Chronic D-Galactose Accelerates Aging-Like Decline in Aerobic Capacity and Cognitive Function Through Inflammation and Oxidative Stress in Central and Peripheral Tissues

Chronic D-Galactose Accelerates Aging-Like Decline in Aerobic Capacity and Cognitive Function Through Inflammation and Oxidative Stress in Central and Peripheral Tissues | 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 Chronic D-Galactose Accelerates Aging-Like Decline in Aerobic Capacity and Cognitive Function Through Inflammation and Oxidative Stress in Central and Peripheral Tissues Daniel Massote de Melo Leite, Bruno Pereira Melo, Maicon Arcanjo da Silva, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8369611/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 Introduction: D-galactose (D-GAL) is an aldohexose naturally present in the body and diet; however, chronic exposure can be deleterious to health and promote aging-related changes. Thus, the effects of D-GAL on aerobic capacity and cognitive function based on central and peripheral hallmarks remain unclear. This study investigated the effects of chronic D-GAL administration on physical performance, memory, inflammatory markers, and oxidative stress in the brain and skeletal muscle of rats. Methods: Male Wistar rats received D-galactose (150 mg·kg⁻¹·day⁻¹) administered intraperitoneally for eight weeks. Muscle strength was assessed using a vertical ladder test, whereas aerobic capacity was evaluated by treadmill testing at weeks 4 and 8. Memory was analyzed using the Novel Object Recognition Test. Biochemical analyses were performed in central and peripheral tissues, including the soleus, extensor digitorum longus (EDL) muscles, hippocampus, and frontal cortex. Results: D-galactose reduced aerobic performance, evidenced by decreased VO₂peak (p = 0.044) and work output (p = 0.011) at week 4, with a further decline in work output at week 8. Chronic D-galactose also impaired long-term memory. Oxidative stress increased, with elevated hydroperoxides in the EDL (p = 0.010) and hippocampus (p = 0.040), and higher TBARS levels in the hippocampus (p = 0.020). Inflammatory modulation was observed, with reduced IL-10 levels in the soleus (p = 0.040), hippocampus (p < 0.001), and frontal cortex (p = 0.044). Conclusion: Chronic D-galactose–induced aging impairs aerobic capacity and memory and is associated with increased oxidative stress and inflammation in skeletal muscle and brain tissue overall in rats. Aerobi capacit decline D-Galactose-induce aging Neuroinflammation Oxidativ stress Recognitio memor impairment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The aging process induces a range of irreversible physiological changes, leading to molecular, cellular, and tissue damage, These effects are particularly evident in the brain, where aging is associated with neurodegeneration of the central nervous system and progressive functional decline, resulting in reduced muscular strength, aerobic capacity, and cognitive function (Garatachea et al. 2015), (Isaac et al. 2021). Scientific evidence has indicates that aging is linked to an increased expression of inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 1 beta (IL-1β), and tumoral necrosis factor alpha (TNF-α) in both, brain (Bellettini-Santos et al. 2023) and skeletal muscle (Yanar et al. 2011). This phenomenon, termed "inflammaging" refers to the chronic, low-grade inflammation associated with aging (Franceschi et al. 2018). It is believed to result from a combination of factors, including cellular senescence, immune system dysregulation , and the accumulation of molecular damage over time (Franceschi et al. 2018), (Santoro et al. 2021). This persistent inflammatory state disrupts tissue homeostasis, impair cellular function, and contributes to the progression of age-related conditions such as cardiovascular diseases, neurodegeneration, and metabolic disorders (López-Otín et al. 2013), (Franceschi et al. 2018). Aging-related diseases are driven by multiple interrelated mechanisms, including: 1) genomic instability, 2) telomere attrition, 3) epigenetic alterations, 4) loss of proteoastasis, 5) dysregulated nutrient sensing, 6) mitochondrial dysfunction, 7) cellular senescence, 8) stem cell exhaustion, and 9) altered intercellular communication (Pantiya et al. 2023). Additionally, oxidative stress plays a key role in aging, characterized by an imbalance between reactive oxygen species (ROS) production of and the capacity of antioxidant defense mechanisms to neutralize them (Cui et al. 2006), (Ayala et al. 2014). This imbalance results in cumulative damage to lipids, proteins, and nucleic acids, exacerbating age-related pathologies such as neurodegenerative disorders, musculoskeletal deterioration and cancer (Garatachea et al. 2015) (Sadigh-Eteghad et al. 2017). Despite significant advancements, the precise etiology of the aging process remains incompletely understood (López-Otín et al. 2013). To explore the cellular and molecular mechanisms underlying aging, , animal models- primarily rodents- have been widely used in laboratory studies (Sadigh-Eteghad et al. 2017), (Azman and Zakaria 2019). However, longitudinal aging studies require long-term maintenance of laboratory animals, which is both costly and time-consuming, considering the lifespan of rodents (~ 24-30 months) (Alejandro 2022), (Cebe et al. 2014). To adress this limitation, aging models based on chronic administration of D-galactose (D-GAL) have been developed (Sadigh-Eteghad et al. 2017), (Azman and Zakaria 2019), (Lin et al. 2020). D-GAL is a naturally occurring aldohexose sugar found in the body and in many foods, such as dairy products and some fruits (Acosta and Gross 1995). However, excessive D-GAL exposure leads to its conversion into aldose and hydroperoxides via galactose oxidase, generating ROS and promoting oxidative stress (Azman and Zakaria 2019). Chronic D-GAL administration induces aging-like characteristics in multiple peripheral tissues, including the heart (Alejandro 2022), liver (Azman et al. 2021), bone (Partadiredja et al. 2019), pancreas and kidney (El‐far et al. 2020). Additionally, chronic D-GAL exposure has been shown to impair memory, with some studies reporting also reduced locomotor performance (Belviranlı and Okudan 2019) while others show significant changes with wheel exercise (Lee et al. 2019). Although aging is known to impair mitochondrial energetics and reduce cardiorespiratory fitness (Mau et al. 2023), the extent to which D-GAL induced-aging recapitulates these hallmarks has not been fully elucidated. Moreover, although the mimetic aging model induced by D-galactose (D-GAL) is well characterized in the literature, the molecular mechanisms underlying the damage caused by D-GAL to peripheral tissues and the central nervous system remain poorly understood. Therefore, this study aimed to evaluate the effects of chronic D-GAL administration on aerobic fitness, oxidative stress, and inflammatory markers in the brain and skeletal muscle, as well as its impact on short and long-term memory in male Wistar rats. 2. Material and Methods 2.1 Animals and Ethics Care Thirty male Wistar rats (220 g, approximately 8 weeks old) were used in the experiments. All experimental procedures were approved by the Ethics Committee Care and Use of Laboratory Animals of the Federal University of Minas Gerais (CEUA – UFMG, protocol number 161/2020) and were conducted following the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The rats were housed in collective cages under controlled temperature (24ºC) and light conditions (12/12 hours) during all experimental study. 2.1.1 Experimental design The rats were habituated to the laboratory environment for seven days. The rats were weighed through a digital scale (FILIZOLA®), randomized, and stratified by body mass into two experimental groups: Control (CON; 327.7 ± 29.7 g; n = 15) and D-galactose (D-GAL; 332.7 ± 32.8 g n = 15). After, both groups were submitted to the Novel Object Recognition Test (NOR) to assess short-term memory (STM) and long-term memory (LTM) for three consecutive days (Bevins and Besheer 2006). After the task, the groups were familiarized with climbing a vertical ladder during three days, and with running on a treadmill for five consecutive days. The rats were subjected to the Maximal Weight Carried Test (MWC) (Novaes Gomes et al. 2014), (Magalhães et al. 2022), and the incremental running test (Prímola-Gomes et al. 2009) to assess the physical performance in terms of maximum strength and aerobic capacity, respectively. The rats allocated into the D-GAL group were submitted to an eight-week aging induction protocol through the administration daily of the intraperitoneal D-galactose dissolved in saline solution (0.9%), while the rats allocated into CON group received only saline solution (Lin et al. 2020). After the aging protocol, the rats of both groups were submitted again to NOR test to assess STM and LTM memory, and physical performance (strength and aerobic capacity test) to investigate the possible deleterious effects of D-galactose aging-associated (Figure 1). The rats were euthanized by decapitation 48 hours after the end of the experimental protocol, the brain and peripheral tissues were removed, weighed, and stockage into -80°C for biochemical analysis. All experimental procedures were performed between 07:00 am and 5:00 pm. There was a sample loss in the D-GAL group, totaling (n = 14). For the memory data, one rat from the D-GAL group and two rats from the CON group did not explore the objects in the training phase and were excluded from the analysis, totaling (n = 13) for each of the groups. For aerobic capacity, there was a sample loss in the D-GAL group (n = 13). For the analysis of relative muscle strength results, TBARS, lipid hydroperoxides, and cytokines, (n = 8) were used as indicated in the graphics results. 2.1.2 Model Aging The model of aging was established through the administration of 150 mg/kg/day of D-galactose powder (Sigma Aldrich, St. Louis, MO, USA, G0750), dissolved in 0.9% saline solution (100 mg . ml -1 ), applied daily via intraperitoneal injection during 8 weeks (Lin et al. 2020). 2.1.3 Novel Object Recognition (NOR) The NOR task is a consolidated test in the literature to assess short and long-term memory (Bevins and Besheer 2006), (Crunfli et al. 2019). It was performed in four stages during three consecutive days. On the first day, familiarization with the arena was performed and consisted of positioning the rats in the center of a square box (70 cm x 70 cm x 40 cm) during free five-minute exploration. On the second day, the training phase was performed. Two identical objects (A) were placed parallel to each other at the ends of the box for free exploration during 10 minutes. Sixty minutes after training phase, short-term memory (STM) was assessed for 5 minutes, with a new object (B) replacing a familiar object (A). Twenty-four hours late (on the third day), long-term memory (LTM) was assessed using a new object (C) in place of object (B) for 5 minutes. At each assessment, the arena and objects were cleaned with 70% alcohol to avoid olfactory cues. Any Maze software was used to analyze the results (ANY Maze Video Tracking System 7.16; STOELTING CO. ©, 2022). The discrimination index was calculated, which consists of the time taken to explore the new object (TN) divided by the sum of the exploration of the new object and the familiar object (TN + TF) (Bevins and Besheer 2006), (Rojas et al. 2013). 