dose of aspirin decreases amyloid load through the activation of peroxisome proliferator-activated receptor alpha (PPAR)-α in the hippocampus of a 5\u0026times;FAD mouse model of memory loss [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Saima Rizwan et al reported that a low-dose aspirin treatment improves memory in the AlCl3-induced mouse model of amnesia in association with an alteration of the opioid system [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. While the circulating platelets have been recognized to contribute to amyloid pathology in the brain upon ageing, the platelet-reducing effect of aspirin has been considered to minimize the neuropathogenic events in the brain of subjects with neurodegenerative disorders [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In corroboration with the previous reports, in the present study, the aspirin-treated animals showed a better performance in NOR, MWM, and cued RAM compared to the control group. Moreover, the behaviour paradigms clearly indicated precognitive properties of aspirin were highly evident as the mice in the treatment group exhibited enhanced working memory. Aspirin-mediated memory enchantment could arise from both COX-dependent or indented pathways that positively contribute to the regulation of hippocampal plasticity.\u003c/p\u003e \u003cp\u003eConsidering the COX inhibition nature, the neuroprotective effect of aspirin treatment has been reported to be mediated via the inhibition of neuroinflammation, and mitochondrial oxidative phosphorylation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The neuroprotective property of aspirin could play a direct role in the prevention of memory decline. Moreover, aspirin has been reported to mitigate free radical production thereby mimicking the antioxidant potential in different organs including the brain [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Patel et al demonstrated that aspirin interacts with PPARα and concedes memory functions through cAMP response element-binding protein (CREB) mediated hippocampal plasticity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, aspirin treatment has been reported to be involved in the epigenetic modification upregulation of the expression of BDNF responsible for preserving the memory function in Benzo[a]pyrene (BaP) treated experimental mice [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, recent experimental data indicated that aspirin may be involved in the regenerative plasticity of the hippocampus [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. In this study, the immunohistochemical assessments of DCX-positive cells and confocal-based BrdU/NeuN revealed a concomitant increase in the neurogenic process at the level of neuronal differentiation of NSCs in the hippocampus of ageing experimental mice. Notably, dendritic arborization of DCX-positive cells was found to be increased in the hippocampal DG of aspirin-treated animals. The augmented dendritic arborization and length signifies the state of neural differentiation and integration of DCX-positive cells in the hippocampal circuit [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Recently, Giacomo Pozzoli et al reported that aspirin promotes the differentiation of SK-N‐SH (N) human neuroblastoma cells through the modulation of the expression of cell cycle checkpoint markers p21\u003csup\u003eWaf1\u003c/sup\u003e and Rb1 independent of the COX pathway [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Considering the facts, it can be presumed that the procognitive effect of aspirin might be resulting from the neuronal differentiation of NSCs augmenting the new neurons in the hippocampus [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNotably, regulation of neurogenesis in the hippocampus has been linked to levels of some neurotransmitters that are crucial for learning and memory. Acetylcholine (ACh) is one of the key neurotransmitters responsible for cognitive function [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Abnormal levels of ACh in the circulation and brain has been identified as a cause of neurocognitive impairments [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. As age increases, a decline in the level of ACh is evident in the circulation and the brain [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Activation of certain enzymes responsible for biochemical pathways of ACh synthesis appears to result in abnormal levels of ACh. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the rate-limiting enzymes that breakdowns ACh into choline and acetate [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. However, AChE appears to break down ACh more than BuChE to a greater extent as BuChE has a lesser affinity for ACh [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Remarkably, increased activities of AChE leading to reduced levels of ACh have been identified as an underlying pathological determinant of cognitive impairments and memory loss in various neurocognitive diseases including AD [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Eventually, increased levels of AChE in the blood have been considered a biomarker of dementia [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Thus, the implementation of AChE inhibitors has been considered to mitigate memory loss in aging and AD [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Recently, few COX inhibitors have been known to diminish the activities of AChE [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Notably, bioinformatics-based docking studies revealed that aspirin binds to the active sites of AChE and acts as a potential inhibitor of its activities [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Ample reports suggest that inhibition of AChE facilitates hippocampal neuroregenerative plasticity in the adult brain [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Considering the aforementioned factors, it can be hypothesized that aspirin treatment might play a role in the regulation of hippocampal regenerative plasticity responsible for memory enhancement in association with the inactivation of AChE. Therefore, this study supports the procognitive and proneurogenic effects of aspirin which can be considered to translate for the treatment regimens to boost neuroregenerative plasticity noticed during various disease conditions with neurocognitive impairments.