The effect of physical activity on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling model

The effect of physical activity on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling 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 The effect of physical activity on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling model abbas kebriaeezadeh, Reza Zaferi, mohammad Sharifzadeh, Javad Mirnajafi-zadeh, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7215509/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jan, 2026 Read the published version in DARU Journal of Pharmaceutical Sciences → Version 1 posted You are reading this latest preprint version Abstract Background A recent body of evidence has suggested regular exercise as a promising complementary therapeutic strategy in the management of epilepsy and its related cognitive impairments. Objectives To put it to the test, our study aimed to comparatively examine the effects of physical exercise, low and high doses of levetiracetam, or the combination of both on amygdala electrical kindling-induced epilepsy in rats, as well as the consequent learning and memory impairments. Methods Male Wistar rats were randomly divided into ten groups (n = 7 per group) as the following: (I) Control (without kindling and exercise), (II) Lev (receiving a high dose of levetiracetam without kindling and exercise), (III) Ex (subjected to exercise without kindling), (IV) Ex-K (subjected to preventive exercise before kindling), (V) K (subjected to kindling without any intervention), (VI) K-Ex (subjected to exercise after kindling), (VII) K-L lev (receiving a low dose of levetiracetam after kindling), (VIII) K-H lev (receiving a high dose of levetiracetam after kindling), (IX) K-Ex-L lev (subjected to exercise and receiving a low dose of levetiracetam after kindling), and (X) K-Ex-H lev (subjected to exercise and receiving a high dose of levetiracetam after kindling). After the kindling procedure and interventions, the seizure parameters, including dADD, S 1 L, S 2 L, S 3 L, S 4 L, S 5 L, Max S5D, and Max ADD, were recorded, and seizure-related behavioral changes were evaluated using the MWM test. Results Our findings showed that in all therapeutic interventional groups, including Ex, L lev, H lev, and their combination (Ex-L lev and Ex-H lev), there was a substantial reduction in parameters, including seizure stages, seizure duration, and dADD. In contrast, there was a significant increase in the mean delay time or latency from electrical stimulation to the onset of stages 1, 2, and 3 of seizure (S 1 L, S2L, and S3L), and all groups were significantly different from the kindling group. Moreover, the kindling-induced spatial memory and learning deficit was remarkably ameliorated by preventive exercise, Ex, L lev, H lev, and their combination. Conclusion Our study reveals that, in conjunction with levetiracetam, regular exercise can ameliorate the intensity and frequency of amygdala electrical kindling-induced epileptic seizures, as well as the consequent spatial memory and learning impairments. Epilepsy Physical exercise amygdala electrical Kindling Levetiracetam Learning Memory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Epilepsy is one of the most common noncommunicable and chronic impairments of cerebral function characterized by the spontaneous and symptomatic recurrent incidence of seizures. Approximately, this neurological dysfunction affects 0.5-1% of the human population worldwide and highly impacts their quality of life by long-term sequelae [ 1 ]. Depending on the seizure origin site, this disorder is divided into two main classes, namely partial and generalized seizure [ 2 ]. In particular, complex partial seizures are the most common phenotype among other types of epilepsy, with a greater number of such epileptic seizures thought to originate from temporal lobes [ 2 , 3 ]. Despite significant advances in pharmacological approaches, epilepsy is not satisfactorily controlled in almost 20–35% of the patients and is considered drug-resistant epilepsy (DRE) [ 4 ]. On the other hand, long-term administration of antiepileptic medications often leads to undesirable adverse effects, such as neurocognitive and neurohormonal disturbances, cardiovascular disorders, osteoporosis, and liver and thyroid dysfunction [ 5 – 7 ]. Hence, experimental and non-pharmacological therapeutics such as regular physical exercise have been extensively investigated for their potential to prevent and/or treat epileptic insults [ 8 , 9 ]. To date, several studies in both animals and humans have demonstrated the favorable impact of regular physical activity programs as a supportive treatment strategy for decreasing epileptic seizure severity and frequency, as well as promoting psychological and cardiovascular health. However, the underlying mechanisms responsible for these beneficial effects have not yet been understood [ 8 – 10 ]. Most of these studies reported reduced ictal activity, delayed development of amygdala kindling, prolonged convulsion threshold, reduced neuronal damage and behavioral alterations, diminished susceptibility to subsequently provoked seizures after exercise, as well as improved neurogenesis and positive plastic neurochemical changes in the hippocampal formation [ 11 ]. From a mechanistic point of view, it has been shown that regular physical exercise induces neurogenesis, increases blood flow to the brain, modulates balance between excitatory and inhibitory neurotransmitter synthesis and activity, and enhances synaptic plasticity, particularly through the production of BDNF, a well-known neurotrophic factor, that plays a crucial role in supporting synaptic formation and plasticity and attenuates detrimental processes, including neuroinflammation, mitochondrial fragmentation, oxidative stress, and neuronal apoptosis [ 12 , 13 ]. Patients who have epilepsy often experience a series of comorbidities, including psychiatric disorders, cognitive dysfunction, behavioral disabilities, and even death if not adequately treated [ 14 – 16 ]. The repeated occurrence of epileptic seizures remarkably reduces learning and memory cognitive functions in affected patients and individuals with temporal lobe epilepsy (TLE) who usually suffer from visuospatial memory complications [ 17 , 18 ]. The occurrence of memory and learning defects in experimentally induced epilepsy in animal models has been confirmed in previous preclinical studies [ 19 ]. The amygdala kindling-induced epilepsy model is one of the widely-used epilepsy models that have considerable similarity with the incidence of epilepsy in humans [ 20 , 21 ]. The kindling method involves focal sub-threshold excitation by repeated and low-frequency electrical stimulation or repetitive administration of sub-convulsive doses of chemicals such as kainic acid (KA), pentylenetetrazol (PTZ), pilocarpine, and metals that can offer insights into the underlying mechanisms and provide new avenues for the development of novel potential therapeutic interventions [ 20 , 22 ]. Levetiracetam, a practical, potent, and relatively safe antiepileptic drug, is one of the most commonly prescribed medications for the treatment and/or control of epilepsy, particularly in benzodiazepines-resistant epilepsies or when first-line treatments are not applicable due to their undesirable effects [ 23 ]. Mechanistically, levetiracetam specifically binds to and inhibits the transmembrane protein located on the synaptic vesicle SV2A, thereby hindering the neurotransmitter priming and intensification of neurotransmission during exaggerated provocation of presynaptic fibers [ 24 ]. Based on previously established findings, the development of kindling was remarkably quicker in SV2A-deficient mice [ 25 ]. Therefore, it can be concluded that levetiracetam reduces the kindling epileptogenesis via modulating the SV2A functions in synaptic transmission [ 26 ]. Based on the mentioned mechanistic aspects of physical exercise, and given the lack of effectiveness of current therapies for epilepsy, a growing body of literature has been interested in the therapeutic potential of regular physical exercise in the prevention or treatment of epilepsy. Thus, the purpose of the current study was to evaluate and compare the effect of physical exercise, low- and high-dose