2.1.4 Vertical ladder familiarization The rats were familiarized with a vertical ladder inclined at 80º (110 cm x 18 cm, with 2 cm between the steps). The vertical ladder familiarization protocol consisted of three trials per day for three days. Initially, the rats were kept in the housing chamber for 60 seconds and then placed on the ladder at 35 cm, 55 cm and 110 cm from the top. Between each trial the animals remained in the housing chamber for 60 seconds (Cassilhas et al. 2012). 2.1.5 Maximal weight carried test (MWC) The maximum weight carried test consisted of the rat initially climbing with a load corresponding to 75% of its body mass attached to the proximal part of the tail, into 50 mL Falcon tubes with fishing weights attached to a plastic-coated steel cable secured by a rubber band (Scotch 3 M). For each successful climb, the rat remained in the ladder housing for 60 seconds and a weight of 30 grams was added. Failure was determined when the rat was unable to continue climbing even after 3 successive gentle stimuli on the tail (Novaes Gomes et al. 2014), (Magalhães et al. 2022). The results were analyzed using the relative maximum load according to the following equation: MWC/BM, where MWC is the maximum load obtained in the strength test and MC, the body mass in kilograms. 2.1.6 Treadmill running familiarization The familiarization protocol for running on a treadmill (Panlab/Havard Apparatus, Cornella Spain) was carried out over five consecutive days for 5 minutes a day at an incremental velocity adjusted every minute (10, 10, 11, 13 and 15 m.min- 1 ) with a 5% incline and an electrical stimulus set at 0.28 mA (Prímola-Gomes et al. 2009). 2.1.7 Incremental running test The incremental treadmill running test (Panlab/Harvard Apparatus) was started at a velocity of 10 m . min -1 , with progressive increments of 1 m . min -1 every 3 minutes. The incline and electrical stimulation were similar to the familiarization protocol. The test was stopped when the animal remained on the electrical stimulation grid for 10 seconds. Peak oxygen consumption (VO 2 peak ) was analyzed using open-flow indirect calorimetry (Gas Analyzer Panlab/Havard Apparatus, Cornella Spain). Aerobic performance was assessed by VO 2 peak and workload (J). The highest VO 2 peak values obtained during the incremental test were taken into account when analyzing the results. Workload was calculated using the equation: = bm × g × s × sinθ × t, where MC is the animal's body mass in kilograms; g: The acceleration of gravity (9.8 m . s -2 ); v: The velocity of the treadmill in meters per minute; sin θ: The angle of inclination of the treadmill (5º) and t: The time spent on each stage in minutes (Soares et al. 2019), (Melo et al. 2022). 2.1.8 Euthanasia and Adiposity index The rats were euthanized by decapitation, without prior sedation, 48 hours after the experimental protocol. The hippocampus, frontal cortex, soleus muscle and extensor digitorum longus muscle (EDL) tissues were removed and stored at -80 °C for later analysis. The retroperitoneal (TAR), mesenteric (TAM) and epididymal (TAE) adipose tissues were removed completely and weighed on a scale (SHIMADZU® Model BL320H, precision 0.001g) to determine the adiposity index (IA%), using the following equation: Adiposity index = (TAR + TAM + TAE) / MC x 100, where MC = Total body mass. 2.1.9 Biochemical analyses - Oxidative stress Approximately 50 mg was taken from brain and skeletal muscles to assess oxidative stress parameters. The fragments were homogenized with ice-cold 1x PBS and centrifuged at 12.000 rpm for 10 minutes. The supernatant was separated for analysis. 2.1.10 Evaluation of lipid peroxidation by thiobarbituric acid reactive substances (TBARS) Thiobarbituric acid-reactive metabolites were measured by adding a solution containing trichloroacetic acid (TCA 15%), thiobarbituric acid (TBA 0.0375%) and hydrochloric acid (HCl 0.25 N) to the organ supernatants. The samples were kept in a boiling water bath for 15 minutes and then cooled. After adding butyl alcohol, the tubes were shaken vigorously. The samples were centrifuged at 3.000 rpm for 10 minutes. 200μL of the supernatant was added to the 96-well plate. The absorbance was measured spectrophotometrically at a wavelength of 535 nm, and the results were normalized by the concentration of protein in the hippocampus, frontal cortex and soleus and EDL muscles (LOWRY et al. 1951). 2.1.11 Evaluation of hydroperoxide concentration This test was carried out using part of the solution prepared by dissolving xylenol orange and ammoniacal ferrous sulphate in H 2 SO 4 diluted in methanol solution containing BHT (butylated hydroxytoluene). For the dosages, the organ supernatants (20 μL each) were added to 180μL of the aforementioned solution, directly into the microplate, in triplicate. The blank was achieved by using phosphate buffer instead of the supernatant. The samples were then kept at room temperature for 30 minutes and the absorbance was measured spectrophotometrically at a wavelength of 560nm. The concentration of hydroperoxides was estimated by the extinction coefficient of hydroperoxides, 4.3 x 10-4M-1cm-1, and by the extinction coefficient of the bluish- purple chromophore produced by xylenol orange when it binds to ferric ions, 1.5 x 10- 4M-1cm-1. The quantification of hydroperoxides in the sample was achieved without TPP and the result was normalized by the protein concentration of each fragment (LOWRY et al. 1951). 2.1.12 Protein dosage The protein concentration in the organ supernatant was measured according to Lowry et al. (1951) (LOWRY et al. 1951). To do this, 250 μL of diluted sample (1:50) was added to 250μL of solution A (one-part copper sulphate, one-part sodium tartrate and 100 parts sodium carbonate) and 25 μL of diluted Folin-Ciocalteau reagent (1:2). The samples were then vortexed and incubated at room temperature for 30 minutes. The samples (200 μL) were then added to the 96-well plate and the absorbance was measured spectrophotometrically at a wavelength of 660 nm. The results were expressed in mg . mL -1 after obtaining the formula from the standard curve made with albumin. 2.1.13 Pro-inflammatory and anti-inflammatory cytokines The concentrations of interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) were assessed in the hippocampus, frontal cortex, soleus, and EDL muscle by means of ELISA (R&D Systems, Inc., Minneapolis, MN, USA; DuoSet kits DY506, DY522, DY501, and DY510, respectively). The manufacturer's recommendations were followed. The cerebral areas were chosen because of their relationship with learning and memory (hippocampus), attention and decision- making (frontal cortex). The muscles were selected according to their metabolic characteristics: soleus muscles because they are related to aerobic performance, with a predominance of oxidative type I fibers (slow-twitch fibers), and the EDL muscles , with predominantly glycolytic type IIa and IIb fibers (fast-twitch fibers) because of their relationship with muscle strength in rodents (Holeček and Mičuda 2017). 2.1.14 Statistical analyses The normality of the data and the homoscedasticity of the residuals were assessed using the Shapiro-Wilk and Levene tests respectively. Data are expressed as mean ± standard error of the mean. Aerobic performance, maximal muscle strength, and short- and long-term memory variables were compared using mixed two-way ANOVA, considering the factors groups (CON vs. D GAL) and time (Before vs. 4th week vs. 8th week). When significant differences were found, Bonferroni post hoc and/or Student's t-test were performed, considering the mean coefficient of variation of the two groups (CON vs. D-GAL) at the three experimental time points. To compare body composition, cytokine concentrations, and markers of oxidative stress between groups (CON vs. D GAL), Student's t-test was used. Pearson's correlation was used to investigate association between TBARS concentrations, and their association with long-term memory in hippocampus. All analyses were performed using R software (version 4.2.2, USA) with Rstudio interface (version 2023.06 + 421). The significance level adopted was 5% (p ≤ 0.05). 3. Results 3.1 Body composition and adiposity index Body weight increased (F 2.54 = 445.55; p < 0.001) similarly in CON and D-GAL groups in the 4- and after 8 weeks of intervention (Figure 2a). When compared to the CON group, D-GAL administration did not alter the adiposity index (4.96 ± 037 vs. 5.03 ± 0.27 %; t 27 = 0.15; p = 0.880; Figure 2B), soleus muscle mass (0.14 ± 0.01 vs. 0.16 ± 0.01 g; t 14 = 1.063; p = 0.305; Figure 2C), and either EDL muscle mass (0.20 ± 0.01 vs. 0.21 ± 0.01 g; t 14 = 0.285; p = 0.779, Figure 2D). 3.1.2 Physical Performance The results of physical performance are expressed in Figure 3a-c. Relative muscle strength increased (F 2.28 = 5.70; p = 0.008) similarly in CON and D-GAL groups in the 4- and after 8 weeks of intervention (Figure 3A). The aging D-GAL-induced administration reduced the aerobic capacity and attenuated the workload after 8 weeks of the intervention. Compared to baseline, D-GAL administration reduced 11.6% the VO 2 peakin 4- (p = 0.008), and 12.2% after 8 weeks ( p = 0.003; Figure 3B). An ANOVA-mixed time effect was found in the workload (F 2.54 = 4.542; p = 0.015; Figure 3C). When compared to the CON group, the aging rats showed the lowest values of VO 2 peak (53.58 ± 1.81 vs. 48.46 ± 1.56 ml . kg -1. min -1 ; p = 0.044; Figure 3b) and, workload (327.61 ± 23.86 vs 248.32 ± 15.41 Joules; p = 0.011; Figure 3C) after 4-week, and also workload (320.86 ± 23.86 vs 268.32 ± 15.24 Joules; p = 0.041; Figure 3C) after 8-week of D-GAL administration. Moreover, the aging D-GAL-induced administration attenuated the workload of the aerobic exercise. The CON group increased 26.8% ( p = 0.005) of the workload in 4-, and 24.2% ( p = 0.005) after 8 weeks, however, this response was not observed in aging rats (Figure 3C). 3.1.3 Skeletal Muscle Analysis The effects of aging on oxidative stress were observed in the skeletal muscle. D-GAL administration increased significantly (t 12 = 2.949; p = 0.01) hydroperoxide levels in the EDL muscle after 8 weeks of administration (Figure 4a). No significant differences were observed in cytokines pro- or anti-inflammatory in the EDL muscle (Figure 4B-E). In the soleus muscle, the aging D-GAL-induced administration reduced IL-10 concentrations (t 13 = 2.232; p = 0.04; Figure 5D), and increased IL-1β/IL-10 ratio (t 12 = 2.679; p = 0.010; Figure 5E), demonstrating a pro-inflammatory milieu in this skeletal muscle. No significant differences were observed in cytokines pro-inflammatory (Figure 5B, C), and either oxidative stress (Figure 5A). 3.1.4 Recognition memory The effects of aging D-GAL-induced administration on recognition memory are expressed in Figures 6A-B. The D-GAL administration promoted cognitive impairment after 8- weeks of the intervention. A mixed ANOVA analysis demonstrated a significant effect of time (F 1.24 = 18.554; p < 0.001) and group (F 1.27 = 11.077; p = 0.002) in the short-term memory recognition index (STM; Figure 6A). Compared to the baseline, the aging rats reduced ( p < 0.001; Figure 6b) the recognition index of the long-term memory test (LTM), demonstrating a cognitive decline in long-term memory. Moreover, compared to the CON group, the aging rats showed the lowest values ( p < 0.001; Figure 6B) on the LTM recognition index after 8- weeks. 