Exercise training increase BDNF/TrkB mRNA, prevents memory loss and ameliorate the hippocampal morphology in multiple sclerosis model

Exercise training increase BDNF/TrkB mRNA, prevents memory loss and ameliorate the hippocampal morphology in multiple sclerosis model | 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 Exercise training increase BDNF/TrkB mRNA, prevents memory loss and ameliorate the hippocampal morphology in multiple sclerosis model Muthanna Hafedh, Muthana Layth Hatem This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4623275/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 Purpose : We aimed to verify the implications of four weeks of consistent exercise on the mRNA of the brain-derived (BDNF) and the tropomyosin-related kinase B (TrkB) receptor that may contribute to ameliorates the hippocampal morphology and induced enhancement of memory in experimental autoimmune encephalomyelitis (EAE) after the diseases recovery stage. Methods : Female Lewis rats were randomly separated into three groups, control group (Co), EAE without exercise (EAE), EAE with exercise (EAE+Ex) , each group contains (10 rats) . All rats underwent to the Morris water maze (MWM) test for four days in week and prop test was done in fifth day and repeated in day 30 post induction. Exercise was prescribed by motorized treadmill starting at 17 m/min and gradually increased, a total duration of 30 minutes per day, five days a week and discontinued two weeks after the induction. EAE was induced subcutaneously after two weeks of exercise as rats injected with an inoculum containing 50 ug guinea pig spinal cord homogenate emulsified in incomplete Freund’s adjuvant and 2mg of heat-mycobacterium at the tail base. Results : EAE+Ex significantly increases the BDNF mRNA (P 0.02) mRNA levels compared to the EAE groups. The Co group increased significantly more than the EAE group (P< 0.01). Swimming speed performance was faster in EAE+Ex (P< 0.02) compared to EAE group lead to improved hippocampus function. Conclusion : It seems that the provision of four weeks of exercise training is capable of increasing the mRNA of BDNF and its receptor along with ameliorating the hippocampus morphological which can have positive effects on memory impairment during EAE disease. Exercise EAE BDNF TrKB Memory Morphology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Multiple sclerosis (MS) affects young people (mean age at onset, 20–30 years) and is characterized by inflammatory demyelination and axonal transection[ 1 ]. It can result in physical deterioration, mental decline and a lower quality of life [ 2 ]. Previous studies have identified a number of mechanisms that play a role in the pathogenesis of MS including the death of neurons as a result of oxidative stress, immune cells invading the central nervous system, the introduction of prolactin, melatonin, and opioids as well as immune cells and neuro-immunomodulatory substances [ 3 , 4 ]. A highly utilized murine model for studying MS is experimental autoimmune encephalomyelitis (EAE)[ 5 ]. The EAE rat model of MS is a model of this disease as it clearly shows hind-limb paralysis, nerve damage and memory dysfunction [ 6 ]. Learning and memory impairment, i.e., impairment that is experienced while acquiring new information, is prevalent disabling and not managed adequately in MS [ 7 ]. A common type of cognitive weakness experienced in MS is memory deficits which are found in 40–60% of patients suffering from the disease [ 8 ]. A critical brain structure that plays a part in the formation of learning and memory is the hippocampus and there is often a relationship between its pathology and memory problems. It shows the highest neuroplasticity within the brain while also showing active neurogenesis all through adulthood [ 9 , 10 ]. Brain-derived neurotrophic factor (BDNF) is a candidate molecular effector of neuroprotective autoimmunity. BDNF is an effective neurotrophic protein that supports neural development and survival produced by immune cells in peripheral blood and MS lesions [ 11 ]. According to a previous study, the neurotrophin BDNF is an important component in the remyelination and/or structural restoration of myelin [ 12 ]. BDNF and its high-affinity receptor (TrkB ) are important molecules in the CNS that affect behavior, mood and cognitive functions like memory and learning [ 13 ]. Long-term learning and memory necessitate synaptic plasticity in which BDNF plays an important role [ 14 ]. Through the TrkB receptor in hippocampus neurons, BDNF promotes synaptic transmission and increases plasticity at the neuromuscular synapse [ 15 ]. Hippocampal-dependent learning requires BDNF-TrkB activation for consolidation which is followed by a delayed stabilization phase that is crucial for retaining that memory [ 16 , 17 ]. Physical activity reduces the dendritic irregularities associated with experimental autoimmune disease [ 18 ]. Leavitt et al. has reported that aerobic exercise improves hippocampus volume and improves cognition in people with multiple sclerosis [ 19 ]. Several treatment approaches are available that concentrate on self-management, physical activity and the quality of life of those suffering from MS. A key treatment for maintaining and enhancing functional status is exercise therapy [ 20 ]. Studies have found that physical activity up-regulates hippocampal BDNF which may be associated with mood states, memory and learning to decrease the deterioration of cognitive function related to MS. Furthermore, anti-oxidant defenses and neurotrophic support may be improved by exercise which could decrease the vulnerability of the CNS to neuronal degeneration [ 21 ]. Through physical training, the levels of BDNF and its TrkB receptor can be positively regulated in various brain structures, particularly in the hippocampus in experimental animals as well as humans[ 22 ]. Furthermore, it has been shown that there is an increase in serum BDNF concentrations right after a single session of light aerobic exercise in people with and without MS[ 23 ]. In the current study, we investigated the effect of four weeks of exercise training on BDNF-TrkB expression in the hippocampus as well as hippocampus morphology and memory in the EAE Model. Experimental subjects Animals 30 female Lewis rats with an age of 8–10 weeks and a weight of (100–125 kg) were purchased from DarouPakhsh Pharmaceutical Company, Iran. Individually housed in special cages, the animals are kept in a controlled