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eConsumption of aspirin, a potent COX inhibitor has been reported to yield diversified results with reference to cognitive functions. The present study demonstrates that aspirin treatment enhances working memory in ageing experimental mice. This study revealed that aspirin promotes neuronal differentiation in the hippocampus in correlation with a reduction in AChE activities in circulation. Considering the facts, better pattern recognition, spatial working memory, and declarative memory functions noticed in the animal in the aspirin treatment group could be largely due to enhanced neuroregenerative plasticity in the hippocampus despite its neuroprotective roles. This study supports the memory-enhancing capacity of aspirin and necessitates further studies to reveal the mechanisms reasonable for pro-neurogenic effects in the brain\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data will be made available from the corresponding author upon reasonable request.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Early Career Research Award (SERB-ECR/2016/000741) from the Science and Engineering Research Board (SERB) under the Department of Science and Technology (DST), Government of India. The authors also thank the research grant (SERB-EEQ/2016/000639) from the Science and Engineering Research Board (SERB) under the Department of Science and Technology (DST), Government of India for the financial support. Dr. Mahesh Kandasamy has been supported by University Grants Commission\u0026ndash;Faculty Recharge Programme (UGC-FRP), New Delhi, India \u0026nbsp;Dr. Anusuyadevi Muthuswamy would like to thank ICMR, New Delhi, India (2019-2605/CMB/Adhoc/BMS) and DST, New Delhi, India (DST/CSRI/2018/343(G)) for the financial support. Authors also thank RUSA 2.0, Biological Sciences, Bharathidasan University for their financial support (TN RUSA: 311/RUSA (2.0) / 2018 dt. 02/12/2020). Divya Bharathi is the recipient of the RUSA 2.0 project fellowship (Ref. No. BDU/RUSA 2.0/TRP/BS/Date 22/04/2021). Jemi Feiona was supported as a project assistant from the project grant SERB EEQ/2016/000639. \u0026nbsp;The authors would like to acknowledge UGC-SAP and DST-FIST for the infrastructure of the Department of Animal Science and Biochemistry, Bharathidasan University. Authors extend acknowledgment to the University Science Instrumentation Centre (USIC)-BDU \u0026amp; DST-PURSE (Phase 1 \u0026amp; 2) for the confocal analysis. The authors would like to thank Dr. Muthu Kumar, USIC-BDU for the excellent technical support in the confocal microscopy. We express sincere thanks to Dr. R. Thirumurugan, Professor and Head, the Department of Animal Science for providing the facility to utilize the Multimode Plate Reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConflict of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMK conceived and designed the experiment. JFAV, DBS, AK, and MK conducted the experiments and analyzed the data. MK \u0026nbsp;JFAV, DBS, AK, and MA wrote, reviewed, and edited the manuscript. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGhlichloo I, Gerriets V (2022) Nonsteroidal Anti-inflammatory Drugs (NSAIDs). In: StatPearls. 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Journal of the Neurological Sciences 347:66\u0026ndash;77. https://doi.org/10.1016/j.jns.2014.09.021\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aspirin, memory, hippocampus, doublecortin, adult neurogenesis, Morris water maze","lastPublishedDoi":"10.21203/rs.3.rs-2789201/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2789201/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAspirin treatment is the most widely used preventive measure against cardiovascular diseases. Aspirin is also expected to provide beneficial effects on the brain. However, the association between aspirin treatment and neurocognitive functions is a subject of debate. Ample reports strongly advocate that a mild dose of aspirin positively modulates hippocampal plasticity responsible for memory. Aspirin is a selective cyclooxygenase (COX)-2 inhibitor but the underlying mechanism through which aspirin modulates neuroplasticity remains unclear. Adult neurogenesis in the hippocampus has been established as an underlying basis of learning and memory. Therefore, aspirin treatment might be linked to the regulation of hippocampal neurogenesis. Thus, this study revisited the effect of low-dose aspirin on learning and memory in correlation with the regulation of hippocampal neurogenesis in the brains of ageing experimental mice. Results from the novel object recognition (NOR) test, Morris water maze (MWM), and cued radial arm maze (cued RAM) revealed that aspirin treatment enhances working memory in experimental ageing mice. Further, the co-immunohistochemical assessments on the brain sections indicated an increased number of doublecortin (DCX) positive immature neurons and bromodeoxyuridine (BrdU)/neuronal nuclei (NeuN) double-positive newly generated neurons in the hippocampi of mice in aspirin-treated group compared to the control group. Recently, enhanced activity of acetylcholinesterase (AChE) in circulation has been identified as an indicative biomarker of dementia. The biochemical assessment in the blood of aspirin-treated mice showed decreased activity of AChE than that of the control group. This study supports the procognitive effects of aspirin which can be translated to treat dementia.\u003c/p\u003e","manuscriptTitle":"A mild dose of aspirin promotes hippocampal neurogenesis and working memory in experimental ageing mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-11 18:28:19","doi":"10.21203/rs.3.rs-2789201/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68be43df-f05f-41cd-ad22-5d567050b013","owner":[],"postedDate":"April 11th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-04-11T18:28:22+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-11 18:28:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2789201","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2789201","identity":"rs-2789201","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}