3.1.5 Hippocampus analysis The results of hippocampus analysis are expressed in Figures 7A-H. The aging D-GAL-induced increased oxidative stress on the hippocampus and, it was associated with impairment of long-term memory. Compared to the CON group, D-GAL administration increased TBARS (t 13 = 3.673; p = 0.020; Figure 7A), and hydroperoxides levels (t 9 = 2.399; p = 0.040; Figure 7B) on the hippocampus after 8-weeks of the intervention. A significant, and inverse correlation (r = -0.773; p < 0.001; r 2 = 0.597; Figure 7H) was found between TBARS levels and the LTM recognition index, demonstrating the influence of oxidative stress on memory loss. Compared to the CON group, the D-GAL administration also reduced TNF-α (t 14 = 3.262; p < 0.011; Figure 7C), and IL-10 (t 14 = 3.005; p < 0.001; Figure 7F) concentrations in the hippocampus after 8- weeks of the intervention. No significant difference was found in the IL-6 (t 14 = 1.780; p = 0.092; Figure 7d), IL-1β (t 12 = 0.3766; p = 0.714; Figure 7E) concentrations, and IL1β/IL-10 ratio (t 13 = 1.326; p = 0.1038; Figure 7F). 3.1.6 Frontal cortex analysis The results of the frontal cortex analysis are expressed in Figures 8A-F. Compared to the CON group, D-GAL administration increased hydroperoxide levels (t 12 = 2.179; p = 0.049; Figure 8B) in the frontal cortex after 8-weeks of the intervention. No significant difference was found in the TBARS concentrations (t 10 = 0.9037; p = 0.384; Figure 8A). When analyzing the cytokines pro- and anti-inflammatory, the aging rats showed the lowest values (t 14 = 2.224; p = 0.044; Figure 8E) of the IL-10 concentration compared to the CON group, in the frontal cortex after 8- weeks. No significant difference was found in the TNF-α (t 14 = 1.046; p = 0.314; Figure 8C), and IL-1β (t 11 = 1.754; p = 0.107; Figure 8D). The aging rats also showed higher values in the IL-1β/IL-10 ratio (t 10 = 4.840; p < 0.001; Figure 8F) when compared to the CON group. 4. Discussion The mimetic aging model induced by D-galactose (D-GAL) is well characterized in the literature; however, the molecular mechanisms underlying the damage caused by D-GAL to peripheral tissues and the central nervous system remain poorly understood In the present study, chronic administration of D-GAL decreased aerobic capacity and impaired short- and long-term memory, without effects on muscle strength. Furthermore, we demonstrated that chronic D-GAL administration increased oxidative stress and inflammatory cytokines in central and peripheral tissues, including the hippocampus, frontal cortex, EDL and soleus. Since these functional and physiological responses are considered key hallmarks of aging, our results emphasize that chronic D-GAL administration may serve as a valid aging model with ecological relevance to older adults. In humans, experimental and review studies have shown that aging is associated with a reduction in VO 2peak (Shephard 2009),(Letnes et al. 2023), arteriovenous difference (Schrage et al. 2007), reduced cardiac output (Betik and Hepple 2008), mitochondrial biogenesis (Betik and Hepple 2008), muscle blood flow, and increased peripheral resistance (Lakatta and Levy 2003), leading to endothelial dysfunction (Schrage et al. 2007) and lower muscle oxidative capacity (Betik and Hepple 2008), which impairs the performance of daily tasks (Chodzko-Zajko et al. 1998),(Pandey et al. 2020). In rodent aging models, a few studies have assessed the ability to perform physical exercise, either through locomotor activity in open-field and Morris water maze behavioral tests (Partadiredja et al. 2019), (Xinghua et al. 2023), (Zhang et al. 2023), or by voluntary wheel running (Belviranlı and Okudan 2019),(Lee et al. 2019). However, the maximum oxidative capacity of these animals has not been evaluated. To our knowledge, this is the first study to demonstrate that D-GAL administration reduces maximal aerobic physical capacity in an experimental aging model. Muscle changes that occur with aging have been linked to a decline in aerobic capacity, often leading to a loss of strength, reduced functional ability, sarcopenia, and mitochondrial dysfunction in the skeletal muscles of older adults (Fan et al. 2016).In the present study, muscle strength increased similarly over time in both groups (D-GAL and CON), with no significant differences between them, indicating that the administration of 150 mg·kg⁻¹·day⁻¹ of D-galactose did not reduce muscle strength. In contrast Chang et al. (Chang et al. 2014) and Wu et al. (Wu et al. 2022) observed a decrease in muscle strength in mice experimentally aged with D-GAL, assessed via the forelimb grip strength test (GRIP test), which is commonly used to indicate neuromuscular alterations. The reduction in strength observed in their studies (with doses of 125 and 100 mg·kg⁻¹·day⁻¹, administered subcutaneously and intraperitoneally for 8 and 6 weeks, respectively) was associated with morphological changes and increased mitochondrial dysfunction in complex I of the gastrocnemius muscle (Chang et al. 2014), as well as elevated levels of malondialdehyde and inflammatory cytokines such as IL-6 and TNF-α (Wu et al. 2022). We used a maximum load test on a vertical ladder to assess muscle strength (Novaes Gomes et al. 2014), (Magalhães et al. 2022). It is possible that this test was not sensitive enough to detect changes in muscle strength. Previous studies in rats have shown that chronic D-GAL administration increases oxidative stress and mitochondrial dysfunction in the gastrocnemius muscle (glycolytic) (Kou et al. 2017) and the soleus muscle (oxidative) (Yanar et al. 2019). Damage to the unsaturated lipids in cell membranes, mediated by the oxidative action of reactive oxygen species (ROS), results in lipid peroxidation and the formation of lipid hydroperoxides, which generate free radicals that induce cytotoxicity, inflammation, and structural changes in various cells (Ayala et al. 2014). In the present study, rats aged with D-GAL exhibited a significant increase in the concentration of lipid hydroperoxides in the EDL, a glycolytic muscle. Previous studies have shown that aging, in both humans and rodents, can lead to increased oxidative stress in glycolytic muscles, which are characterized by, as predominance of type II muscle fibers, and low mitochondrial content. Oxidative damage has been observed in the vastus lateralis of elderly individuals (Lexell et al. 1988), apoptosis in the superficial vastus lateralis of rats (Phillips and Leeuwenburgh 2005), and mitochondrial dysfunction in the gastrocnemius of mice treated with D-GAL (Chang et al. 2014). However, studies evaluating oxidative stress through hydroperoxides in the EDL muscle in D-GAL-induced aging models are scarce. We expected the soleus muscle to exhibited an increase in lipid peroxidation (compared to the CON group) due to its higher mitochondrial content and greater oxidative stress production from oxidative metabolism (Holeček and Mičuda 2017). Previous findings have shown that D-GAL, administered at a dose of 60 mg·kg⁻¹·day⁻¹ via intraperitoneal injection for 6 weeks in rats, increased lipid hydroperoxide concentrations in in the soleus muscle, resulting in mitochondrial dysfunction, increased carbonylated protein content (a marker of oxidative damage), and impaired of muscle redox homeostasis (Yanar et al. 2019). Although we did not observe changes in the hydroperoxide concentration chronic D-GAL administration in the present study also reduced IL-10 concentrations in the soleus muscle (a muscle with slow-twitch fibers) inducing a decrease in the anti-inflammatory capacity of this tissue. The reduction of IL-10 in skeletal muscle as a result of the aging has been linked to greater macrophage activation´s, antigen release, and inflammatory cytokine production (Hacham et al. 2004), (Moore et al. 2001),(Mittal and Roche 2015),(Dagdeviren et al. 2017). In line with our results, higher concentrations of IL-10 in blood serum have been directly correlated with better aerobic fitness (VO 2max ) in the elderly (Rosado-Pérez and Mendoza-Núñez 2018). Additionally, when analyzing the pro-inflammatory ratio between IL-1β / IL-10 in the soleus muscle, we found an inflammatory milieu in this tissue. The chronic low-grade pro-inflammatory state observed in aging may be explained by the increased activation of the transcription factor NF-kβ, which regulates the production of pro-inflammatory cytokines activated by increased oxidative stress, impaired autophagy, DNA damage and the accumulation of senescent cells in various tissues (Bektas et al. 2017),(Franceschi et al. 2018). Regarding central responses, our study demonstrated that short- and long-term memory were negatively affected by chronic D-GAL administration. These effects may be associated with increased oxidative stress and an inflammatory environment in the hippocampus and frontal cortex, suggesting that chronic D-GAL administration induces an ecological model of neurodegeneration linked to aging. The effects of different doses and duration of chronic D-GAL administration on cognitive and memory performance have been explored in the literature.-GAL administration at a dose of 150 mg.kg -1 .day -1 for 8- (Duan et al. 2017),(Zhang et al. 2021), 6- (Sun et al. 2018) or 10 weeks (Nam et al. 2019) induced cognitive impairment and memory loss, as assessed by the object recognition test. The hippocampus and frontal cortex are structures involved in neurogenesis, memory and learning (Garcez et al. 2021). Oxidative stress and inflammation, have been identified as the primary physiological mechanism underlying memory impairment and neurodegenerative diseases. In the present study, we showed that chronic D-GAL administration increased TBARS and lipid hydroperoxide concentrations in the hippocampus and lipid hydroperoxide in the frontal cortex underscoring the effects of oxidative stress on aging-associated neurodegeneration. These findings corroborate the literature, which has shown higher hippocampal TBARS and MDA concentrations in rats administered 100 mg.kg -1 .day -1 of D-GAL for 8- (Garcez et al. 2021) and 6 weeks (Budni et al. 2016) and at a dose of 150 mg.kg -1 .day -1 for 8 weeks (Banji et al. 2014). Accompanied by oxidative stress, several studies have also reported that chronic systemic administration of intraperitoneal or subcutaneous D-GAL can induce neuroinflammation (Sadigh-Eteghad et al. 2017), (Azman and Zakaria 2019), (Lin et al. 2020), (Kumari et al. 2022). The mechanisms by which D-GAL promotes neuroinflammation are related to excessive levels of this sugar in the body, which are catalyzed by the enzyme galactose oxidase into aldose and hydrogen peroxide. These compounds generate ROS through oxidative metabolism and glycosylation, activating NF-KB and its transcriptional action on inflammatory cytokines (Kumari et al. 2022). In the present study, when we analyzed the IL-1β / IL-10 ratio in the frontal cortex, we found increased inflammation in the D-GAL group, indicating an inflammatory milieu (Kumari et al. 2022). Previous studies have shown that high concentrations of IL-1β in the brains of aged animals induced by lipopolysaccharide, a potent immune response activator, had detrimental effects on long-term memory due to neuroinflammation associated with microglia hyperactivity and greater induction of IL-1β induction (Henry et al. 2009),(Huang et al. 2008),(Huang and Sheng 2010). We demonstrated an inverse correlation between increased TBARS in the hippocampus and long-term memory. Review studies have shown that aging through D-GA administration with varying dosages, durations, and strains, which increases oxidative stress, can promote memory impairment (Sadigh-Eteghad et al. 2017),(Azman and Zakaria 2019) similar to what is observed in elderly individuals. Oxidative stress is considered a major causative factor of neurodegenerative diseases (Rehman et al. 2017). The cellular redox imbalance caused by aging and oxidative metabolism leads to increased ROS production, lipid oxidation, DNA damage and increased oxidative stress, impairing cell function, promoting neuronal death, and leading to memory impairment (Hermann et al. 2014),(Rehman et al. 2017),(Budni et al. 2016). 