environment (12/12 h light and dark) and fed standard rodent chow and water. The animals were acclimated to their new surroundings for one week before being divided into one of three groups (10 rats each): control (Co), EAE only (EAE) and an EAE exercise group (EAE+Ex). Estrous cycle evaluation. As reported previously [1], Female rats estrous cycles were observed every day. The rats with estrous cycles that began on the same day were chosen. Between 7 and 8 in the morning, vaginal lavage was carried out with a mild manual restraint. A plastic pipette with the tip inserted inside the animal vagina and 10μl of normal saline (0.9%) was added to collect vaginal excrement. The glass slides were covered with vaginal fluid. Each rat had one drop of vaginal fluid removed with a clean tip. Morris Water Maze The Morris Water Maze (MWM) is considered to be the gold standard to assess spatial learning as well as memory capacity [24]. This approach is based on the observation that animals have developed an efficient manner of navigating their surroundings and escaping danger that allows them to attain the desired outcome with the least amount of effort [25, 26]. The MWM used in this study was a black circular tank with a diameter of about 136 cm and a height of 60cm filled with water (21 o C) at a depth of 25 cm. Maze was divided into four quadrants: northeast, northwest, southeast, and southwest as well as starting positions: north, south, west, and east. A concealed circular platform with a diameter of 10 cm was situated in the SW quadrant's center, 1.5 cm below the water's surface. Each test was recorded and assessed using a video camera connected to a tracking device (EthoVision XT) situated above the pool to calculate the time in each quadrant and swimming velocity of each rat. Animals were allowed to adapt to the WMM test for two days. The MWM test was implemented across five consecutive days: four days for learning and one day as prop test. All sessions began at 8:00 a.m. For the training days, each rat was placed at one of the four predetermined beginning locations in the water, facing the tank's wall, and then given free reign to swim until they reached the platform that was concealed inside the tank. Every trial required every rat to locate the hidden platform within 60 seconds. If the rat discovered the platform, it was permitted to stay there for 30 seconds. If the rat did not find the platform within 60 seconds, it was placed on platform 30 seconds. The prop test was performed on day 5 to assess memory. On the prop test day, the platform was removed from the tank and the time it took the rat to reach the area in which the platform was previously placed was measured as well as time spent in that specific area. The test was discontinued at 60 seconds. Assessments were completed at baseline prior to EAE induction and again 30 days post inoculation. Induction and Clinical Disability Assessment As previously described [27], 50 ug of guinea pig spinal cord homogenate and 2 mg of heat-mycobacterium (Difco H37Ra) were added to incomplete Freund's adjuvant (Sigma). A subcutaneous injection of 50 ul of the inoculation was given into the base of the tail. The presence of any indicators of impairment was assessed using EAE scores previously described [28]. Exercise Training and Treatment For seven days, the animals practiced running for 15 minutes each day at a belt pace of 5 to 10 meters per minute without slop. Rats were made to run by administering an electric shock (0.3 mA). As described by Kim and colleagues [29] exercise training was started 2 weeks prior EAE immunization (preconditioning protocol) and continued until 2 weeks post EAE immunization. Exercise was prescribed starting at 17 m/min and increased each week (week 1: 17 m/min, week 2: 18 m/min, week 3: 19 m/min, and week 4: 20 m/min) for a total duration of 30 minutes per day, five days a week. Rats in the non-exercise groups were placed on the treadmill lane with the machine turned off and were subjected to the same schedule as the rats in the exercise groups. Anaesthesia and euthanasia Rats were euthanized by ketamine and xylazine (3/1) and the animal brains were extracted. Hippocampal tissue was quickly dissected and frozen in liquid nitrogen immediately. Frozen tissues were homogenized in the Trizol reagent at a 1 ml Trizol/100 mg tissue ratio. The left hippocampal was dissected and frozen at 80°C for gene expression analysis. The right hippocampal was also dissected for morphology investigation, post-fixed in 4% formaldehyde and immersed in double-distilled water. Gene expression analysis. Real-time polymerase chain reaction (PCR) was utilized to analyse TrKB and BDNF mRNA in the hippocampus. Total RNA was extracted from samples using the TRIzol kit (Invitrogen, USA) in accordance with the manufacturer's instructions. After RNA purification, TrKB and BDNF gene primers were made using the NCBI website ( Table 1. ) . Real-time PCR was carried out using the Applied Biosystems TM Real-Time PCR apparatus with Master Mix and SYBR Green I (Fluka, Switzerland) as the starting materials. Using a standard curve, the efficacy of each gene was determined. GAPDH was used as a checkpoint. Table 1. The nucleotide sequences of sense and antisense primers. Cresyl violet staining method Cresyl violet staining was used to count the number of neurons in the right hippocampus. Brain slices are dried in three sequential steps: 70% alcohol (for 10 min), 95% alcohol (with a few drops of 10% acetic acid; for 2-3 min) and finally 100% alcohol after creating 5 m-long microtome incisions and undergoing tissue processing (for 10 min). Slices were washed in xylene for five minutes, colored with cresyl violet, covered with DPX, and then sealed with a cover slip. Statistical analyses The mean and standard error are used to represent the data. The data were analyzed using a two-way variance analysis (ANOVA). Repeated measures were used in the MWM and significance was assessed using P values <0.05. The normality and homogeneity of the data was determined using Tukey post-hoc test. Results Exercise training increased BDNF and TrKB mRNA in hippocampus The ANOVA revealed significant difference between the three groups. Post hoc-analysis received that the EAE significantly reduced both BDNF mRNA (P0.01) compared to Co group. Although, EAE+Ex group showed a higher BDNF mRNA (P>0.05) and TrkB mRNA (P>0.02) compared to EAE group, respectively. No significant difference was observed between the Co and