levetiracetam, and their combination on epileptic seizures and consequent learning and memory impairment in amygdala electrical kindled rat model. Materials and Methods Animals Seventeen male Wistar rats weighing 220 ± 20 g (about 8–10 weeks old, specific pathogen-free or SPF grades) were provided by the animal house of the Faculty of Pharmacy at Tehran University of Medical Sciences (Tehran, Iran). The animals were housed in a specific-pathogen-free cage with a 12/12 h light-dark cycle at a temperature of 24 ± 2°C and relative humidity of 30–70% in an air-conditioned room. The rats were fed with a rodent diet and water ad libitum during the experiments. All experiments were approved by the Research Ethics Committees of Biosafety & Laboratory, Tehran University of Medical Sciences & Health Services (Ethical code: IR.TUMS.TIPS.REC.1397.015). Ethical principles and considerations, as delineated in the National Institute of Health (NIH), were complied with by all animals and at all steps of the research to minimize pain and distress. Experimental design As presented in Table 1 , rats were randomly divided into 10 groups (7 rats/group). A 10-day recovery period from the stereotaxic procedure to electrode implantation was employed to perform amygdala kindling in rats. Before performing behavioral tests, rats were re-stimulated for 1 day, a process that served as a challenge. After performing all procedures and interventions, as well as completing the behavioral tests, all groups were sacrificed using standard CO 2 chambers. Table 1 Experimental design for evaluation of levetiracetam administration plus physical exercise on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling model. Group No. Interventions Group 1 Control: rats were not submitted to physical exercise and kindling and received only saline trough i.p. administration Group 2 Lev: rats were not submitted to physical exercise and kindling and received only high dose of levetiracetam trough i.p. administration for 4-weeks Group 3 Ex: rats were subjected to physical exercise but were not submitted to kindling after surgery and recovery period Group 4 Ex-K: rats were submitted to 4-weeks physical exercise before surgery and then subjected to kindling Group 5 K: rats were submitted to kindling after surgery and recovery period and then received saline trough i.p. administration Group 6 K-Ex: rats were submitted to 4-weeks physical exercise after surgery and kindling Group 7 K-L lev: rats were received to 4-weeks low dose of levetiracetam (27 mg/kg BW, once a day) trough i.p. administration after surgery and kindling Group 8 K-H lev: rats were received to 4-weeks high dose of levetiracetam (54 mg/kg BW, once a day) trough i.p. administration after surgery and kindling Group 9 K-Ex-L lev: rats were submitted to 4-weeks physical exercise plus treatment with low dose of levetiracetam (27 mg/kg BW, once a day) trough i.p. administration after surgery and kindling Group 10 K-Ex-H lev: rats were submitted to 4-weeks physical exercise plus treatment with high dose of levetiracetam (54 mg/kg BW, once a day) trough i.p. administration after surgery and kindling Physical exercise program After recovery from stereotaxic surgery and acclimatization to the treadmill, rats were subjected to forced physical exercise once a day for 30 minutes, 5 days a week, for 4 weeks. The exercise program included running on a treadmill as described in the following: (I) 5 minutes at 2 meters per minute, (II) 5 minutes at 5 meters per minute, and (III) the last 20 minutes at an 8 meters per minute speed [ 27 ]. It should be noted that such a physical exercise protocol was not performed for all groups. The rats belonging to the Control, Lev, Ex, K, K-L Lev, and K-H Lev groups were placed on the treadmill for 30 minutes daily for 4 weeks at a speed of 0 m/s. The incline of the treadmill was 0%, and a piece of foam or synthetic sponge was placed at the tag end of the treadmill to protect the rats from sticking at the tag end of the device. Rats were monitored during physical exercise to evaluate the signs of potential stress or pain. At the end of the physical exercise period and one day after the challenging rats, the behavioral test MWM was carried out. Surgical procedure for electrode implantation Following deep general anesthesia by i.p. administration of 100 mg/kg BW ketamine 10% (Alfasan, Netherlands) and 10 mg/kg BW xylazine 2% (Alfasan, Netherlands), the animals were placed and fixed on the stereotaxic device as their skull was in a perfectly horizontal position. The animals were fixed on the stereotaxic instrument, protected by the placement of a bar behind the incisors and two bars at the temples. The dental bar was adjusted so that the coordinates of Bregma and Lambda were parallel in height. The scalp was incised vertically from the area between the two eyes to the end of the occipital bone, and the bone was dried with a tampon so that the Bregma and Lambda could be seen. After specifying the position of the right basolateral nucleus of the amygdala (BLA), the coordinates of the electrode implementation were determined based on the paxinos atlas [ 28 ]. The coordinates were extracted from the appropriate atlas section to access this region: the AP = − 2/5, L = + 4/8, and V= -7.5. During surgery, the rats were monitored for evaluation of consciousness signs using previously established methods such as checking paw and eye reflexes, as well as dose repetition and adjustment if necessary. The stainless bipolar electrodes (A-M Systems Inc, USA) with 127 µm diameter were implanted. In order to surface recording, four stainless steel screws were placed and adjusted on the skull using dental acrylics, one in the right forehead as electrical ground, and two others on the occipital and right head regions for EEG recording and were coupled with a stimulator and EEG amplifier. After surgery and electrode implantation, the surgical site was disinfected using a 10% povidone-iodine solution. Finally, the animals were housed individually in separate cages, and a 10-day recovery period was considered for wound healing and familiarization before the kindling process and interventions. The kindling process After a 10-day recovery period from surgery, the animals were transferred to a plexiglass cage (dimensions: 50×50×50 cm, made of transparent plexiglass), and the kindling procedure was started. The attached miniature plastic socket to the animals' skull was connected to a flexible cable with a noise-resistant shield. Rats were allowed to move freely within the recording cage. EEG recording was performed using a monitoring system with eTrace experiment software (version 1.39, Parto Danesh Institute, Tehran, Iran), and offline analysis was carried out using eTrace analysis software (Parto Danesh Institute, Tehran, Iran) on a computer. For performing kindling stimulations, the after-discharge threshold (ADT) was assessed for each rat at the beginning of the kindling process. For this purpose, the BLA region of animals was stimulated based on the following parameters: 20 µA single-phase square pulse; frequency = 50 Hz; wave width = 1 ms, and duration = 2 s. If afterdischarge (AD) waves were recorded (for at least 15 seconds), this current intensity was known as the threshold intensity; otherwise, the intensity was increased by 10 µA at 20-minute intervals until AD waves were recorded, which was designated as the ADT. Afterward, the semi-rapid kindling model (six times stimulation per day with the same intensity of ADT at 20-minute intervals and approximately the same time frame during the experiment) was used to induce seizures in rats. Seizure-related behavioral symptoms were classified and recorded based on Racine's scale [ 29 ]. Rats that exhibited stage 5 seizures during stimulation for three consecutive days and whose seizure parameters remained relatively invariant over continuous stimulations were considered ideally kindled subjects. Seizure parameters recording The comparable and measurable parameters following the kindling process in the current investigation included: (I) daily afterdischarge duration (dADD), (II) delay time from electrical stimulation to the onset of stage 1 of the seizure (stage 1 latency, S 1 L), (III) delay time from electrical stimulation to stage 2 of the seizure (stage 2 