5. Conclusion Chronic D-GAL administration led to the reduction in aerobic capacity and impairments in long-term memory, which were likely associated with increased oxidative stress and inflammatory cytokines in central and peripheral tissues, including the hippocampus, frontal cortex, and skeletal muscle soleus. These responses can be considered characteristic of the aging process and, therefore, may represent a mimetic aging model with ecological validity observed in older adults. Declarations Acknowledgments We would like to express our sincere gratitude to Federal University of Minas Gerais (UFMG)/ Exercise Physiology Laboratory – LAFISE, Neuropsychology Laboratory – ICB – (UFMG), Experimental Physical Training Laboratory – (UFVJM), Cell Biology Laboratory – ICB – (UFMG), Lipids, Atherosclerosis, and Nutritional Biochemistry Laboratory – LABIN – (UFMG). Special thanks to Minas Gerais State Research Support Foundation (FAPEMIG) (CDS-APQ-03546-15 and CDS-APQ-00417-15 and BPD-00009-22), CAPES – Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (001- CAPES PRINT) and CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico. (Process # 310014/2021-2) from Brazil. We also appreciate the contributions of colleagues their insightful feedback and assistance. Declaration of Competing Interest The authors declare that they have no conflict of interest. Data availability statements The experimental data and the simulation results that support the findings of this study are available in google docs with the identifier: https://docs.google.com/spreadsheets/d/1nqYZRKHWea7_pHsCsDs52C5l4H26si1G/edit?usp=sharing&ouid=103532559065939165088&rtpof=true&sd=true Author contributions Daniel Massote de Melo Leite, Bruno Pereira Melo, Maicon Arcanjo da Silva, Guilherme Guisso Pizzol, Paola Caroline Lacerda Leocádio, Paola Caroline Lacerda Leocádio and Danusa Dias Soares performed the research. Daniel Massote de Melo Leite Bruno Pereira Melo and Danusa Dias Soares coordinated the review and wrote the initial draft of the manuscript. All authors revised it critically and approved the final version of the manuscript. CRediT authorship contribution statement Daniel Massote de Melo Leite: Investigation, Writing – original draft, Visualization, Validation, Data curation, Conceptualization, Methodology. Bruno Pereira Melo: Investigation, Writing – original draft, Visualization, Data curation, Conceptualization. Maicon Arcanjo da Silva: Investigation, Visualization, Conceptualization. Guilherme Guisso Pizzol: Investigation, Visualization, Conceptualization. Paola Caroline Lacerda Leocádio: Writing – research & editing, Investigation, Supervision, Methodology. Jacqueline Isaura Alvarez Leite: Writing – research & editing, Visualization, Investigation, Conceptualization, Supervision, Methodology. Danusa Dias Soares: Writing – original draft, Writing – research & editing, Visualization, Investigation, Validation, Conceptualization, Supervision, Data curation, Methodology. References Acosta PB, Gross KC (1995) Hidden sources of galactose in the environment. Eur J Pediatr 154:. https://doi.org/10.1007/BF02143811 Alejandro SP (2022) ER stress in cardiac aging, a current view on the D-galactose model. 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1","display":"","copyAsset":false,"role":"figure","size":124305,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design. HAB: Habituation; CON: Control group; D-GAL: D Galactose group; NOR: Novel object recognition; STM: Short-term memory; LTM: Long-term memory; MWC: Maximal weight carried test; VO2 peak: Volume peak of oxygen; Work: Work in joule; Mg: Milligram; Kg: Kilogram\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/5a811ee477197936f784ba4e.png"},{"id":98885333,"identity":"66b839fd-a8cc-4e80-8140-2f7d9d123a8b","added_by":"auto","created_at":"2025-12-23 14:54:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":102593,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight, adiposity index, and muscle mass before, 4, and after 8 weeks of intervention.\u003c/p\u003e\n\u003cp\u003ea) \u0026nbsp;Body weight; b) Adiposity index, c) Muscle mass (Soleus), and d) Muscle mass (EDL). Mean ± EPM. Mixed Anova analysis for body weight. Student´s Test for adiposity index and muscle mass. CON (n = 15); D-GAL (n = 14) for body weight and adiposity index; CON (n = 8); D-GAL (n = 8) for muscle mass (Soleus and EDL)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/da9265b7ff62e8395f59ec74.png"},{"id":98885332,"identity":"f02b9670-aa62-4f30-bf4d-20432f5cedcf","added_by":"auto","created_at":"2025-12-23 14:54:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112410,"visible":true,"origin":"","legend":"\u003cp\u003eRelative muscle strength, VO\u003csub\u003e2\u003c/sub\u003epeak, and Workload (J) during the 4 and after 8 weeks of D-GAL. a) Relative muscle strength; b) VO\u003csub\u003e2\u003c/sub\u003epeak; c) Workload (J). Mean ± SEM. Mixed Anova followed by Student´s T test for Workload (J) and Bonferroni post-hoc analysis for Relative muscle strength and VO\u003csub\u003e2\u003c/sub\u003epeak. \u003csup\u003e\u003cstrong\u003e*\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e \u003c/strong\u003eSignificant difference \u003cem\u003evs \u003c/em\u003eCON (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u0026nbsp; \u003csup\u003e\u003cstrong\u003e#\u003c/strong\u003e\u003c/sup\u003e Significant difference \u003cem\u003evs. \u003c/em\u003ebefore (\u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt; \u003c/em\u003e0.05); CON (n = 15); D-GAL (n = ≤ 14) and CON (n = 8); D-GAL (n = 8) for Relative muscle strength\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/1f483afef3b5573fe03dfc1e.png"},{"id":99309325,"identity":"37a6ef20-ee9e-4511-b224-e64e531b6529","added_by":"auto","created_at":"2025-12-31 16:10:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72213,"visible":true,"origin":"","legend":"\u003cp\u003eHydroperoxides, cytokine concentrations of TNF-α, IL-1β and IL-10 in the EDL muscle after 8 weeks of D-GAL. a) Hydroperoxides – EDL; b) TNF-α – EDL; c) IL-1β – EDL; d) IL-10 – EDL; e) Ratio IL-1β / IL-10 – EDL. Mean ± SEM. Student's T test. \u003cstrong\u003e*\u003c/strong\u003e Significant difference \u003cem\u003evs. \u003c/em\u003eCON. Hydroperoxides (\u003cem\u003ep \u003c/em\u003e= 0.01)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/334144b452a7a3f71b1718fc.png"},{"id":98885345,"identity":"1270c827-4d1a-4be3-9c72-7dd8ea13b060","added_by":"auto","created_at":"2025-12-23 14:54:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75402,"visible":true,"origin":"","legend":"\u003cp\u003eHydroperoxides and cytokine concentration of TNF-α, IL-1β, and IL-10 in the soleus muscle after 8 weeks of D-GAL. a) Hydroperoxides - Soleus; b) TNF-α – Soleus; c) IL-1β – Soleus; d) IL-10 – Soleus; f) Ratio IL-1β / IL-10 – Soleus. Mean ± SEM. Student's T-test.\u003cstrong\u003e *\u003c/strong\u003e Significant difference \u003cem\u003evs. \u003c/em\u003eCON. IL-10 (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05) and IL-1β / IL-10 ratio (\u003cem\u003ep\u003c/em\u003e = 0.01).CON (n = ≤ 8); D-GAL (n = ≤ 8)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/ca4283b84253b673b693c209.png"},{"id":99309464,"identity":"3f3f8bb9-7f8a-4be7-919b-bb633071732b","added_by":"auto","created_at":"2025-12-31 16:10:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":67914,"visible":true,"origin":"","legend":"\u003cp\u003eShort (STM) and long-term memory (LTM) before and after 8 weeks of D-GAL. a)\u0026nbsp;STM - NOR; b) LTM - NOR. Mean ± SEM. Mixed Anova followed by Bonferroni post-hoc analysis. \u003cstrong\u003e*** \u003c/strong\u003eSignificant difference \u003cem\u003evs. \u003c/em\u003eCON (\u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt; \u003c/em\u003e0.001), \u003csup\u003e\u003cstrong\u003e### \u003c/strong\u003e\u003c/sup\u003eSignificant difference \u003cem\u003evs. \u003c/em\u003ebefore D-GAL (\u003cem\u003ep\u003c/em\u003e \u003cem\u003e\u0026lt; \u003c/em\u003e0.001).\u003cstrong\u003e \u003c/strong\u003eCON (n = 13); D-GAL (n = 13)\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/739275ddfe31c09133a23a76.png"},{"id":99309476,"identity":"8a406be5-e86e-4af3-a3dd-ca3b310ed7a5","added_by":"auto","created_at":"2025-12-31 16:10:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":116412,"visible":true,"origin":"","legend":"\u003cp\u003eThiobarbituric acid reactive substances (TBARS), hydroperoxides and cytokines concentrations and Pearson´s correlation in the hippocampus after 8 weeks of D-GAL. a) \u0026nbsp;TBARS - Hippocampus; b) Hydroperoxides – Hippocampus; c) TNF-α – Hippocampus; d) IL-6 – Hippocampus; e) IL-1β – Hippocampus; f) IL-10 – Hippocampus; f) Ratio IL-1β / IL-10 – Hippocampus; h) Pearson's correlation Long Term Memory (LTM) versus TBARS – Hippocampus. Mean ± SEM. Student's T-test and Pearson´s correlation.\u003cstrong\u003e *\u003c/strong\u003e Significant difference \u003cem\u003evs. \u003c/em\u003eCON. TBARS (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); Hydroperoxides (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05); TNF-α (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01) and IL-10 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). CON (n = ≤ 8); D-GAL (n = ≤ 8)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/5662ca54f10c3401c1ecb990.png"},{"id":98885355,"identity":"dc76de38-9d03-4bbc-bf3d-6a6faa203d68","added_by":"auto","created_at":"2025-12-23 14:54:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":88137,"visible":true,"origin":"","legend":"\u003cp\u003eThiobarbituric acid reactive substances (TBARS), hydroperoxides and cytokines concentrations in the frontal cortex after 8 weeks of D-GAL. a) \u0026nbsp;TBARS - Frontal cortex; b) Hydroperoxides – Frontal cortex; c) TNF-α – Frontal cortex; d) IL-1β – Frontal cortex; e) IL-10 – Frontal cortex; f) IL-1β / IL-10 ratio - Frontal cortex. \u0026nbsp;Mean ± SEM. Student's T-test.\u003cstrong\u003e *\u003c/strong\u003e Significant difference \u003cem\u003evs. \u003c/em\u003eCON. Hydroperoxides (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05); IL-10 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) and IL-1β / IL-10 ratio (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). CON (n = ≤ 8); D-GAL (n = ≤ 8)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/000137722ef715ff106874e3.png"},{"id":99322729,"identity":"2aa813f1-63e2-4280-abbc-3d86b1f3aa50","added_by":"auto","created_at":"2025-12-31 16:44:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1531776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8369611/v1/3a1ed1fb-19ef-42c6-91be-0e675868d16c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eChronic D-Galactose Accelerates Aging-Like Decline in Aerobic Capacity and Cognitive Function Through Inflammation and Oxidative Stress in Central and Peripheral Tissues\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe aging process induces a range of \u0026nbsp;irreversible physiological changes, leading to molecular, cellular, and tissue damage, These effects are particularly evident in the brain,\u0026nbsp;where aging is associated with \u0026nbsp;neurodegeneration of the central nervous system and progressive \u0026nbsp;functional decline, resulting in reduced muscular strength, aerobic capacity, and cognitive function (Garatachea et al. 2015), (Isaac et al. 2021).