the EAE+Ex group. (Figure 1). Exercise helps preserve memory in EAE Comparison between post test and prior test was following the animal recovery from the EAE disease and ability the animals to move as evidenced by their swimming speed. The prop test was used by removing the platform from the tank. Two-way ANOVA found significant differences among groups. Post-hoc results didn’t find any significant difference within the Co group in prior text compared to the post test. Further, swimming speed in the prop test was considerably slower in the EAE group prior text compared to post text (P0.02) compared to the prior test). Fig2 . Between group comparisons found no difference in swimming speed between the Co group and the EAE+Ex group. Co group showed a significant difference (P>0.01) compared to the EAE. EAE-Ex group inducted significant (P>0.02) compared to the EAE group, respectively. ( Fig.3) Hippocampal Morphology There were no histological changes in the hippocampal morphology in Co group and normal dendritic spine density, dendritic spine length and the number of cells in the hippocampal region were normal (Fig4.A). Animals in the EAE group had unstructured hippocampal tissue with decreasing dendritic spine density and length as well as several cells in the hippocampal region (Fig.4.B) . Further, EAE+Ex group had slightly higher dendritic spine density, dendritic spine length and hippocampal cell number (Fig.4.C). Discussion For some hippocampus-dependent processes, increasing exercise-induced hippocampal neurogenesis is advantageous. The increased production of certain transmitters, growth factors, or neurotrophic factors during exercise, however, may regulate overall dynamic balance [ 25 ]. In line with this, data from this study demonstrated that the mRNA of BDNF and its TrkB receptor in the hippocampus were increased following the four weeks of exercise training in the EAE + Ex group after day 30 dpi. Our findings are consistent with the previous studies [ 29 , 30 ] that suggested that exercise training leads to an increase in the expression of BDNF mRNA in the hippocampus compared to a sedentary EAE group. An increase in BDNF mRNA is one of the persistent and potent effects of exercise on the brain[ 31 ]. BDNF/TrkB expression and activation is important for neurophysiological processes like neuroprotection, neuromaturation and maintenance of neurons. According to previous studies, EAE and similar neurodegenerative disorders are related to both activation and malfunction of the BDNF-TrkB pathway. It was noted in exercise literature that there was a higher shortage of TrKB in the animal model of MS disease. With the passage of time, there is a decrease in BDNF and TrkB expression in the hippocampus and temporal cortex in humans which puts them at a higher risk of experiencing various neurodegenerative pathologies [ 32 ].The findings of this study indicate that memory deficits occurred in the preliminary stage of the disease (30 dpi) in the EAE group. Our findings support the notion that memory deficits in EAE are common in the early stages of MS [ 33 ] because the volume of the CA1 region first decreases during the early stages of hippocampal injury, which then spreads to the CA2, CA3, and dentate gyrus [ 34 ]. The present study showed an improvement in memory in the EAE + Ex group, this was accompanied by enhanced swimming speed activity. In line with this study, our previous findings indicated that exercise training improved memory while also promoting adrenal gland recovery during the EAE stage [ 1 ]. Furthermore, studies have shown that in the absence of exercise training, cognitive impairment was observed in the EAE model on day 90 after induction rather than day 30 and persisted until the end of motor recovery [ 35 ]. Furthermore, Jun-Jun Sun and colleagues found that cognitive impairment occurred later in the course of EAE (55 dpi) as opposed to the early stage (13 dpi) of the illness. Although Kurkowska and colleagues [ 28 ] found no cognitive abnormalities in EAE animals compared to controls, the researchers believe that a relapse of MS may start the neurodegenerative process inside the hippocampus, and that this may not always be associated with memory difficulties (90). In line with this thinking, the present study findings also showed that the hippocampal tissue had significant damage in rats in the EAE group, while the EAE-Ex group showed significantly different modulation of hippocampal morphology. Cell proliferation is thought to be about two times higher during the acute phase of the disease compared to controls [ 36 ]. Additionally, Huehnchen and colleagues (2011) observed that not only in the acute phase of the disease but also in the chronic phase, there were more DCX + and BrdU + cells, as an indication of cell proliferation, in the DG of EAE-Ex than in controls. However, this outcome does not imply that the EAE-Ex group will return to normal [ 37 ]. According to a study conducted by Magalon et al. (2007), environmental enrichment promotes neural progenitor migration into demyelinating lesions and enhances functional recovery [ 38 ]. Information that is newly obtained from external sources is initially stored as short-term memory after which it is converted into long-term memory through recurring exposure [ 39 ]. It is also indicated by our previous findings that a significant role is played by glucocorticoid therapy in the improvement in learning and memory in the EAE model, whereas these effects are supported by exercise[ 40 ]. In other neurological disease such as Alzheimer disease, the endurance training showed delays Alzheimer’s disease and reduces dementia in the elderly [ 41 ]. Exercise also has a close relationship with cognitive function. Through numerous cellular and molecular mechanisms, it promotes angiogenesis, synaptogenesis, and neurogenesis which enhance memory and brain plasticity [ 42 , 43 ].Exercise also determined the positive changes in the hippocampal tissue and learning and memory also showed improved because adverse changes in the structure of the hippocampus play an essential role in the impairment of learning, and memory [ 41 ]. This regulation will bring about an improvement in the long term potentiation (LTP) following the recovery in the EAE model. It has been shown in various studies that memory consolidation following training is enhanced with the dose-dependent administration of glucocorticoids [ 44 ]. In addition, LTP induction is related to the activation of