latency, S 2 L), (IV) delay time from electrical stimulation to stage 3 of seizure (stage 3 latency, S 3 L), (V) delay time from electrical stimulation to stage 4 of seizure (stage 4 latency, S 4 L), (VI) delay time from electrical stimulation to stage 5 of seizure (stage 5 latency, S 5 L), (VII) maximum duration of stage5 of seizure (Max S5D: Stage 5 Duration), (VIII) seizure duration and stages that developed in the animal in response to six times a day stimulation (both on the kindling and testing days), and (IX) The longest-lasting ADD per each day (Max ADD). Spatial learning and memory test After the post-intervention 1-day challenge, the spatial learning and memory assessment was performed using a Morris water maze (MWM) as described previously [ 30 ]. To evaluate the cognitive learning function, rats were subjected to training on four consecutive days to locate a hidden escape platform in a water-filled (23 ± 2°C) circular pool (150 cm in diameter, 60 cm in depth), with four trials per day. A memory test was performed on day 5 (probe test), with the removed platform removed from the maze. The movements of the rats were analyzed and processed by The Ethovision XT 11.5 video tracking software (Noldus Information Technology, Wageningen, Netherlands). Four parameters, including time delay in finding the platform (latency), traveled distance, the subjects' swimming speed, and time spent in the target quadrant, were evaluated on training days and the probe day, respectively (the first two parameters on training days and the last two parameters on the probe day). Statistical Analysis The results of our study were presented as Mean ± SEM and analyzed by Graph pad prism software (GraphPad, version 10.1, La Jolla, CA, USA). One-way and two-way ANOVA and Tukey's post hoc tests were used for statistical testing and comparisons for seizure parameters and MWM test results, respectively. An unpaired t-test was used to compare spent time, traveled distance, and probe in the control, Ex, H lev, and K groups. The P < 0.05 was considered statistically significant. Results Comparative analysis of seizure parameters As shown in Fig. 1 , one-way ANOVA analysis with Tukey's post hoc test revealed that the number of days required for full kindling of the amygdala in animals did not differ significantly among the different interventional groups. There was only an apparent and relative difference between the group subjected to preventive physical exercise (Ex-K, pre-kindling exercise) and the other groups. Still, this observed difference was not substantially meaningful. In addition, the threshold current intensity for the development of AD did not significantly differ among the various groups and was indeed 80–160 µA for all fully kindled rats in all groups. In the context of seizure parameters recording findings, two-way ANOVA analysis with Tukey's post hoc tests results demonstrated that there was a logical correlation between the all 10 experimental groups in the observation of total seizure stages (Fig. 2 A), the maximum seizure stages (Fig. 2 B), maximum seizure duration (Fig. 2 C), total seizure duration (Fig. 2 D), total AD duration (Fig. 2 E), and maximum AD duration (Fig. 2 F). Notably, only Ex-K group, which was submitted to preventive physical training before kindling, exhibited a marked difference with the K group on all days. As depicted in Fig. 3 , one-way ANOVA analysis with Tukey's post hoc test results indicated that there was a logical and positive correlation among all 10 experimental groups regarding the maximum duration of stage 5 of the seizure (Max S5D: Stage 5 Duration) (Fig. 3 A). There was also a significant correlation among all 10 experimental groups in the context of the mean delay time or latency from electrical stimulation to the onset of stage 1 of the seizure (stage 1 latency, S 1 L) (Fig. 3 B), stage 2 of the seizure (stage 2 latency, S 2 L) (Fig. 3 C), stage 3 of the seizure (stage 3 latency, S 3 L) (Fig. 3 D), stage 4 of the seizure (stage 4 latency, S 4 L) (Fig. 3 E), and stage 5 of the seizure (stage 5 latency, S 5 L) (Fig. 3 F). Notably, only the Ex-K group, which was submitted to preventive physical exercise before kindling, exhibited a marked difference from the K group on all days. The effects of treatments on seizure intensity following the kindling process (post-kindling alterations) As mentioned in the experimental design section, to evaluate the potential anticonvulsant effect of the treatments, rats underwent the rekindling process (six times at a 20-minute interval) on the day after the last intervention, and then various seizure parameters were investigated. In this regard, two-way ANOVA analysis with Tukey's post hoc test results showed that in all therapeutic interventional groups including physical exercise (Ex), low dose of levetiracetam (L lev), high dose of levetiracetam (H lev), and their combination (Ex-L lev and Ex-H lev), there was a substantial reduction in parameters, including seizure stages (Fig. 4 A), seizure duration (Fig. 4 B), dADD (Fig. 4 C), the mean delay time or latency from electrical stimulation to the onset of stage1 of seizure (stage 1 latency, S 1 L) (Fig. 4 D), stage 2 of seizure (stage 2 latency, S 2 L) (Fig. 4 E), and stage 3 of seizure (stage 3 latency, S 3 L) (Fig. 4 F) on the challenging (rekindling) day compared to same parameters during the main kindling process. All groups were significantly different from the kindling group. Spatial learning and memory As is evident from Fig. 5 , the one-way ANOVA analysis with Tukey's post hoc test results indicated that the speed of experimental animals did not differ significantly among all groups on the training days and probe day, indicating no difference in the level of gross motor activity among the different groups. The t-test analysis showed that the mean escape latency time of animals at consecutive 4-training days of MWM assay was not significantly different in the high-dose levetiracetam (H Lev) and physical exercise (Ex) groups compared to the control group (Co) (Fig.s 6A and 6B, respectively). On the other hand, and as a notable finding, there was a remarkable difference in control and preventive physical exercise groups compared to the kindling group (Fig. 6 C and 6 D, respectively). The t-test analysis showed that the mean travel distance of animals at consecutive 4-training days of MWM assay was not significantly different in the high-dose levetiracetam (H Lev) and physical exercise (Ex) groups compared to the control group (Co) (Fig. 7 A and 7 B, respectively). On the other hand, and as a notable finding, there was a remarkable difference in control and preventive physical exercise groups compared to kindling groups (Fig. 7 C and 7 D, respectively). As represented in Fig. 8 , the t-test analysis showed that the time spent in the target quadrant (Q1) of animals on the probe trial day of the MWM assay was not significantly different in the high-dose levetiracetam (H Lev) and physical exercise (Ex) groups compared to the control group (Co) (Fig. 8 A and 8 B, respectively). On the other hand, and as a notable finding, there was a remarkable difference in control and preventive physical exercise groups compared to kindling groups (Fig. 8 C and 8 D, respectively). The results indicated that animals in the kindling group (K) spent less time in the target quadrant (Q1) than the control group, and animals subjected to preventive physical exercise before kindling (Ex-K) spent more time in the target quadrant (Q1) than the kindling (K) group. According to Fig. 9 , two-way ANOVA analysis with Tukey's post hoc test results demonstrated that in physical exercise (Ex), low dose of levetiracetam (L lev), high dose of levetiracetam (H lev), and their combination (Ex-L lev and Ex-H lev) groups, there was a significant reduction in the mean escaped latency time (Fig. 9 A) and the mean the distance (Fig. 9 B) of animals at consecutive 4-training days as well as a considerable increase in time spent in target quadrant (Q1) (Fig. 9 C) of animals at probe trial day compared to kindling (K) group as reported by MWM assay. Discussion Given the relatively high prevalence of epilepsy, noticeable investigations are being conducted on its etiologic factors and safe novel approaches to prevent and/or treat the disease. To date, a