\u003c/p\u003e\n\u003cp\u003eScientific evidence has indicates that aging is linked to an increased expression of inflammatory cytokines, such as interleukin 6 (IL-6), interleukin 1 beta (IL-1β), and tumoral necrosis factor alpha (TNF-α) in both, brain (Bellettini-Santos et al. 2023) and skeletal muscle (Yanar et al. 2011). This phenomenon, termed \"inflammaging\" \u0026nbsp;refers to the chronic, low-grade inflammation \u0026nbsp;associated with aging (Franceschi et al. 2018). It is believed to result from a combination of factors, including cellular senescence, immune system dysregulation , and \u0026nbsp;the accumulation of \u0026nbsp;molecular damage over time (Franceschi et al. 2018), (Santoro et al. 2021). This \u0026nbsp;persistent inflammatory state disrupts tissue homeostasis, impair cellular function, and contributes to the progression of age-related conditions such as cardiovascular diseases, neurodegeneration, and metabolic disorders (López-Otín et al. 2013), (Franceschi et al. 2018).\u003c/p\u003e\n\u003cp\u003eAging-related diseases\u0026nbsp;are driven by multiple interrelated mechanisms, including: 1) genomic instability, 2) telomere attrition, 3) epigenetic alterations, 4) loss of proteoastasis, 5) dysregulated nutrient sensing, 6) mitochondrial dysfunction, 7) cellular senescence, 8) stem cell exhaustion, and 9) altered intercellular communication (Pantiya et al. 2023). Additionally, oxidative stress plays a key role in aging, \u0026nbsp;characterized by an imbalance between reactive oxygen species (ROS) production of and the capacity of antioxidant defense mechanisms to neutralize them (Cui et al. 2006), (Ayala et al. 2014). This imbalance results in cumulative damage to lipids, proteins, and nucleic acids, exacerbating age-related pathologies such as neurodegenerative disorders, musculoskeletal deterioration and cancer (Garatachea et al. 2015) (Sadigh-Eteghad et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite significant advancements, the precise etiology of the aging process remains incompletely understood (López-Otín et al. 2013). To explore the cellular and molecular mechanisms underlying aging, , animal models- primarily \u0026nbsp; rodents- \u0026nbsp;have been widely used in laboratory studies (Sadigh-Eteghad et al. 2017), (Azman and Zakaria 2019). However, longitudinal aging studies require long-term maintenance of laboratory animals, \u0026nbsp; which is both \u0026nbsp;costly and time-consuming, considering the lifespan of rodents (~ 24-30 months) (Alejandro 2022), (Cebe et al. 2014). To adress this limitation, aging models based on \u0026nbsp;chronic administration of D-galactose (D-GAL)\u0026nbsp;have been developed\u0026nbsp;(Sadigh-Eteghad et al. 2017),\u0026nbsp;(Azman and Zakaria 2019),\u0026nbsp;(Lin et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD-GAL is a naturally occurring aldohexose sugar found in the body and in many foods, such as dairy products and some fruits (Acosta and Gross 1995). However, excessive D-GAL exposure \u0026nbsp;leads to its conversion into aldose and hydroperoxides via galactose oxidase, generating ROS\u0026nbsp;and promoting oxidative stress\u0026nbsp;(Azman and Zakaria 2019). Chronic D-GAL administration induces aging-like \u0026nbsp;characteristics in multiple peripheral tissues, including the heart\u0026nbsp;(Alejandro 2022), liver\u0026nbsp;(Azman et al. 2021), bone\u0026nbsp;(Partadiredja et al. 2019), pancreas and kidney\u0026nbsp;(El‐far et al. 2020). Additionally, chronic D-GAL exposure has been shown to impair memory, with some studies reporting also reduced locomotor performance\u0026nbsp;(Belviranlı and Okudan 2019)\u0026nbsp;while others show significant changes with wheel exercise\u0026nbsp;(Lee et al. 2019).\u003c/p\u003e\n\u003cp\u003eAlthough aging is known to impair mitochondrial energetics and reduce cardiorespiratory fitness (Mau et al. 2023), the extent to which \u0026nbsp;D-GAL induced-aging recapitulates these hallmarks has not been fully elucidated. Moreover, although the mimetic aging model induced by D-galactose (D-GAL) is well characterized in the literature, the molecular mechanisms underlying the damage caused by D-GAL to peripheral tissues and the central nervous system remain poorly understood. Therefore, this study aimed to evaluate the effects of chronic D-GAL administration on aerobic fitness, oxidative stress, and inflammatory markers in the brain and skeletal muscle, as well as its impact on short and long-term memory in male Wistar rats.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Animals and Ethics Care\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty male Wistar rats (220 g, approximately 8 weeks old) were used in the experiments. All experimental procedures were approved by the Ethics Committee Care and Use of Laboratory Animals of the Federal University of Minas Gerais (CEUA – UFMG, protocol number 161/2020) and were conducted following the Brazilian National Council for the Control of Animal Experimentation (CONCEA). The rats were housed in collective cages under controlled temperature (24ºC) and light conditions (12/12 hours) during all experimental study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.1 Experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were habituated to the laboratory environment for seven days. The rats were weighed through a digital scale (FILIZOLA®), randomized, and stratified by body mass into two experimental groups: Control (CON; 327.7 ± 29.7 g; n = 15) and D-galactose (D-GAL; 332.7 ± 32.8 g n = 15). After, both groups were submitted to the Novel Object Recognition Test (NOR) to assess short-term memory (STM) and long-term memory (LTM) for three consecutive days (Bevins and Besheer 2006). After the task, the groups were familiarized with climbing a vertical ladder during three days, and with running on a treadmill for five consecutive days. The rats were subjected to the Maximal Weight Carried Test (MWC) (Novaes Gomes et al. 2014), (Magalhães et al. 2022), and the incremental running test (Prímola-Gomes et al. 2009) to assess the physical performance in terms of maximum strength and aerobic capacity, respectively. The rats allocated into the D-GAL group were submitted to an eight-week aging induction protocol through the administration daily of the intraperitoneal D-galactose dissolved in saline solution (0.9%), while the rats allocated into CON group received only saline solution (Lin et al. 2020). After the aging protocol, the rats of both groups were submitted again to NOR test to assess STM and LTM memory, and physical performance (strength and aerobic capacity test) to investigate the possible deleterious effects of D-galactose aging-associated (Figure 1). The rats were euthanized by decapitation 48 hours after the end of the experimental protocol, the brain and peripheral tissues were removed, weighed, and stockage into -80°C for biochemical analysis. All experimental procedures were performed between 07:00 am and 5:00 pm.\u003c/p\u003e\n\u003cp\u003eThere was a sample loss in the D-GAL group, totaling (n = 14). For the memory data, one rat from the D-GAL group and two rats from the CON group did not explore the objects in the training phase and were excluded from the analysis, totaling (n = 13) for each of the groups. For aerobic capacity, there was a sample loss in the D-GAL group (n = 13). For the analysis of relative muscle strength results, TBARS, lipid hydroperoxides, and cytokines, (n = 8) were used as indicated in the graphics results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.2 Model Aging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe model of aging was established through the administration of 150 mg/kg/day of D-galactose powder (Sigma Aldrich, St. Louis, MO, USA, G0750), dissolved in 0.9% saline solution (100 mg\u003csup\u003e.\u003c/sup\u003eml\u003csup\u003e-1\u003c/sup\u003e), applied daily via intraperitoneal injection during 8 weeks (Lin et al. 2020).\u003c/p\u003e\n\u003cp id=\"_Toc162260540\"\u003e\u003cstrong\u003e2.1.3 Novel Object Recognition (NOR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NOR task is a consolidated test in the literature to assess short and long-term memory (Bevins and Besheer 2006), (Crunfli et al. 2019). It was performed in four stages during three consecutive days. On the first day, familiarization with the arena was performed and consisted of positioning the rats in the center of a square box (70 cm x 70 cm x 40 cm) during free five-minute exploration. \u003c/p\u003e\n\u003cp\u003eOn the second day, the training phase was performed. Two identical objects (A) were placed parallel to each other at the ends of the box for free exploration during 10 minutes. Sixty minutes after training phase, short-term memory (STM) was assessed for 5 minutes, with a new object (B) replacing a familiar object (A).\u003c/p\u003e\n\u003cp\u003eTwenty-four hours late (on the third day), long-term memory (LTM) was assessed using a new object (C) in place of object (B) for 5 minutes. At each assessment, the arena and objects were cleaned with 70% alcohol to avoid olfactory cues.\u003c/p\u003e\n\u003cp\u003eAny Maze software was used to analyze the results (ANY Maze Video Tracking System 7.16; STOELTING CO. ©, 2022). The discrimination index was calculated, which consists of the time taken to explore the new object (TN) divided by the sum of the exploration of the new object and the familiar object (TN + TF) (Bevins and Besheer 2006), (Rojas et al. 2013).\u003c/p\u003e\n\u003cp id=\"_Toc162260541\"\u003e\u003cstrong\u003e2.1.4 Vertical ladder familiarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were familiarized with a vertical ladder inclined at 80º (110 cm x 18 cm, with 2 cm between the steps). The vertical ladder familiarization protocol consisted of three trials per day for three days. Initially, the rats were kept in the housing chamber for 60 seconds and then placed on the ladder at 35 cm, 55 cm and 110 cm from the top. Between each trial the animals remained in the housing chamber for 60 seconds (Cassilhas et al. 2012). \u003c/p\u003e\n\u003cp id=\"_Toc162260542\"\u003e\u003cstrong\u003e2.1.5 Maximal weight carried test (MWC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe maximum weight carried test consisted of the rat initially climbing with a load corresponding to 75% of its body mass attached to the proximal part of the tail, into 50 mL Falcon tubes with fishing weights attached to a plastic-coated steel cable secured by a rubber band (Scotch 3 M). For each successful climb, the rat remained in the ladder housing for 60 seconds and a weight of 30 grams was added. Failure was determined when the rat was unable to continue climbing even after 3 successive gentle stimuli on the tail (Novaes Gomes et al. 2014), (Magalhães et al. 2022). The results were analyzed using the relative maximum load according to the following equation: MWC/BM, where MWC is the maximum load obtained in the strength test and MC, the body mass in kilograms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.6 Treadmill running familiarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe familiarization protocol for running on a treadmill (Panlab/Havard Apparatus, Cornella Spain) was carried out over five consecutive days for 5 minutes a day at an incremental velocity adjusted every minute (10, 10, 11, 13 and 15 m.min-\u003csup\u003e1\u003c/sup\u003e) with a 5% incline and an electrical stimulus set at 0.28 mA (Prímola-Gomes et al. 2009).