a significant number of signaling cascades, including those that are activated by BDNF [ 45 ]. The long-held belief that the number of neurons in the brain remains constant during prenatal and neonatal development has been disproved by evidence showing adult brains can manufacture new neurons through a process known as neurogenesis, which can reduce the harmful consequences of neurodegeneration [ 46 ]. Conclusion According to the findings of this study, BDNF and TrkB mRNA levels increased after four weeks of exercise training, which also improved swimming speed. Furthermore, four weeks of exercise training provided a protective effect on the maintenance of the hippocampus morphological structure during EAE disease. Declarations Acknowledgements We thankful to every one support us in this work . especially thanks for Dr Alaa salim from biology department in Basrah university who arranged the histological methodology for this study. Ethical considerations The experiments were carried out in accordance with the Helsinki Declaration's ethical guidelines (laboratory animals) and were funded by the Basra University of Medicine's Animals Lab (IR.Basrah.4107.022). Data Sharing Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. Author contributions MH, Investigation, writing first draft, project administration, data analysis. MLH, exercise training protocol. Conflict of interest Authors have no conflict of interest. funding sources This research did not receive any specific grant. References Hafedh, M. and A. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4623275","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":327664109,"identity":"0e4bcf1f-fa5f-4d75-9dd9-0c2a8ff002fb","order_by":0,"name":"Muthanna Hafedh","email":"data:image/png;base64,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","orcid":"","institution":"General Directorate of Education Basrah","correspondingAuthor":true,"prefix":"","firstName":"Muthanna","middleName":"","lastName":"Hafedh","suffix":""},{"id":327664110,"identity":"de3d03e6-be80-4d39-a919-9cdf116856e2","order_by":1,"name":"Muthana Layth Hatem","email":"","orcid":"","institution":"University of missan, missan","correspondingAuthor":false,"prefix":"","firstName":"Muthana","middleName":"Layth","lastName":"Hatem","suffix":""}],"badges":[],"createdAt":"2024-06-22 22:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4623275/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4623275/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60876533,"identity":"559601a1-dcae-431e-9e22-3fa67a1175fa","added_by":"auto","created_at":"2024-07-23 06:08:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21987,"visible":true,"origin":"","legend":"\u003cp\u003eExercise effect on BDNF and TrkB mRNA expression in EAE. Four-week exercise program demonstrated the BDNF expression in the hippocampus in three groups. Expression levels and represent the mean and standard deviation. Asterisks indicate statistically significant differences between the EAE+Ex (P\u0026gt; 0.05), compared to EAE as well as significant differences between the Co group (P\u0026lt; 0.01). \u0026nbsp;compared to the EAE group (P\u0026gt;0.02). As depicted in fig. B. TrKB expression was downregulated in the EAE group compared to the Co group (P\u0026gt;0.05). Values are expressed as a percentage of control expression levels and represent the mean and standard deviation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4623275/v1/3eeb31297166369daf9cd59d.png"},{"id":60876537,"identity":"2e1d9a97-ba10-4bab-a5ad-caabe22a1260","added_by":"auto","created_at":"2024-07-23 06:08:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21734,"visible":true,"origin":"","legend":"\u003cp\u003eSwimming speed in Co group showed no significantly change in post test compared to prior test. As well as showed in the results the swimming speed in the prop test was considerably slower in the EAE group prior text compared to post text (P\u0026lt;0.01) ***. The performance of the probe test in EAE+Ex group was significantly faster to reach to the platform area in post text (P\u0026gt;0.02)* compared to the prior test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4623275/v1/386fa24cc4ca1cef65327eaf.png"},{"id":60876534,"identity":"3c649a76-ac0f-42d1-86d9-783cfc51ffa5","added_by":"auto","created_at":"2024-07-23 06:08:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13765,"visible":true,"origin":"","legend":"\u003cp\u003eillustrate no significantly different of swimming speed in EAE+Ex compared to Co group. Although, a significantly of swimming speed (P\u0026gt;0.01) in Co group ** compared to EAE group. A significantly higher in EAE+Ex group (P\u0026gt;0.02) compared to EAE\u003csup\u003e# \u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4623275/v1/ae4b92a28036fa5dc372e7eb.png"},{"id":60876536,"identity":"2e0139e5-7c22-43e2-b463-3007b5340dc2","added_by":"auto","created_at":"2024-07-23 06:08:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212741,"visible":true,"origin":"","legend":"\u003cp\u003eHistological representation of hippocampus morphology. Histological section of the Co group (A) of rats show normal structure and hippocampus morphology. EAE inoculation (B) led to unstructured hippocampal tissue with decreased dendritic spine density and length. EAE-Ex presented with slightly increased dendritic spine density, dendritic spine length, and number of cells in the hippocampal region Cresyl violet stain (indicated by black arrows).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4623275/v1/843bba5f2565c91b60068a64.png"},{"id":83493219,"identity":"f90b52f0-9e88-45f5-b350-272dcfde8252","added_by":"auto","created_at":"2025-05-27 10:53:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":999577,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4623275/v1/6b57ae71-4198-45b4-893a-c34ab34f5a33.