growing number of preclinical studies and clinical trials have demonstrated that physical exercise exhibits beneficial and valuable therapeutic properties against epileptic seizures, as well as behavioral and cognitive complications, through various mechanisms [ 31 – 33 ]. In this regard, Aguiar et al. reported that a mild-intensity physical exercise program attenuated aging-associated impairments in long-term memory and spatial learning, as assessed by the Morris Water Maze (MWM) assay. Notably, the reported cognitive-improving features of mild-intensity exercise were mainly mediated by the activation of the AKT and CREB pro-survival signaling pathway, which resulted in a marked elevation of the mRNA expression and protein levels of BDNF in the hippocampus of aging animals [ 34 ]. In a study conducted by Gomes et al., physical exercise significantly diminished seizure frequency and reversed impaired long-term memory in adulthood following early-life status epilepticus induced by pilocarpine in rats, according to water maze task observations. Moreover, physical exercise enhanced cell proliferation, promoted anti-apoptotic responses, and normalized BDNF levels in the brains of rats, leading to improved hippocampal neuroplasticity. [ 35 ]. These results are in line with our study that revealed there was a significant reduction in the mean escaped latency time, and the mean distance traveled of animals at consecutive 4-training days as well as a considerable increase in time spent in the target quadrant (Q1) of animals at the probe trial day in rats subjected to regular physical exercise compared to kindling (K) group as reported by MWM assay. In another study, exposure to immediate and 60-day delayed exercise (daily 15 min swimming for 30 days) enhanced dendritic branch points and intersections (DDBPI) and neuron numbers (NN) in the basolateral complex of the amygdala, CA3, CA1, and various regions of limbic system and motor cortex in kainic acid-induced TLE rat model that highlights the helpful effect of regular exercise, even delayed exposure, in improvement of neural plasticity in regions controlling the motor coordination and emotional regulation [ 36 ]. In this study, levetiracetam was chosen due to several reasons, including its broad therapeutic index, unique mode of action, acceptable pharmacokinetic feature, fewer drug-drug interactions in comparison to other antiepileptic drugs, lower adverse effects, and proven efficacy in different kindling models, especially against amygdala kindling [ 37 , 38 ]. Moreover, considering the positive impact of physical exercise on epilepsy, two selected doses of levetiracetam (27 mg/kg BW as a low dose and 54 mg/kg BW as a high dose) alone and in combination with a physical exercise program were used in the current study to compare and investigate their potential effect on kindling-induced epilepsy. The protective role of levetiracetam against the induction of partial seizure by KA, pilocarpine, and PTZ, as well as its inhibitory effect against kindling (both electrically and chemically), was established in previous animal studies [ 39 ]. Our results indicated that the development of kindling was partially delayed in animals subjected to pre-kindling physical exercise, highlighting the promising and beneficial role of exercise in preventing epilepsy. Indeed, it should be emphasized that the various parameters of the exercise program, including the intensity, frequency, and duration (acute or chronic), as well as the patient's quality of life, are the main contributors to exercise-induced health effects on patients suffering from epileptic seizures which are in line with previous studies [ 8 , 40 , 41 ]. The findings of the present study showed that there was a logical and positive correlation among all 10 experimental groups in regards to modifying the seizure parameters, including the maximum duration of stage 5 of the seizure (Max S5D: Stage 5 Duration), the mean delay time or latency from electrical stimulation to the onset of stages 1, 2, 3, 4 and 5 of the seizure (S 1 L, S 2 L, S 3 L, S 4 L, and S 5 L). Notably, only the Ex-K group, which was subjected to preventive physical exercise before kindling, exhibited a marked difference from the K group on all days. Therefore, considering the abovementioned findings, there was no significant difference in the recorded seizure parameters among the various experimental groups, and it can be inferred that the degree of nervous system excitability and the susceptibility to the development of epileptic seizures in the abovementioned subgroups did not differ remarkably from each other at the onset of the experiment. Moreover, our results demonstrated that there was a statistically significant correlation among the treatment interventional groups (Ex, L-lev, H-lev, Ex-L lev, and Ex-H lev) in the reduction of total seizure stages, the maximum seizure stages, maximum seizure duration, total seizure duration, total AD duration, and maximum AD duration. Notably, only the Ex-K group, which was subjected to preventive physical exercise before kindling, exhibited a marked difference from the K group on all days. These findings highlight the potential role of physical exercise as a preventive treatment or prophylactic agent in reducing seizure duration and shortening seizure stages. These findings also suggest that if physical exercise is used as a preventative measure, it may be able to reduce ADD in seizures. More specifically, the results of our study demonstrated a significant difference between groups subjected to exercise and receiving low- and high-doses of levetiracetam, as well as their combination, compared to the control group in seizure duration, ADD, S 1 L, S 2 L, and S 3 L parameters of seizure. These data indicate a consequential role of all therapeutic interventions (Ex, L-lev, H-lev, Ex-L lev, and Ex-H lev) in reducing seizure duration and ADD in seizure as well as in increasing S 1 L, S 2 L, and S 3 L. Given that there was a notable difference in the groups that received combinational therapy (Ex-L lev and Ex-H lev) compared to the groups that received monotherapy (Ex, L-lev and H-lev), which indicates synergism between physical exercise and levetiracetam, it is conceivable that exercise as a complementary non-pharmacological treatment option could be able to probably enhance medication adherence and pharmacological approaches (antiepileptic drugs) effectiveness in the management of seizure and dependent comorbidities. To date, several studies have been conducted, with results consistent with our current study. For example, Jia et al. reported that a combination of sodium valproate with low-intensity physical exercise alleviated epileptic seizures and related comorbidities like anxiety, depression, and cognitive impairment through down-regulation of TLR4 and NF-κB-dependent neuroinflammatory cytokines, including TNF-α, IL-1β, and IL-6 in the hippocampus of KA-lesioned murine model [ 42 ]. In a similar investigation, it has been revealed that combined therapy of regular swimming exercise plus sodium valproate administration results in prolongation of seizure latency and decline of seizure severity and frequency, confirmed by reduction of epileptiform discharge numbers as well as the numbers of total spike using EEG recording and visual observation methods in PTZ-kindled rat models. Additionally, learning and memory skills were not influenced in groups receiving combination therapy, as indicated by the passive avoidance test [ 43 ]. Barzroodi Pour et al. reported that the addition of regular exercise to the administration of carbamazepine (CBZ) resulted in a significant reduction of seizure severity as well as a remarkable increase in the mean latency in PTZ-kindled rats. As a notable finding, they also demonstrated that the distribution of glutamate decarboxylase (GAD), an enzyme responsible for the conversion of glutamate into GABA, was elevated in both the CA3 and CA1 regions of the hippocampus and the cortical area in groups subjected to the combination therapy of exercise and CBZ. Based on our results and supporting evidence, it can be inferred that regular exercise has the potential to enhance the efficacy of antiepileptic medications and reduce the required doses of these medications, as well as minimize associated adverse