\u003c/p\u003e\n\u003cp id=\"_Toc162260544\"\u003e\u003cstrong\u003e2.1.7 Incremental running test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe incremental treadmill running test (Panlab/Harvard Apparatus) was started at a velocity of 10 m\u003csup\u003e.\u003c/sup\u003emin\u003csup\u003e-1\u003c/sup\u003e, with progressive increments of 1 m\u003csup\u003e.\u003c/sup\u003emin\u003csup\u003e-1\u003c/sup\u003e every 3 minutes. The incline and electrical stimulation were similar to the familiarization protocol. The test was stopped when the animal remained on the electrical stimulation grid for 10 seconds. Peak oxygen consumption (VO\u003csub\u003e2\u003c/sub\u003e\u003csub\u003epeak\u003c/sub\u003e) was analyzed using open-flow indirect calorimetry (Gas Analyzer Panlab/Havard Apparatus, Cornella Spain). Aerobic performance was assessed by VO\u003csub\u003e2\u003c/sub\u003e\u003csub\u003epeak\u003c/sub\u003e and workload (J). The highest VO\u003csub\u003e2\u003c/sub\u003e\u003csub\u003epeak\u003c/sub\u003e values obtained during the incremental test were taken into account when analyzing the results. Workload was calculated using the equation: = bm × g × s × sinθ × t, where MC is the animal's body mass in kilograms; g: The acceleration of gravity (9.8 m\u003csup\u003e.\u003c/sup\u003es\u003csup\u003e-2\u003c/sup\u003e); v: The velocity of the treadmill in meters per minute; sin θ: The angle of inclination of the treadmill (5º) and t: The time spent on each stage in minutes (Soares et al. 2019), (Melo et al. 2022).\u003c/p\u003e\n\u003cp id=\"_Toc162260545\"\u003e\u003cstrong\u003e2.1.8 Euthanasia and Adiposity index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe rats were euthanized by decapitation, without prior sedation, 48 hours after the experimental protocol. The hippocampus, frontal cortex, soleus muscle and extensor digitorum longus muscle (EDL) tissues were removed and stored at -80 °C for later analysis. The retroperitoneal (TAR), mesenteric (TAM) and epididymal (TAE) adipose tissues were removed completely and weighed on a scale (SHIMADZU® Model BL320H, precision 0.001g) to determine the adiposity index (IA%), using the following equation: Adiposity index = (TAR + TAM + TAE) / MC x 100, where MC = Total body mass.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.9 Biochemical analyses\u003c/strong\u003e\u003cstrong\u003e - \u003c/strong\u003e\u003cstrong\u003eOxidative stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 50 mg was taken from brain and skeletal muscles to assess oxidative stress parameters. The fragments were homogenized with ice-cold 1x PBS and centrifuged at 12.000 rpm for 10 minutes. The supernatant was separated for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.10 Evaluation of lipid peroxidation by thiobarbituric acid reactive substances (TBARS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThiobarbituric acid-reactive metabolites were measured by adding a solution containing trichloroacetic acid (TCA 15%), thiobarbituric acid (TBA 0.0375%) and hydrochloric acid (HCl 0.25 N) to the organ supernatants. The samples were kept in a boiling water bath for 15 minutes and then cooled. After adding butyl alcohol, the tubes were shaken vigorously. The samples were centrifuged at 3.000 rpm for 10 minutes. 200μL of the supernatant was added to the 96-well plate. The absorbance was measured spectrophotometrically at a wavelength of 535 nm, and the results were normalized by the concentration of protein in the hippocampus, frontal cortex and soleus and EDL muscles (LOWRY et al. 1951).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.11 Evaluation of hydroperoxide concentration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis test was carried out using part of the solution prepared by dissolving xylenol orange and ammoniacal ferrous sulphate in H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e diluted in methanol solution containing BHT (butylated hydroxytoluene). For the dosages, the organ supernatants (20 μL each) were added to 180μL of the aforementioned solution, directly into the microplate, in triplicate. The blank was achieved by using phosphate buffer instead of the supernatant. The samples were then kept at room temperature for 30 minutes and the absorbance was measured spectrophotometrically at a wavelength of 560nm. The concentration of hydroperoxides was estimated by the extinction coefficient of hydroperoxides, 4.3 x 10-4M-1cm-1, and by the extinction coefficient of the bluish- purple chromophore produced by xylenol orange when it binds to ferric ions, 1.5 x 10- 4M-1cm-1. The quantification of hydroperoxides in the sample was achieved without TPP and the result was normalized by the protein concentration of each fragment (LOWRY et al. 1951).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.12 Protein dosage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein concentration in the organ supernatant was measured according to Lowry \u003cem\u003eet al.\u003c/em\u003e (1951) (LOWRY et al. 1951). To do this, 250 μL of diluted sample (1:50) was added to 250μL of solution A (one-part copper sulphate, one-part sodium tartrate and 100 parts sodium carbonate) and 25 μL of diluted Folin-Ciocalteau reagent (1:2). The samples were then vortexed and incubated at room temperature for 30 minutes. The samples (200 μL) were then added to the 96-well plate and the absorbance was measured spectrophotometrically at a wavelength of 660 nm. The results were expressed in mg\u003csup\u003e.\u003c/sup\u003e mL\u003csup\u003e-1\u003c/sup\u003e after obtaining the formula from the standard curve made with albumin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.1.13 Pro-inflammatory and anti-inflammatory cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentrations of interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) were assessed in the hippocampus, frontal cortex, soleus, and EDL muscle by means of ELISA (R\u0026amp;D Systems, Inc., Minneapolis, MN, USA; DuoSet kits DY506, DY522, DY501, and DY510, respectively). The manufacturer's recommendations were followed. The cerebral areas were chosen because of their relationship with learning and memory (hippocampus), attention and decision- making (frontal cortex). The muscles were selected according to their metabolic characteristics: soleus muscles because they are related to aerobic performance, with a predominance of oxidative type I fibers (slow-twitch fibers), and the EDL muscles , with predominantly glycolytic type IIa and IIb fibers (fast-twitch fibers) because of their relationship with muscle strength in rodents (Holeček and Mičuda 2017).\u003c/p\u003e\n\u003cp id=\"_Toc162260547\"\u003e\u003cstrong\u003e2.1.14 Statistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe normality of the data and the homoscedasticity of the residuals were assessed using the Shapiro-Wilk and Levene tests respectively. Data are expressed as mean ± standard error of the mean. Aerobic performance, maximal muscle strength, and short- and long-term memory variables were compared using mixed two-way ANOVA, considering the factors groups (CON vs. D GAL) and time (Before vs. 4th week vs. 8th week). When significant differences were found, Bonferroni post hoc and/or Student's t-test were performed, considering the mean coefficient of variation of the two groups (CON vs. D-GAL) at the three experimental time points. To compare body composition, cytokine concentrations, and markers of oxidative stress between groups (CON vs. D GAL), Student's t-test was used. Pearson's correlation was used to investigate association between TBARS concentrations, and their association with long-term memory in hippocampus. All analyses were performed using R software (version 4.2.2, USA) with Rstudio interface (version 2023.06 + 421). The significance level adopted was 5% (p ≤ 0.05).\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Body composition and adiposity index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBody weight increased (F\u003csub\u003e2.54\u0026nbsp;\u003c/sub\u003e= 445.55; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) similarly in CON and D-GAL groups in the 4- and after 8 weeks of intervention (Figure 2a). When compared to the CON group, D-GAL administration did not alter the adiposity index (4.96 ± 037 vs. 5.03 ± 0.27 %; t\u003csub\u003e27\u003c/sub\u003e = 0.15; \u003cem\u003ep\u003c/em\u003e = 0.880; Figure 2B), soleus muscle mass (0.14 ± 0.01 vs. 0.16 ± 0.01 g; t\u003csub\u003e14\u003c/sub\u003e = 1.063; \u003cem\u003ep\u003c/em\u003e = 0.305; Figure 2C), and either EDL muscle mass (0.20 ± 0.01 vs. 0.21 ± 0.01 g; t\u003csub\u003e14\u003c/sub\u003e = 0.285; \u003cem\u003ep\u003c/em\u003e = 0.779, Figure 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.2 Physical Performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of physical performance are expressed in Figure 3a-c. Relative muscle strength increased (F\u003csub\u003e2.28\u0026nbsp;\u003c/sub\u003e= 5.70; \u003cem\u003ep\u003c/em\u003e = 0.008) similarly in CON and D-GAL groups in the 4- and after 8 weeks of intervention (Figure 3A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe aging D-GAL-induced administration reduced the aerobic capacity and attenuated the workload after 8 weeks of the intervention. Compared to baseline, D-GAL administration reduced 11.6% the VO\u003csub\u003e2\u003c/sub\u003epeakin 4- (p = 0.008), and 12.2% after 8 weeks (\u003cem\u003ep\u003c/em\u003e = 0.003; Figure 3B). An ANOVA-mixed time effect was found in the workload (F\u003csub\u003e2.54\u003c/sub\u003e = 4.542; \u003cem\u003ep\u003c/em\u003e = 0.015; Figure 3C).\u003c/p\u003e\n\u003cp\u003eWhen compared to the CON group, the aging rats showed the lowest values of VO\u003csub\u003e2\u003c/sub\u003epeak (53.58 ± 1.81 vs. 48.46 ± 1.56 ml\u003csup\u003e.\u003c/sup\u003ekg\u003csup\u003e-1.\u003c/sup\u003emin\u003csup\u003e-1\u003c/sup\u003e; \u003cem\u003ep\u003c/em\u003e = 0.044; Figure 3b) and, workload (327.61 ± 23.86 vs 248.32 ± 15.41 Joules; \u003cem\u003ep\u003c/em\u003e = 0.011; Figure 3C) after 4-week, and also workload (320.86 ± 23.86 vs 268.32 ± 15.24 Joules; p = 0.041; Figure 3C) after 8-week of D-GAL administration. Moreover, the aging D-GAL-induced administration attenuated the workload of the aerobic exercise. The CON group increased 26.8% (\u003cem\u003ep\u003c/em\u003e = 0.005) of the workload in 4-, and 24.2% (\u003cem\u003ep\u003c/em\u003e = 0.005) after 8 weeks, however, this response was not observed in aging rats (Figure 3C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.3 Skeletal Muscle\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAnalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of aging on oxidative stress were observed in the skeletal muscle. D-GAL administration increased significantly (t\u003csub\u003e12\u003c/sub\u003e = 2.949; \u003cem\u003ep\u003c/em\u003e = 0.01) hydroperoxide levels in the EDL muscle after 8 weeks of administration (Figure 4a). No significant differences were observed in cytokines pro- or anti-inflammatory in the EDL muscle (Figure 4B-E).