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exercise training increase BDNF/TrkB mRNA, prevents memory loss and ameliorate the hippocampal morphology in multiple sclerosis model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMultiple sclerosis (MS) affects young people (mean age at onset, 20\u0026ndash;30 years) and is characterized by inflammatory demyelination and axonal transection[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It can result in physical deterioration, mental decline and a lower quality of life [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous studies have identified a number of mechanisms that play a role in the pathogenesis of MS including the death of neurons as a result of oxidative stress, immune cells invading the central nervous system, the introduction of prolactin, melatonin, and opioids as well as immune cells and neuro-immunomodulatory substances [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA highly utilized murine model for studying MS is experimental autoimmune encephalomyelitis (EAE)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The EAE rat model of MS is a model of this disease as it clearly shows hind-limb paralysis, nerve damage and memory dysfunction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Learning and memory impairment, i.e., impairment that is experienced while acquiring new information, is prevalent disabling and not managed adequately in MS [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A common type of cognitive weakness experienced in MS is memory deficits which are found in 40\u0026ndash;60% of patients suffering from the disease [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A critical brain structure that plays a part in the formation of learning and memory is the hippocampus and there is often a relationship between its pathology and memory problems. It shows the highest neuroplasticity within the brain while also showing active neurogenesis all through adulthood [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBrain-derived neurotrophic factor (BDNF) is a candidate molecular effector of neuroprotective autoimmunity. BDNF is an effective neurotrophic protein that supports neural development and survival produced by immune cells in peripheral blood and MS lesions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. According to a previous study, the neurotrophin BDNF is an important component in the remyelination and/or structural restoration of myelin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBDNF and its high-affinity receptor (TrkB ) are important molecules in the CNS that affect behavior, mood and cognitive functions like memory and learning [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Long-term learning and memory necessitate synaptic plasticity in which BDNF plays an important role [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Through the TrkB receptor in hippocampus neurons, BDNF promotes synaptic transmission and increases plasticity at the neuromuscular synapse [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hippocampal-dependent learning requires BDNF-TrkB activation for consolidation which is followed by a delayed stabilization phase that is crucial for retaining that memory [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Physical activity reduces the dendritic irregularities associated with experimental autoimmune disease [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Leavitt et al. has reported that aerobic exercise improves hippocampus volume and improves cognition in people with multiple sclerosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral treatment approaches are available that concentrate on self-management, physical activity and the quality of life of those suffering from MS. A key treatment for maintaining and enhancing functional status is exercise therapy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Studies have found that physical activity up-regulates hippocampal BDNF which may be associated with mood states, memory and learning to decrease the deterioration of cognitive function related to MS. Furthermore, anti-oxidant defenses and neurotrophic support may be improved by exercise which could decrease the vulnerability of the CNS to neuronal degeneration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Through physical training, the levels of BDNF and its TrkB receptor can be positively regulated in various brain structures, particularly in the hippocampus in experimental animals as well as humans[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, it has been shown that there is an increase in serum BDNF concentrations right after a single session of light aerobic exercise in people with and without MS[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the current study, we investigated the effect of four weeks of exercise training on BDNF-TrkB expression in the hippocampus as well as hippocampus morphology and memory in the EAE Model.\u003c/p\u003e"},{"header":"Experimental subjects","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e30 female Lewis rats with an age of 8\u0026ndash;10 weeks and a weight of (100\u0026ndash;125 kg) were purchased from DarouPakhsh Pharmaceutical Company, Iran. Individually housed in special cages, the animals are kept in a controlled environment (12/12 h light and dark) and fed standard rodent chow and water. The animals were acclimated to their new surroundings for one week before being divided into one of three groups (10 rats each): control (Co), EAE only (EAE) and an EAE exercise group (EAE+Ex).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstrous cycle evaluation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs reported previously\u0026nbsp;[1], Female rats estrous cycles were observed every day. The rats with estrous cycles that began on the same day were chosen. Between 7 and 8 in the morning, vaginal lavage was carried out with a mild manual restraint. A plastic pipette with the tip inserted inside the animal vagina and 10\u0026mu;l of normal saline (0.9%) was added to collect vaginal excrement. The glass slides were covered with vaginal fluid. Each rat had one drop of vaginal fluid removed with a clean tip.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorris Water Maze\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Morris Water Maze (MWM) is considered to \u0026nbsp;be the gold standard to assess spatial learning as well as memory capacity \u0026nbsp;[24].\u0026nbsp;This approach is based on the observation that animals have developed an efficient manner of navigating their surroundings and escaping danger that allows them to attain the desired outcome with the least amount of effort\u0026nbsp;[25, 26].