outcomes. The improvement of GABA and the downstream signaling pathway may also contribute to the synergistic effect of exercise and antiepileptic medications [ 44 ]. Cognitive impairments are prominent manifestations in patients who have epilepsy, while limited successful and effective medicines are available at present [ 45 ]. As another significant finding of our study, preventive physical exercise attenuated the spatial learning and memory deficits following amygdala electrical kindling-induced epileptic seizures. The results of the present study demonstrated that in all therapeutic interventional groups including Ex, L lev, H lev, and their combination (Ex-L lev and Ex-H lev), there was a significant reduction in the mean escaped latency time and the mean distance traveled of animals at consecutive 4-training days as well as a considerable increase in time spent in target quadrant (Q1) of animals at probe trial day compared to kindling (K) group as reported by MWM assay. More specifically, there was a notable difference between the groups that received combinational therapy (Ex-L lev and Ex-H lev) and the groups that received monotherapy (Ex, L-lev, and H-lev), indicating the synergistic effect of physical exercise and levetiracetam. Notably, these findings align with reports from previous investigations [ 43 , 44 ]. In line with our study, Gorantla et al. reported that both immediate and delayed exposures to swimming exercise enhanced the percent bias, correct responses, and number of changes based on T-maze testing observations in the kainate-induced TLE model of rats. These findings suggest that even delayed exposure to exercise was beneficial in improving seizure-associated cognitive and behavioral dysfunctions [ 36 ]. In another investigation, cognitive function was enhanced in PTZ-induced epileptic rats subjected to both long-term forced swimming and endurance running, as confirmed by improved performance in the passive avoidance test and object recognition test. Moreover, the decreased levels of synaptic proteins and impaired function of GABAergic neurons were ameliorated in rats subjected to exercise. Furthermore, antagonizing CA3 GABAergic neurons hindered the reversal effects of physical activity on seizures and related cognitive skill dysfunction. These data suggest that long-term and regular exercise can improve cognitive skills by normalizing GABAergic function and synaptic plasticity in the CA3 area of the hippocampus [ 47 ]. A study conducted by Almeida et al. demonstrated that seizure-associated memory deficits are mitigated by a 4-week resistance exercise program, as indicated by inhibitory avoidance test records. They reported that IGF-1 levels increased in pilocarpine-lesioned epileptic rats, and resistance exercise restored them to control levels. On the other hand, reductions in BDNF levels, as well as hypoactivation of ERK and mTOR, were observed in epileptic rats, and these values were restored to control levels by resistance exercise [ 48 ]. In a study evaluating the effect of treadmill exercise on behavioral rehabilitation in pilocarpine-induced epileptic rat models, cognitive tests (Y-maze, Open-field, and Novel object recognition assays) confirmed the enhancement of spatial and object memory following endurance exercise, and electrophysiology-based studies indicated the conservation of hippocampal neuronal plasticity as a consequence of physical activity. Investigations of the potential underlying mechanisms of these observed effects affirmed that exercise protects parvalbumin-expressing interneurons in the hippocampus, probably through the quenching of neuroinflammation and upgrading the integrity of the BBB [ 49 ]. Collectively, the aforementioned findings suggest that regular physical exercise may contribute to neuroprotection and attenuate the progression of kindling. Conclusion Finally, based on the findings of the current study, exercise appears to have an anticonvulsant effect in combination with levetiracetam, a well-proven and highly effective medication for treating various types of epilepsy. This effect was confirmed across all seizure parameters examined in our investigation. Moreover, exercise can improve kindling-induced memory and spatial learning deficits as effectively as levetiracetam. On the other hand, no significant difference was observed between the therapeutic effects of low and high doses of levetiracetam, so it can be inferred that the full therapeutic effect can be achieved even with a low dose, and this effect does not accelerate upon increasing the dose; however, the combination of exercise with low- and high-doses of levetiracetam enhances the therapeutic efficacy in all seizure parameters and improves memory and spatial learning cognitive functions. These beneficial effects should be taken into consideration for the potential clinical translation of physical exercise as a non-pharmacological intervention in combination with conventional antiepileptic medications for more efficacious control or treatment of seizures. Declarations Ethics approval All experiment practices were approved by the Research Ethics Committees of Biosafety & laboratory, Tehran University of Medical Sciences & Health Services (Ethical code: IR.TUMS.TIPS.REC.1397.015). Conflict of interest The authors declare no conflict of interest. Funding This research was supported by a grant from the Tehran University of Medical Sciences, Tehran, Iran. Author Contribution Abbas Kebriaeezadeh suppervising research and electrical kindling design Mohammad Sharizadeh supervising and reseach design for MAZ and memory study Ghoran Taghizadeh research design for physical activity testReza Zafari researcher Hassan Gheibi Co researcherMamoud Rezaei co researcher Acknowledgement Tehran University of Medical Sciences Data availability The data that support the findings of this study are available on request from the corresponding author. References Milligan TA. Epilepsy: a clinical overview. Am J Med (2021) 134(7):840–7. Blair RDG. Temporal lobe epilepsy semiology. Epilepsy Res Treat (2012) 2012(1):751510. Henning O et al. Temporal lobe epilepsy. Tidsskr Den Nor legeforening (2023) 143. Li Z, Cao W, Sun H, Wang X, Li S, Ran X, Zhang H. 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The effects of regular swimming exercise during sodium valproate treatment on seizure behaviors and EEG recordings in pentylenetetrazole-kindled rats. Epilepsy Res (2022) 179:106830. Barzroodi Pour M, Bayat M, Navazesh A, Soleimani M, Karimzadeh F. Exercise improved the antiepileptic effect of carbamazepine through gaba enhancement in epileptic rats. Neurochem Res (2021) 46:2112–30. Chen J et al. Activation of medial septum cholinergic neurons restores cognitive function in temporal lobe epilepsy. Neural Regen Res (2023) 18(11):2459–65. Sabaghi A, Heirani A, Kiani A, Yousofvand N, Sabaghi S. The Reduction of Seizure Intensity and Attenuation of Memory Deficiency and Anxiety-Like Behavior through Aerobic Exercise by Increasing the BDNF in Mice with Chronic Epilepsy. Neurochem J (2020) 14:197–203. Lin X-Y, Cui Y, Wang L, Chen W. Chronic exercise buffers the cognitive dysfunction and decreases the susceptibility to seizures in PTZ-treated rats. Epilepsy Behav (2019) 98:173–87. Allendorfer JB, Arida RM. Role of physical activity and exercise in alleviating cognitive impairment in people with epilepsy. Clin Ther (2018) 40(1):26–34. Yu H, Shao M, Luo X, Pang C, So K-F, Yu J, Zhang L. Treadmill exercise improves hippocampal neural plasticity and relieves cognitive deficits in a mouse model of epilepsy. Neural Regen Res (2024) 19(3):657–62. Additional Declarations No competing interests reported. Supplementary Files GraphicalAbstract1.pdf Cite Share Download PDF Status: Published Journal Publication published 27 Jan, 2026 Read the published version in DARU Journal of Pharmaceutical Sciences → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7215509","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":509435965,"identity":"c7616327-b2cd-4630-8455-40cafe708a7b","order_by":0,"name":"abbas