\u003c/p\u003e\n\u003cp\u003eIn the soleus muscle, the aging D-GAL-induced administration reduced IL-10 concentrations (t\u003csub\u003e13\u003c/sub\u003e = 2.232;\u003cem\u003e\u0026nbsp;p\u003c/em\u003e = 0.04; Figure 5D), and increased IL-1β/IL-10 ratio (t\u003csub\u003e12\u003c/sub\u003e = 2.679; \u003cem\u003ep\u003c/em\u003e = 0.010; Figure 5E), demonstrating a pro-inflammatory \u003cem\u003emilieu\u003c/em\u003e in this skeletal muscle. No significant differences were observed in cytokines pro-inflammatory (Figure 5B, C), and either oxidative stress (Figure 5A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.4 Recognition memory\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of aging D-GAL-induced administration on recognition memory are expressed in Figures 6A-B. The D-GAL administration promoted cognitive impairment after 8- weeks of the intervention. A mixed ANOVA analysis demonstrated a significant effect of time (F\u003csub\u003e1.24\u003c/sub\u003e = 18.554; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001) and group (F\u003csub\u003e1.27\u003c/sub\u003e = 11.077; \u003cem\u003ep\u003c/em\u003e = 0.002) in the short-term memory recognition index (STM; Figure 6A). Compared to the baseline, the aging rats reduced (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Figure 6b) the recognition index of the long-term memory test (LTM), demonstrating a cognitive decline in long-term memory. Moreover, compared to the CON group, the aging rats showed the lowest values (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Figure 6B) on the LTM recognition index after 8- weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.5 Hippocampus analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of hippocampus analysis are expressed in Figures 7A-H. The aging D-GAL-induced increased oxidative stress on the hippocampus and, it was associated with impairment of long-term memory. Compared to the CON group, D-GAL administration increased TBARS (t\u003csub\u003e13\u003c/sub\u003e = 3.673; \u003cem\u003ep\u003c/em\u003e = 0.020; Figure 7A), and hydroperoxides levels (t\u003csub\u003e9\u003c/sub\u003e = 2.399; \u003cem\u003ep\u003c/em\u003e = 0.040; Figure 7B) on the hippocampus after 8-weeks of the intervention. A significant, and inverse correlation (r = -0.773; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; r\u003csup\u003e2\u003c/sup\u003e = 0.597; Figure 7H) was found between TBARS levels and the LTM recognition index, demonstrating the influence of oxidative stress on memory loss.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompared to the CON group, the D-GAL administration also reduced TNF-α (t\u003csub\u003e14\u003c/sub\u003e = 3.262; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.011; Figure 7C), and IL-10 (t\u003csub\u003e14\u0026nbsp;\u003c/sub\u003e= 3.005; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Figure 7F) concentrations in the hippocampus after 8- weeks of the intervention. No significant difference was found in the IL-6 (t\u003csub\u003e14\u003c/sub\u003e = 1.780; \u003cem\u003ep\u003c/em\u003e = 0.092; Figure 7d), IL-1β (t\u003csub\u003e12\u003c/sub\u003e = 0.3766; \u003cem\u003ep\u003c/em\u003e = 0.714; Figure 7E) concentrations, and IL1β/IL-10 ratio (t 13 = 1.326; \u003cem\u003ep\u003c/em\u003e = 0.1038; Figure 7F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.1.6 Frontal cortex analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the frontal cortex analysis are expressed in Figures 8A-F. Compared to the CON group, D-GAL administration increased hydroperoxide levels (t\u003csub\u003e12\u003c/sub\u003e = 2.179; \u003cem\u003ep\u003c/em\u003e = 0.049; Figure 8B) in the frontal cortex after 8-weeks of the intervention. No significant difference was found in the TBARS concentrations (t\u003csub\u003e10\u003c/sub\u003e = 0.9037; \u003cem\u003ep\u003c/em\u003e = 0.384; Figure 8A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen analyzing the cytokines pro- and anti-inflammatory, the aging rats showed the lowest values (t\u003csub\u003e14\u003c/sub\u003e = 2.224; \u003cem\u003ep\u003c/em\u003e = 0.044; Figure 8E) of the IL-10 concentration compared to the CON group, in the frontal cortex after 8- weeks. No significant difference was found in the TNF-α (t\u003csub\u003e14\u003c/sub\u003e = 1.046; \u003cem\u003ep\u003c/em\u003e = 0.314; Figure 8C), and IL-1β (t\u003csub\u003e11\u003c/sub\u003e = 1.754; \u003cem\u003ep\u003c/em\u003e = 0.107; Figure 8D). The aging rats also showed higher values in the IL-1β/IL-10 ratio (t\u003csub\u003e10\u003c/sub\u003e = 4.840; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; Figure 8F) when compared to the CON group.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe mimetic aging model induced by D-galactose (D-GAL) is well characterized in the literature; however, the molecular mechanisms underlying the damage caused by D-GAL to peripheral tissues and the central nervous system remain poorly understood\u003c/p\u003e\n\u003cp\u003eIn the present study, chronic administration of D-GAL decreased aerobic capacity and impaired short- and long-term memory, without effects on muscle strength. Furthermore, we demonstrated that chronic D-GAL administration increased oxidative stress and inflammatory cytokines in central and peripheral tissues, including the hippocampus, frontal cortex, EDL and soleus. Since these functional and physiological responses are considered key hallmarks of aging, our results emphasize that chronic D-GAL administration may serve as a valid aging model with ecological relevance to older adults.\u003c/p\u003e\n\u003cp\u003eIn humans, experimental and review studies have shown that aging is associated with a reduction in VO\u003csub\u003e2peak\u0026nbsp;\u003c/sub\u003e(Shephard 2009),(Letnes et al. 2023), arteriovenous difference (Schrage et al. 2007), reduced cardiac output (Betik and Hepple 2008), mitochondrial biogenesis (Betik and Hepple 2008), muscle blood flow, and increased peripheral resistance (Lakatta and Levy 2003), leading to endothelial dysfunction (Schrage et al. 2007) and lower muscle oxidative capacity (Betik and Hepple 2008), which impairs \u0026nbsp;the performance of daily tasks (Chodzko-Zajko et al. 1998),(Pandey et al. 2020). \u0026nbsp;In rodent aging models, a few studies have assessed the \u0026nbsp;ability to perform physical exercise, either through locomotor activity in open-field and Morris water maze behavioral tests (Partadiredja et al. 2019), (Xinghua et al. 2023), (Zhang et al. 2023), or by voluntary wheel running (Belviranlı and Okudan 2019),(Lee et al. 2019). However, the maximum oxidative capacity of these animals has not been evaluated. To our knowledge, this is the first study to demonstrate that D-GAL administration reduces maximal aerobic physical capacity in an experimental aging model.\u003c/p\u003e\n\u003cp\u003eMuscle changes that occur with aging have been linked to a decline in aerobic capacity, often leading to a loss of strength, reduced functional ability, sarcopenia, and mitochondrial dysfunction in the skeletal muscles of older adults (Fan et al. 2016).In the present study, muscle strength increased similarly over time in both groups (D-GAL and CON), with no significant differences between them, indicating that the administration of 150 mg·kg⁻¹·day⁻¹ of D-galactose did not reduce muscle strength. In contrast Chang et al. (Chang et al. 2014) and Wu et al. (Wu et al. 2022) observed a decrease in muscle strength in mice experimentally aged with D-GAL, assessed via the forelimb grip strength test (GRIP test), which is commonly used to indicate neuromuscular alterations. The reduction in strength observed in their studies \u0026nbsp;(with doses of 125 and 100 mg·kg⁻¹·day⁻¹, administered subcutaneously and intraperitoneally for 8 and 6 weeks, respectively) was associated with morphological \u0026nbsp; changes and increased mitochondrial dysfunction in complex I of the gastrocnemius muscle (Chang et al. 2014), as well as elevated levels of malondialdehyde and \u0026nbsp;inflammatory cytokines such as IL-6 and TNF-α (Wu et al. 2022). We used a maximum load test on a vertical ladder to assess muscle strength (Novaes Gomes et al. 2014), (Magalhães et al. 2022). It is possible that this test was not sensitive enough to detect changes in muscle strength. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies in rats have shown that chronic D-GAL administration increases \u0026nbsp;oxidative stress and mitochondrial dysfunction in the gastrocnemius muscle (glycolytic) (Kou et al. 2017) and the soleus muscle (oxidative) (Yanar et al. 2019). Damage to the unsaturated lipids in cell membranes, \u0026nbsp;mediated by the oxidative action of reactive oxygen species (ROS), results \u0026nbsp;in lipid peroxidation and the formation of lipid hydroperoxides, which generate free radicals that induce cytotoxicity, inflammation, and structural changes \u0026nbsp;in various cells (Ayala et al. 2014).\u003c/p\u003e\n\u003cp\u003eIn the present study, rats aged with D-GAL exhibited a significant increase in the concentration of lipid hydroperoxides in the EDL, a glycolytic muscle. Previous studies have shown that aging, in both humans and rodents, can lead to increased oxidative stress in glycolytic muscles, which are characterized by, as predominance of type II muscle fibers, and low mitochondrial content. Oxidative damage has been observed in the vastus lateralis of elderly individuals (Lexell et al. 1988), apoptosis in the superficial vastus lateralis of rats (Phillips and Leeuwenburgh 2005), and mitochondrial dysfunction in the gastrocnemius of mice treated with \u0026nbsp;D-GAL (Chang et al. 2014). However, studies evaluating oxidative stress through hydroperoxides in the EDL muscle in D-GAL-induced aging models are scarce.\u003c/p\u003e\n\u003cp\u003eWe expected the soleus muscle to exhibited \u0026nbsp;an increase in lipid peroxidation (compared to the CON group) due to its higher mitochondrial content and greater oxidative stress production from \u0026nbsp;oxidative metabolism (Holeček and Mičuda 2017). Previous findings have shown that D-GAL, administered at a dose of 60 mg·kg⁻¹·day⁻¹ via intraperitoneal injection for 6 weeks in rats, increased lipid hydroperoxide concentrations in \u0026nbsp;in the soleus muscle, resulting in mitochondrial dysfunction, increased carbonylated protein content (a marker of oxidative damage), and impaired \u0026nbsp;of muscle redox homeostasis (Yanar et al. 2019).\u003c/p\u003e\n\u003cp\u003eAlthough we did not observe changes in the hydroperoxide concentration chronic D-GAL administration in the present study also reduced IL-10 concentrations in the soleus muscle (a muscle with slow-twitch fibers) inducing a decrease in the anti-inflammatory capacity of this tissue. The reduction of IL-10 in skeletal muscle as a result of the aging \u0026nbsp;has been linked to \u0026nbsp;greater macrophage activation´s, antigen release, and inflammatory cytokine production (Hacham et al. 2004), (Moore et al. 2001),(Mittal and Roche 2015),(Dagdeviren et al. 