\u0026nbsp;The MWM used in this study was a black circular tank with a diameter of about 136 cm and a height of 60cm filled with water (21\u003csup\u003eo\u003c/sup\u003eC) at a depth of 25 cm. Maze was divided into four quadrants: northeast, northwest, southeast, and southwest as well as starting positions: north, south, west, and east. A concealed circular platform with a diameter of 10 cm was situated in the SW quadrant\u0026apos;s center, 1.5 cm below the water\u0026apos;s surface. Each test was recorded and assessed using a video camera connected to a tracking device (EthoVision XT) situated above the pool to calculate the time in each quadrant and swimming velocity of each rat. \u0026nbsp;Animals were allowed to adapt to the WMM test for two days. The MWM test was implemented across five consecutive days: four days for learning and one day as prop test. All sessions began at 8:00 a.m. For the training days, each rat was placed at one of the four predetermined beginning locations in the water, facing the tank\u0026apos;s wall, and then given free reign to swim until they reached the platform that was concealed inside the tank. Every trial required every rat to locate the hidden platform within 60 seconds. If the rat discovered the platform, it was permitted to stay there for 30 seconds. If the rat did not find the platform within 60 seconds, it was placed on platform 30 seconds. The prop test was performed on day 5 to assess memory. On the prop test day, the platform was removed from the tank and the time it took the rat to reach the area in which the platform was previously placed was measured as well as time spent in that specific area. The test was discontinued at 60 seconds. Assessments were completed at baseline prior to EAE induction and again 30 days post inoculation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction and Clinical Disability Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously described\u0026nbsp;[27], 50 ug of guinea pig spinal cord homogenate and 2 mg of heat-mycobacterium (Difco H37Ra) were added to incomplete Freund\u0026apos;s adjuvant (Sigma). A subcutaneous injection of 50 ul of the inoculation was given into the base of the tail. The presence of any indicators of impairment was assessed using EAE scores previously described\u0026nbsp;[28]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExercise Training and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor seven days, the animals practiced running for 15 minutes each day at a belt pace of 5 to 10 meters per minute without slop. Rats were made to run by administering an electric shock (0.3 mA). As described by Kim and colleagues\u0026nbsp;[29]\u0026nbsp;exercise training was started 2 weeks prior EAE immunization (preconditioning protocol) and continued until 2 weeks post EAE immunization. Exercise was prescribed starting at 17 m/min and increased each week (week 1: 17 m/min, week 2: 18 m/min, week 3: 19 m/min, and week 4: 20 m/min) for a total duration of 30 minutes per day, five days a week. Rats in the non-exercise groups were placed on the treadmill lane with the machine turned off and were subjected to the same schedule as the rats in the exercise groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnaesthesia and euthanasia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were euthanized by ketamine and xylazine (3/1) and the animal brains were extracted. Hippocampal tissue was quickly dissected and frozen in liquid nitrogen immediately. Frozen tissues were homogenized in the Trizol reagent at a 1 ml Trizol/100 mg tissue ratio. The left hippocampal was dissected and frozen at 80\u0026deg;C for gene expression analysis. The right hippocampal was also dissected for morphology investigation, post-fixed in 4% formaldehyde and immersed in double-distilled water.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReal-time polymerase chain reaction (PCR) was utilized to analyse TrKB and BDNF mRNA in the hippocampus. Total RNA was extracted from samples using the TRIzol kit (Invitrogen, USA) in accordance with the manufacturer\u0026apos;s instructions. After RNA purification, TrKB and BDNF gene primers were made using the NCBI website \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e. Real-time PCR was carried out using the Applied Biosystems TM Real-Time PCR apparatus with Master Mix and SYBR Green I (Fluka, Switzerland) as the starting materials. Using a standard curve, the efficacy of each gene was determined. GAPDH was used as a checkpoint.\u003c/p\u003e\n\u003cp\u003eTable 1. The nucleotide sequences of sense and antisense primers.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCresyl violet staining method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Cresyl violet staining was used to count the number of neurons in the right hippocampus. Brain slices are dried in three sequential steps: 70% alcohol (for 10 min), 95% alcohol (with a few drops of 10% acetic acid; for 2-3 min) and finally 100% alcohol after creating 5 m-long microtome incisions and undergoing tissue processing (for 10 min). Slices were washed in xylene for five minutes, colored with cresyl violet, covered with DPX, and then sealed with a cover slip.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mean and standard error are used to represent the data. The data were analyzed using a two-way variance analysis (ANOVA). Repeated measures were used in the MWM and significance was assessed using P values \u0026lt;0.05. The normality and homogeneity of the data was determined using Tukey post-hoc test.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExercise training increased BDNF and TrKB mRNA in hippocampus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ANOVA revealed significant difference between the three groups. Post hoc-analysis received that the EAE significantly reduced both BDNF mRNA (P\u0026lt;0.02) and TrkB mRNA (P\u0026gt;0.01) compared to Co group. Although, EAE+Ex group showed a higher BDNF mRNA (P\u0026gt;0.05) and TrkB mRNA (P\u0026gt;0.02) compared to EAE group, respectively. No significant difference was observed between the Co and the EAE+Ex group. \u003cstrong\u003e(Figure 1).