kebriaeezadeh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYBACCQYeBoYEIIMfLsRMrBbJNpK0gIDBMWIdJtnee+zDgxobOeP7PaYbfzDYyTOw8z7Aq0Wa51zyjIRjacZmx3jMbvMwJBs2MLMb4NUiJ5FjzJDAdjhxG0gL0CMJDMxs+B0mJ/8GqOXf//rNbTxmN38w1BPWIi3BY8yQ2HYgwYCNx+wGD8Nhwloke/KSGRL7kg1nHEsru81jcNywjZAWieNnDzP++GYnz998eNvNHxXV8vz8RAc3GADDioAdo2AUjIJRMAqIAQDcdzjEo6iHVAAAAABJRU5ErkJggg==","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"abbas","middleName":"","lastName":"kebriaeezadeh","suffix":""},{"id":509435966,"identity":"e57af11d-fcb2-463c-b022-1bf70a17e696","order_by":1,"name":"Reza Zaferi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Reza","middleName":"","lastName":"Zaferi","suffix":""},{"id":509435967,"identity":"c6cc5c62-b879-4154-94b3-06717358df3e","order_by":2,"name":"mohammad Sharifzadeh","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"mohammad","middleName":"","lastName":"Sharifzadeh","suffix":""},{"id":509435968,"identity":"7958d075-4330-40f0-8056-d2e3e2be98a1","order_by":3,"name":"Javad Mirnajafi-zadeh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Javad","middleName":"","lastName":"Mirnajafi-zadeh","suffix":""},{"id":509435969,"identity":"4d573238-372c-4aa1-a5b0-7e0357b5364b","order_by":4,"name":"Ghorban Taghizadeh","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ghorban","middleName":"","lastName":"Taghizadeh","suffix":""},{"id":509435970,"identity":"47b7ebbf-88f3-445f-ab8f-63540452588e","order_by":5,"name":"Hassan Gheibi","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"Gheibi","suffix":""},{"id":509435971,"identity":"f8405789-250f-4812-b2fb-d63939509c5b","order_by":6,"name":"Mahmoud Rezaei","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"","lastName":"Rezaei","suffix":""}],"badges":[],"createdAt":"2025-07-25 15:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7215509/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7215509/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s40199-025-00583-w","type":"published","date":"2026-01-27T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90696083,"identity":"5c44d89e-b086-4bc2-840d-9933a0671b8c","added_by":"auto","created_at":"2025-09-05 20:19:32","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96822,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different interventions on number of days needed for access full kindling of the amygdala in rats.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/56b77ef455418cbef564f160.jpg"},{"id":90696084,"identity":"615276d1-bdda-4de9-8476-8381221360ca","added_by":"auto","created_at":"2025-09-05 20:19:32","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":308903,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation among the study experimental groups in the observation of total seizure stages (A), the maximum seizure stages (B), maximum seizure duration (C), total seizure duration (D), total AD duration (E), and maximum AD duration (F). P\u0026lt; 0.0001**** is considered significant changes in relation to kindling group.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/64b9273fe05d5c628d91a41e.jpg"},{"id":90696469,"identity":"8d14bbd8-ebbd-40da-98b6-3cd28472937b","added_by":"auto","created_at":"2025-09-05 20:27:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":275685,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation among the study experimental groups in the Max S5D: Stage 5 Duration (A), mean delay time or latency from electrical stimulation to onset of stage1 of seizure (stage 1 latency, S\u003csub\u003e1\u003c/sub\u003eL) (B), stage2 of seizure (stage 2 latency, S\u003csub\u003e2\u003c/sub\u003eL) (C), stage3 of seizure (stage 3 latency, S\u003csub\u003e3\u003c/sub\u003eL) (D), seizure (stage 4 latency, S\u003csub\u003e4\u003c/sub\u003eL) (E), and stage5 of seizure (stage 5 latency, S\u003csub\u003e5\u003c/sub\u003eL) (F). P \u0026lt; 0.0001**** is considered significant changes in relation to kindling group.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/fa7ad8247d990b396a27158e.jpg"},{"id":90696087,"identity":"c9623e47-f5fc-431b-8f70-72a3dd3857e8","added_by":"auto","created_at":"2025-09-05 20:19:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176477,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of treatments effects on seizure parameters including seizure stages (A), seizure duration (B), dADD (C), mean delay time or latency from electrical stimulation to onset of stage1 of seizure (stage 1 latency, S\u003csub\u003e1\u003c/sub\u003eL) (D), stage2 of seizure (stage 2 latency, S\u003csub\u003e2\u003c/sub\u003eL) (E) and stage3 of seizure (stage 3 latency, S\u003csub\u003e3\u003c/sub\u003eL) (F) following the kindling process (post-kindling alterations). P\u0026lt; 0. 01 **, P \u0026lt; 0.001 *** and P \u0026lt; 0.0001**** are considered significant changes in relation to kindling group.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/f5fd07d289cf735788d4f2e7.jpg"},{"id":90696090,"identity":"e5833b03-1259-49b7-a796-bb74050197a3","added_by":"auto","created_at":"2025-09-05 20:19:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":161603,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of experimental animal speeds among all groups in MWM test.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/e089761befde3f152597659a.jpg"},{"id":90696092,"identity":"e3eead9e-4e5d-4c82-8a7d-81afe2ab27f6","added_by":"auto","created_at":"2025-09-05 20:19:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":176958,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative tracings of the mean time to reach the hidden platform (escaped latency) at consecutive 4-training days in MWM test. P\u0026lt; 0.0001**** is considered significant changes in relation to kindling group.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/6c3293c4d6de0a9f68a2adca.jpg"},{"id":90696096,"identity":"a4201c49-a872-4f2e-a23c-0a90ba47f112","added_by":"auto","created_at":"2025-09-05 20:19:33","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":103769,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative tracings of the mean distance traveled at consecutive 4-training days in MWM test. P\u0026lt; 0.0001**** is considered significant changes in relation to kindling group.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/4265a3d017a7e01da70cbdc3.jpg"},{"id":90696660,"identity":"673a602f-cf6b-499a-bca4-4fe68d6dfd5c","added_by":"auto","created_at":"2025-09-05 20:35:33","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":97435,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative tracings of the time spent in target quadrant (Q1) at probe trial day in MWM test. P\u0026lt; 0. 01 ** and P \u0026lt; 0.001 *** are considered significant changes in relation to kindling group.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/6fe6bf602d067ca4c6a2f476.jpg"},{"id":90696097,"identity":"fa7c15db-19cb-44c5-9874-42999b1624e5","added_by":"auto","created_at":"2025-09-05 20:19:33","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":131996,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative tracings of the mean time (escaped latency) and mean distance traveled to reach the hidden platform at consecutive 4-training days and time spent in target quadrant (Q1) at probe trial day affected by therapeutic interventions including physical exercise (Ex), low dose of levetiracetam (L lev), high dose of levetiracetam (H lev) and their combination (Ex-L lev and Ex-H lev) in MWM test. P\u0026lt; 0. 01 ** P \u0026lt; 0.001 *** and P\u0026lt; 0.0001**** are considered significant changes in relation to kindling group.\u003c/p\u003e","description":"","filename":"Picture9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/717c6c210430e34306375cff.jpg"},{"id":101690388,"identity":"8c7a53f8-0984-473a-be5e-ccd0655c42f0","added_by":"auto","created_at":"2026-02-02 16:00:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2295812,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/3ee041f0-3a75-4525-9eb1-7f2066eb2c64.pdf"},{"id":90696470,"identity":"ab90f079-7956-40b8-b3d8-48fa03976430","added_by":"auto","created_at":"2025-09-05 20:27:32","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":141927,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7215509/v1/e3fe3bc1a69c5f89c2a80436.