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn line with our results, higher concentrations of IL-10 in blood serum have been directly correlated with better aerobic fitness (VO\u003csub\u003e2max\u003c/sub\u003e) in the elderly (Rosado-Pérez and Mendoza-Núñez 2018). Additionally, when analyzing the pro-inflammatory ratio between IL-1β / IL-10 in the soleus muscle, we found an inflammatory \u003cem\u003emilieu\u003c/em\u003e in this tissue. The chronic low-grade pro-inflammatory state observed \u0026nbsp;in aging may \u0026nbsp;be explained by the increased activation of the transcription factor NF-kβ, which regulates the production of pro-inflammatory cytokines activated by \u0026nbsp; increased oxidative stress, impaired autophagy, DNA damage and the accumulation of senescent cells in various tissues \u0026nbsp;(Bektas et al. 2017),(Franceschi et al. 2018).\u003c/p\u003e\n\u003cp\u003eRegarding central responses, our study demonstrated that short- and long-term memory were negatively affected by chronic D-GAL administration. These effects may be associated with increased oxidative stress and an inflammatory environment in the hippocampus and frontal cortex, suggesting that chronic D-GAL administration induces an ecological model of neurodegeneration linked to aging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effects of different doses and duration of chronic D-GAL administration on cognitive and memory performance have been explored in the literature.-GAL administration \u0026nbsp;at a dose of 150 mg.kg\u003csup\u003e-1\u003c/sup\u003e.day\u003csup\u003e-1\u003c/sup\u003e for 8- (Duan et al. 2017),(Zhang et al. 2021), 6- (Sun et al. 2018) or 10 weeks (Nam et al. 2019) induced cognitive impairment and memory loss, as assessed \u0026nbsp;by the object recognition test. The hippocampus and frontal cortex are structures involved in neurogenesis, memory and learning (Garcez et al. 2021). Oxidative stress and inflammation, have been identified \u0026nbsp; as the primary physiological mechanism underlying memory impairment and neurodegenerative diseases. In the present study, we showed that chronic D-GAL administration increased TBARS and lipid hydroperoxide concentrations in the hippocampus and lipid hydroperoxide in the frontal cortex underscoring the effects of oxidative stress on aging-associated neurodegeneration. These findings \u0026nbsp;corroborate the literature, which has shown \u0026nbsp;higher hippocampal TBARS and MDA concentrations in rats administered 100 mg.kg\u003csup\u003e-1\u003c/sup\u003e.day\u003csup\u003e-1\u003c/sup\u003e of D-GAL for 8- (Garcez et al. 2021) and 6 weeks (Budni et al. 2016) and at a dose of 150 mg.kg\u003csup\u003e-1\u003c/sup\u003e.day\u003csup\u003e-1\u003c/sup\u003e for 8 weeks (Banji et al. 2014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccompanied by oxidative stress, several studies have also \u0026nbsp;reported that chronic systemic administration of intraperitoneal or subcutaneous D-GAL can induce neuroinflammation (Sadigh-Eteghad et al. 2017), (Azman and Zakaria 2019), (Lin et al. 2020), (Kumari et al. 2022). The mechanisms by which D-GAL promotes neuroinflammation are related to excessive levels of this sugar in the body, which \u0026nbsp; are catalyzed by the enzyme galactose oxidase into aldose and hydrogen peroxide. These compounds generate ROS \u0026nbsp;through oxidative metabolism and glycosylation, activating \u0026nbsp;NF-KB and its transcriptional action on inflammatory cytokines (Kumari et al. 2022). In the present study, when we analyzed the \u0026nbsp;IL-1β / IL-10 ratio in the frontal cortex, we found increased inflammation in the D-GAL group, indicating an inflammatory \u003cem\u003emilieu\u003c/em\u003e (Kumari et al. 2022). Previous studies have shown that high concentrations of IL-1β in the brains of aged animals induced by lipopolysaccharide, a potent immune response activator, had detrimental effects on long-term memory due to neuroinflammation associated with \u0026nbsp;microglia hyperactivity \u0026nbsp;and greater induction of IL-1β induction (Henry et al. 2009),(Huang et al. 2008),(Huang and Sheng 2010).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe demonstrated an inverse correlation between increased TBARS in the hippocampus and long-term memory. Review studies have shown that aging through D-GA administration with varying \u0026nbsp;dosages, durations, and strains, \u0026nbsp;which increases oxidative stress, can promote memory impairment (Sadigh-Eteghad et al. 2017),(Azman and Zakaria 2019) similar to what is observed \u0026nbsp;in elderly individuals. Oxidative stress is considered \u0026nbsp;a major causative factor of neurodegenerative diseases (Rehman et al. 2017). \u0026nbsp;The cellular redox imbalance caused by aging and oxidative metabolism leads \u0026nbsp;to increased ROS production, lipid \u0026nbsp;oxidation, DNA damage and increased oxidative stress, impairing cell function, promoting neuronal death, and leading to memory impairment (Hermann et al. 2014),(Rehman et al. 2017),(Budni et al. 2016).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eChronic D-GAL administration led to the reduction in aerobic capacity and impairments in long-term memory, which were likely associated with increased oxidative stress and inflammatory cytokines in central and peripheral tissues, including the hippocampus, frontal cortex, and skeletal muscle soleus. These responses can be considered characteristic of the aging process and, therefore, may represent a mimetic aging model with ecological validity observed in older adults.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to Federal University of Minas Gerais (UFMG)/ Exercise Physiology Laboratory \u0026ndash; LAFISE, Neuropsychology Laboratory \u0026ndash; ICB \u0026ndash; (UFMG), Experimental Physical Training Laboratory \u0026ndash; (UFVJM), Cell Biology Laboratory \u0026ndash; ICB \u0026ndash; (UFMG), Lipids, Atherosclerosis, and Nutritional Biochemistry Laboratory \u0026ndash; LABIN \u0026ndash; (UFMG). Special thanks to Minas Gerais State Research Support Foundation (FAPEMIG) (CDS-APQ-03546-15 and CDS-APQ-00417-15 and BPD-00009-22), CAPES \u0026ndash; Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal do Ensino Superior (001- CAPES PRINT) and CNPq - Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico. (Process # 310014/2021-2) from Brazil. We also appreciate the contributions of colleagues their insightful feedback and assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data and the simulation results that support the findings of this study are available in google docs with the identifier: https://docs.google.com/spreadsheets/d/1nqYZRKHWea7_pHsCsDs52C5l4H26si1G/edit?usp=sharing\u0026amp;ouid=103532559065939165088\u0026amp;rtpof=true\u0026amp;sd=true\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDaniel Massote de Melo Leite, Bruno Pereira Melo, Maicon Arcanjo da Silva, Guilherme Guisso Pizzol, Paola Caroline Lacerda Leoc\u0026aacute;dio, Paola Caroline Lacerda Leoc\u0026aacute;dio and Danusa Dias Soares performed the research. Daniel Massote de Melo Leite Bruno Pereira Melo and Danusa Dias Soares coordinated the review and wrote the initial draft of the manuscript. All authors revised it critically and approved the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDaniel Massote de Melo Leite: Investigation, Writing \u0026ndash; original draft, Visualization, Validation, Data curation, Conceptualization, Methodology. Bruno Pereira Melo: Investigation, Writing \u0026ndash; original draft, Visualization, Data curation, Conceptualization. Maicon Arcanjo da Silva: Investigation, Visualization, Conceptualization. Guilherme Guisso Pizzol: Investigation, Visualization, Conceptualization. Paola Caroline Lacerda Leoc\u0026aacute;dio: Writing \u0026ndash; research \u0026amp; editing, Investigation, Supervision, Methodology. Jacqueline Isaura Alvarez Leite: Writing \u0026ndash; research \u0026amp; editing, Visualization, Investigation, Conceptualization, Supervision, Methodology. \u0026nbsp;Danusa Dias Soares: Writing \u0026ndash; original draft, Writing \u0026ndash; research \u0026amp; editing, Visualization, Investigation, Validation, Conceptualization, Supervision, Data curation, Methodology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAcosta PB, Gross KC (1995) Hidden sources of galactose in the environment. Eur J Pediatr 154:. https://doi.org/10.1007/BF02143811\u003c/li\u003e\n\u003cli\u003eAlejandro SP (2022) ER stress in cardiac aging, a current view on the D-galactose model. Exp Gerontol 169:. https://doi.org/10.1016/J.EXGER.2022.111953\u003c/li\u003e\n\u003cli\u003eAyala A, Mu\u0026ntilde;oz MF, Arg\u0026uuml;elles S (2014) Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. 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Brain Sci 13:. https://doi.org/10.3390/BRAINSCI13030422\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Aerobi, capacit, decline, D-Galactose-induce, aging, Neuroinflammation, Oxidativ, stress, Recognitio, memor, impairment","lastPublishedDoi":"10.21203/rs.3.rs-8369611/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8369611/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction: \u003c/strong\u003eD-galactose (D-GAL) is an aldohexose naturally present in the body and diet; however, chronic exposure can be deleterious to health and promote aging-related changes. Thus, the effects of D-GAL on aerobic capacity and cognitive function based on central and peripheral hallmarks remain unclear. This study investigated the effects of chronic D-GAL administration on physical performance, memory, inflammatory markers, and oxidative stress in the brain and skeletal muscle of rats.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Male Wistar rats received D-galactose (150 mg·kg⁻¹·day⁻¹) administered intraperitoneally for eight weeks. Muscle strength was assessed using a vertical ladder test, whereas aerobic capacity was evaluated by treadmill testing at weeks 4 and 8. Memory was analyzed using the Novel Object Recognition Test. Biochemical analyses were performed in central and peripheral tissues, including the soleus, extensor digitorum longus (EDL) muscles, hippocampus, and frontal cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e D-galactose reduced aerobic performance, evidenced by decreased VO₂peak (p = 0.044) and work output (p = 0.011) at week 4, with a further decline in work output at week 8. Chronic D-galactose also impaired long-term memory. Oxidative stress increased, with elevated hydroperoxides in the EDL (p = 0.010) and hippocampus (p = 0.040), and higher TBARS levels in the hippocampus (p = 0.020). Inflammatory modulation was observed, with reduced IL-10 levels in the soleus (p = 0.040), hippocampus (p \u0026lt; 0.001), and frontal cortex (p = 0.044).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eChronic D-galactose–induced aging impairs aerobic capacity and memory and is associated with increased oxidative stress and inflammation in skeletal muscle and brain tissue overall in rats.\u003c/p\u003e","manuscriptTitle":"Chronic D-Galactose Accelerates Aging-Like Decline in Aerobic Capacity and Cognitive Function Through Inflammation and Oxidative Stress in Central and Peripheral Tissues","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-23 14:54:47","doi":"10.21203/rs.3.rs-8369611/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":"2ae46806-fcac-476e-a887-39ade046c457","owner":[],"postedDate":"December 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-27T09:54:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-23 14:54:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8369611","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8369611","identity":"rs-8369611","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}