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExercise helps preserve memory in EAE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComparison between post test and prior test was following the animal recovery from the EAE disease and ability the animals to move as evidenced by their swimming speed. The prop test was used by removing the platform from the tank. Two-way ANOVA found significant differences among groups. Post-hoc results didn\u0026rsquo;t find any significant difference within the Co group in prior text compared to the post test. Further, swimming speed in the prop test was considerably slower in the EAE group prior text compared to post text (P\u0026lt;0.01). In contract, EAE+Ex group was able to improve swimming speed time to reach to the platform area in \u0026nbsp;post text (P\u0026gt;0.02) compared to the prior test). \u003cstrong\u003eFig2\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eBetween group comparisons found no difference in swimming speed between the Co group and the EAE+Ex group. Co group showed a significant difference (P\u0026gt;0.01) compared to the EAE. EAE-Ex group inducted significant (P\u0026gt;0.02) compared to the EAE group, respectively. (\u003cstrong\u003eFig.3)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHippocampal Morphology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere were no histological changes in the hippocampal morphology in Co group and normal dendritic spine density, dendritic spine length and the number of cells in the hippocampal region were normal \u003cstrong\u003e(Fig4.A).\u0026nbsp;\u003c/strong\u003eAnimals in the EAE group had unstructured hippocampal tissue with decreasing dendritic spine density and length as well as several cells in the hippocampal region \u003cstrong\u003e(Fig.4.B)\u003c/strong\u003e . Further, EAE+Ex group had slightly higher dendritic spine density, dendritic spine length and hippocampal cell number \u003cstrong\u003e(Fig.4.C).\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFor some hippocampus-dependent processes, increasing exercise-induced hippocampal neurogenesis is advantageous. The increased production of certain transmitters, growth factors, or neurotrophic factors during exercise, however, may regulate overall dynamic balance [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In line with this, data from this study demonstrated that the mRNA of BDNF and its TrkB receptor in the hippocampus were increased following the four weeks of exercise training in the EAE\u0026thinsp;+\u0026thinsp;Ex group after day 30 dpi. Our findings are consistent with the previous studies [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] that suggested that exercise training leads to an increase in the expression of BDNF mRNA in the hippocampus compared to a sedentary EAE group. An increase in BDNF mRNA is one of the persistent and potent effects of exercise on the brain[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. BDNF/TrkB expression and activation is important for neurophysiological processes like neuroprotection, neuromaturation and maintenance of neurons. According to previous studies, EAE and similar neurodegenerative disorders are related to both activation and malfunction of the BDNF-TrkB pathway. It was noted in exercise literature that there was a higher shortage of TrKB in the animal model of MS disease. With the passage of time, there is a decrease in BDNF and TrkB expression in the hippocampus and temporal cortex in humans which puts them at a higher risk of experiencing various neurodegenerative pathologies [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].The findings of this study indicate that memory deficits occurred in the preliminary stage of the disease (30 dpi) in the EAE group. Our findings support the notion that memory deficits in EAE are common in the early stages of MS [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] because the volume of the CA1 region first decreases during the early stages of hippocampal injury, which then spreads to the CA2, CA3, and dentate gyrus [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The present study showed an improvement in memory in the EAE\u0026thinsp;+\u0026thinsp;Ex group, this was accompanied by enhanced swimming speed activity. In line with this study, our previous findings indicated that exercise training improved memory while also promoting adrenal gland recovery during the EAE stage [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Furthermore, studies have shown that in the absence of exercise training, cognitive impairment was observed in the EAE model on day 90 after induction rather than day 30 and persisted until the end of motor recovery [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, Jun-Jun Sun and colleagues found that cognitive impairment occurred later in the course of EAE (55 dpi) as opposed to the early stage (13 dpi) of the illness. Although Kurkowska and colleagues [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] found no cognitive abnormalities in EAE animals compared to controls, the researchers believe that a relapse of MS may start the neurodegenerative process inside the hippocampus, and that this may not always be associated with memory difficulties (90). In line with this thinking, the present study findings also showed that the hippocampal tissue had significant damage in rats in the EAE group, while the EAE-Ex group showed significantly different modulation of hippocampal morphology. Cell proliferation is thought to be about two times higher during the acute phase of the disease compared to controls [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Additionally, Huehnchen and colleagues (2011) observed that not only in the acute phase of the disease but also in the chronic phase, there were more DCX\u0026thinsp;+\u0026thinsp;and BrdU\u0026thinsp;+\u0026thinsp;cells, as an indication of cell proliferation, in the DG of EAE-Ex than in controls. However, this outcome does not imply that the EAE-Ex group will return to normal [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. According to a study conducted by Magalon et al. (2007), environmental enrichment promotes neural progenitor migration into demyelinating lesions and enhances functional recovery [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Information that is newly obtained from external sources is initially stored as short-term memory after which it is converted into long-term memory through recurring exposure [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. It is also indicated by our previous findings that a significant role is played by glucocorticoid therapy in the improvement in learning and memory in the EAE model, whereas these effects are supported by exercise[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In other neurological disease such as Alzheimer disease, the endurance training showed delays Alzheimer\u0026rsquo;s disease and reduces dementia in the elderly [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExercise also has a close relationship with cognitive function. Through numerous cellular and molecular mechanisms, it promotes