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of physical activity on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is one of the most common noncommunicable and chronic impairments of cerebral function characterized by the spontaneous and symptomatic recurrent incidence of seizures. Approximately, this neurological dysfunction affects 0.5-1% of the human population worldwide and highly impacts their quality of life by long-term sequelae [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Depending on the seizure origin site, this disorder is divided into two main classes, namely partial and generalized seizure [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In particular, complex partial seizures are the most common phenotype among other types of epilepsy, with a greater number of such epileptic seizures thought to originate from temporal lobes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite significant advances in pharmacological approaches, epilepsy is not satisfactorily controlled in almost 20\u0026ndash;35% of the patients and is considered drug-resistant epilepsy (DRE) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOn the other hand, long-term administration of antiepileptic medications often leads to undesirable adverse effects, such as neurocognitive and neurohormonal disturbances, cardiovascular disorders, osteoporosis, and liver and thyroid dysfunction [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Hence, experimental and non-pharmacological therapeutics such as regular physical exercise have been extensively investigated for their potential to prevent and/or treat epileptic insults [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To date, several studies in both animals and humans have demonstrated the favorable impact of regular physical activity programs as a supportive treatment strategy for decreasing epileptic seizure severity and frequency, as well as promoting psychological and cardiovascular health. However, the underlying mechanisms responsible for these beneficial effects have not yet been understood [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Most of these studies reported reduced ictal activity, delayed development of amygdala kindling, prolonged convulsion threshold, reduced neuronal damage and behavioral alterations, diminished susceptibility to subsequently provoked seizures after exercise, as well as improved neurogenesis and positive plastic neurochemical changes in the hippocampal formation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. From a mechanistic point of view, it has been shown that regular physical exercise induces neurogenesis, increases blood flow to the brain, modulates balance between excitatory and inhibitory neurotransmitter synthesis and activity, and enhances synaptic plasticity, particularly through the production of BDNF, a well-known neurotrophic factor, that plays a crucial role in supporting synaptic formation and plasticity and attenuates detrimental processes, including neuroinflammation, mitochondrial fragmentation, oxidative stress, and neuronal apoptosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePatients who have epilepsy often experience a series of comorbidities, including psychiatric disorders, cognitive dysfunction, behavioral disabilities, and even death if not adequately treated [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The repeated occurrence of epileptic seizures remarkably reduces learning and memory cognitive functions in affected patients and individuals with temporal lobe epilepsy (TLE) who usually suffer from visuospatial memory complications [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The occurrence of memory and learning defects in experimentally induced epilepsy in animal models has been confirmed in previous preclinical studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The amygdala kindling-induced epilepsy model is one of the widely-used epilepsy models that have considerable similarity with the incidence of epilepsy in humans [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The kindling method involves focal sub-threshold excitation by repeated and low-frequency electrical stimulation or repetitive administration of sub-convulsive doses of chemicals such as kainic acid (KA), pentylenetetrazol (PTZ), pilocarpine, and metals that can offer insights into the underlying mechanisms and provide new avenues for the development of novel potential therapeutic interventions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLevetiracetam, a practical, potent, and relatively safe antiepileptic drug, is one of the most commonly prescribed medications for the treatment and/or control of epilepsy, particularly in benzodiazepines-resistant epilepsies or when first-line treatments are not applicable due to their undesirable effects [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Mechanistically, levetiracetam specifically binds to and inhibits the transmembrane protein located on the synaptic vesicle SV2A, thereby hindering the neurotransmitter priming and intensification of neurotransmission during exaggerated provocation of presynaptic fibers [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Based on previously established findings, the development of kindling was remarkably quicker in SV2A-deficient mice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, it can be concluded that levetiracetam reduces the kindling epileptogenesis \u003cem\u003evia\u003c/em\u003e modulating the SV2A functions in synaptic transmission [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBased on the mentioned mechanistic aspects of physical exercise, and given the lack of effectiveness of current therapies for epilepsy, a growing body of literature has been interested in the therapeutic potential of regular physical exercise in the prevention or treatment of epilepsy. Thus, the purpose of the current study was to evaluate and compare the effect of physical exercise, low- and high-dose levetiracetam, and their combination on epileptic seizures and consequent learning and memory impairment in amygdala electrical kindled rat model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eAnimals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSeventeen male Wistar rats weighing 220\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g (about 8\u0026ndash;10 weeks old, specific pathogen-free or SPF grades) were provided by the animal house of the Faculty of Pharmacy at Tehran University of Medical Sciences (Tehran, Iran). The animals were housed in a specific-pathogen-free cage with a 12/12 h light-dark cycle at a temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 30\u0026ndash;70% in an air-conditioned room. The rats were fed with a rodent diet and water \u003cem\u003ead libitum\u003c/em\u003e during the experiments. All experiments were approved by the Research Ethics Committees of Biosafety \u0026amp; Laboratory, Tehran University of Medical Sciences \u0026amp; Health Services (Ethical code: IR.TUMS.TIPS.REC.1397.015). Ethical principles and considerations, as delineated in the National Institute of Health (NIH), were complied with by all animals and at all steps of the research to minimize pain and distress.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExperimental design\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, rats were randomly divided into 10 groups (7 rats/group). A 10-day recovery period from the stereotaxic procedure to electrode implantation was employed to perform amygdala kindling in rats. Before performing behavioral tests, rats were re-stimulated for 1 day, a process that served as a challenge. After performing all procedures and interventions, as well as completing the behavioral tests, all groups were sacrificed using standard CO\u003csub\u003e2\u003c/sub\u003e chambers.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental design for evaluation of levetiracetam administration plus physical exercise on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling model.