angiogenesis, synaptogenesis, and neurogenesis which enhance memory and brain plasticity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].Exercise also determined the positive changes in the hippocampal tissue and learning and memory also showed improved because adverse changes in the structure of the hippocampus play an essential role in the impairment of learning, and memory [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This regulation will bring about an improvement in the long term potentiation (LTP) following the recovery in the EAE model. It has been shown in various studies that memory consolidation following training is enhanced with the dose-dependent administration of glucocorticoids [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, LTP induction is related to the activation of a significant number of signaling cascades, including those that are activated by BDNF [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The long-held belief that the number of neurons in the brain remains constant during prenatal and neonatal development has been disproved by evidence showing adult brains can manufacture new neurons through a process known as neurogenesis, which can reduce the harmful consequences of neurodegeneration [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAccording to the findings of this study, BDNF and TrkB mRNA levels increased after four weeks of exercise training, which also improved swimming speed. Furthermore, four weeks of exercise training provided a protective effect on the maintenance of the hippocampus morphological structure during EAE disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thankful to every one support us in this work . especially thanks for Dr Alaa salim from biology department in Basrah university who arranged the histological methodology for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were carried out in accordance with the Helsinki Declaration's ethical guidelines (laboratory animals) and were funded by the Basra University of Medicine's Animals Lab (IR.Basrah.4107.022).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing Statement:\u003c/strong\u003e The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMH, Investigation, writing first draft,\u0026nbsp;project administration, data analysis. MLH, exercise training protocol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003efunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHafedh, M. and A. Parnow, \u003cem\u003eExercise training improves memory and produces changes in the adrenal gland morphology in the experimental autoimmune encephalomyelitis.\u003c/em\u003e Endocrine Regulations, 2022. \u003cstrong\u003e56\u003c/strong\u003e(1): p. 31-37.\u003c/li\u003e\n\u003cli\u003eMcGinley, M.P., C.H. Goldschmidt, and A.D. Rae-Grant, \u003cem\u003eDiagnosis and treatment of multiple sclerosis: a review.\u003c/em\u003e Jama, 2021. \u003cstrong\u003e325\u003c/strong\u003e(8): p. 765-779.\u003c/li\u003e\n\u003cli\u003eMahmoodi, M., F. 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Roozendaal, \u003cem\u003eStress, glucocorticoids and memory: implications for treating fear-related disorders.\u003c/em\u003e Nature Reviews Neuroscience, 2017. \u003cstrong\u003e18\u003c/strong\u003e(1): p. 7-19.\u003c/li\u003e\n\u003cli\u003eKafitz, K.W., et al., \u003cem\u003eNeurotrophin-evoked rapid excitation through TrkB receptors.\u003c/em\u003e Nature, 1999. \u003cstrong\u003e401\u003c/strong\u003e(6756): p. 918-921.\u003c/li\u003e\n\u003cli\u003eVan Praag, H., et al., \u003cem\u003eExercise enhances learning and hippocampal neurogenesis in aged mice.\u003c/em\u003e Journal of Neuroscience, 2005. \u003cstrong\u003e25\u003c/strong\u003e(38): p. 8680-8685.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Exercise, EAE, BDNF, TrKB, Memory, Morphology","lastPublishedDoi":"10.21203/rs.3.rs-4623275/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4623275/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e: We aimed to verify the implications of four weeks of consistent exercise on the mRNA of the brain-derived (BDNF) and the tropomyosin-related kinase B (TrkB) receptor that may contribute to ameliorates the hippocampal morphology and induced enhancement of memory in experimental autoimmune encephalomyelitis (EAE) after the diseases recovery stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Female Lewis rats were randomly separated into three groups, control group (Co), EAE without exercise (EAE), EAE with exercise (EAE+Ex) , each group contains (10 rats) . All rats underwent to the Morris water maze (MWM) test for four days in week and prop test was done in fifth day and repeated in day 30 post induction. Exercise was prescribed by motorized treadmill starting at 17 m/min and gradually increased, a total duration of 30 minutes per day, five days a week and discontinued two weeks after the induction. EAE was induced subcutaneously after two weeks of exercise as rats injected with an inoculum containing 50 ug guinea pig spinal cord homogenate emulsified in incomplete Freund’s adjuvant and 2mg of heat-mycobacterium at the tail base.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: EAE+Ex significantly increases the BDNF mRNA (P\u0026lt; 0.05) and TrkB (P\u0026gt; 0.02) mRNA levels compared to the EAE groups. The Co group increased significantly more than the EAE group (P\u0026lt; 0.01). Swimming speed performance was faster in EAE+Ex (P\u0026lt; 0.02) compared to EAE group lead to improved hippocampus function.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: It seems that the provision of four weeks of exercise training is capable of increasing the mRNA of BDNF and its receptor along with ameliorating the hippocampus morphological which can have positive effects on memory impairment during EAE disease.\u003c/p\u003e","manuscriptTitle":"Exercise training increase BDNF/TrkB mRNA, prevents memory loss and ameliorate the hippocampal morphology in multiple sclerosis model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 06:08:10","doi":"10.21203/rs.3.rs-4623275/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":"cfdf49ea-95a2-44d5-84a5-554917c8ef07","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-05-27T10:53:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-23 06:08:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4623275","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4623275","identity":"rs-4623275","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}