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eInterventions\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eControl: rats were not submitted to physical exercise and kindling and received only saline trough i.p. administration\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLev: rats were not submitted to physical exercise and kindling and received only high dose of levetiracetam trough i.p. administration for 4-weeks\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEx: rats were subjected to physical exercise but were not submitted to kindling after surgery and recovery period\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEx-K: rats were submitted to 4-weeks physical exercise before surgery and then subjected to kindling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK: rats were submitted to kindling after surgery and recovery period and then received saline trough i.p. administration\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK-Ex: rats were submitted to 4-weeks physical exercise after surgery and kindling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK-L lev: rats were received to 4-weeks low dose of levetiracetam (27 mg/kg BW, once a day) trough i.p. administration after surgery and kindling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK-H lev: rats were received to 4-weeks high dose of levetiracetam (54 mg/kg BW, once a day) trough i.p. administration after surgery and kindling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK-Ex-L lev: rats were submitted to 4-weeks physical exercise plus treatment with low dose of levetiracetam (27 mg/kg BW, once a day) trough i.p. administration after surgery and kindling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup 10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK-Ex-H lev: rats were submitted to 4-weeks physical exercise plus treatment with high dose of levetiracetam (54 mg/kg BW, once a day) trough i.p. administration after surgery and kindling\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhysical exercise program\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter recovery from stereotaxic surgery and acclimatization to the treadmill, rats were subjected to forced physical exercise once a day for 30 minutes, 5 days a week, for 4 weeks. The exercise program included running on a treadmill as described in the following: (I) 5 minutes at 2 meters per minute, (II) 5 minutes at 5 meters per minute, and (III) the last 20 minutes at an 8 meters per minute speed [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It should be noted that such a physical exercise protocol was not performed for all groups. The rats belonging to the Control, Lev, Ex, K, K-L Lev, and K-H Lev groups were placed on the treadmill for 30 minutes daily for 4 weeks at a speed of 0 m/s. The incline of the treadmill was 0%, and a piece of foam or synthetic sponge was placed at the tag end of the treadmill to protect the rats from sticking at the tag end of the device. Rats were monitored during physical exercise to evaluate the signs of potential stress or pain. At the end of the physical exercise period and one day after the challenging rats, the behavioral test MWM was carried out.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSurgical procedure for electrode implantation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFollowing deep general anesthesia by i.p. administration of 100 mg/kg BW ketamine 10% (Alfasan, Netherlands) and 10 mg/kg BW xylazine 2% (Alfasan, Netherlands), the animals were placed and fixed on the stereotaxic device as their skull was in a perfectly horizontal position. The animals were fixed on the stereotaxic instrument, protected by the placement of a bar behind the incisors and two bars at the temples. The dental bar was adjusted so that the coordinates of Bregma and Lambda were parallel in height. The scalp was incised vertically from the area between the two eyes to the end of the occipital bone, and the bone was dried with a tampon so that the Bregma and Lambda could be seen. After specifying the position of the right basolateral nucleus of the amygdala (BLA), the coordinates of the electrode implementation were determined based on the paxinos atlas [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The coordinates were extracted from the appropriate atlas section to access this region: the AP\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;2/5, L\u0026thinsp;=\u0026thinsp;+\u0026thinsp;4/8, and V= -7.5. During surgery, the rats were monitored for evaluation of consciousness signs using previously established methods such as checking paw and eye reflexes, as well as dose repetition and adjustment if necessary. The stainless bipolar electrodes (A-M Systems Inc, USA) with 127 \u0026micro;m diameter were implanted. In order to surface recording, four stainless steel screws were placed and adjusted on the skull using dental acrylics, one in the right forehead as electrical ground, and two others on the occipital and right head regions for EEG recording and were coupled with a stimulator and EEG amplifier. After surgery and electrode implantation, the surgical site was disinfected using a 10% povidone-iodine solution. Finally, the animals were housed individually in separate cages, and a 10-day recovery period was considered for wound healing and familiarization before the kindling process and interventions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe kindling process\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter a 10-day recovery period from surgery, the animals were transferred to a plexiglass cage (dimensions: 50\u0026times;50\u0026times;50 cm, made of transparent plexiglass), and the kindling procedure was started. The attached miniature plastic socket to the animals' skull was connected to a flexible cable with a noise-resistant shield. Rats were allowed to move freely within the recording cage. EEG recording was performed using a monitoring system with eTrace experiment software (version 1.39, Parto Danesh Institute, Tehran, Iran), and offline analysis was carried out using eTrace analysis software (Parto Danesh Institute, Tehran, Iran) on a computer. For performing kindling stimulations, the after-discharge threshold (ADT) was assessed for each rat at the beginning of the kindling process. For this purpose, the BLA region of animals was stimulated based on the following parameters: 20 \u0026micro;A single-phase square pulse; frequency\u0026thinsp;=\u0026thinsp;50 Hz; wave width\u0026thinsp;=\u0026thinsp;1 ms, and duration\u0026thinsp;=\u0026thinsp;2 s. If afterdischarge (AD) waves were recorded (for at least 15 seconds), this current intensity was known as the threshold intensity; otherwise, the intensity was increased by 10 \u0026micro;A at 20-minute intervals until AD waves were recorded, which was designated as the ADT. Afterward, the semi-rapid kindling model (six times stimulation per day with the same intensity of ADT at 20-minute intervals and approximately the same time frame during the experiment) was used to induce seizures in rats. Seizure-related behavioral symptoms were classified and recorded based on Racine's scale [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Rats that exhibited stage 5 seizures during stimulation for three consecutive days and whose seizure parameters remained relatively invariant over continuous stimulations were considered ideally kindled subjects.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSeizure parameters recording\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe comparable and measurable parameters following the kindling process in the current investigation included: (I) daily afterdischarge duration (dADD), (II) delay time from electrical stimulation to the onset of stage 1 of the seizure (stage 1 latency, S\u003csub\u003e1\u003c/sub\u003eL), (III) delay time from electrical stimulation to stage 2 of the seizure (stage 2 latency, S\u003csub\u003e2\u003c/sub\u003eL), (IV) delay time from electrical stimulation to stage 3 of seizure (stage 3 latency, S\u003csub\u003e3\u003c/sub\u003eL), (V) delay time from electrical stimulation to stage 4 of seizure (stage 4 latency, S\u003csub\u003e4\u003c/sub\u003eL), (VI) delay time from electrical stimulation to stage 5 of seizure (stage 5 latency, S\u003csub\u003e5\u003c/sub\u003eL), (VII) maximum duration of stage5 of seizure (Max S5D: Stage 5 Duration), (VIII) seizure duration and stages that developed in the animal in response to six times a day stimulation (both on the kindling and testing days), and (IX) The longest-lasting ADD per each day (Max ADD).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpatial learning and memory test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter the post-intervention 1-day challenge, the spatial learning and memory assessment was performed using a Morris water maze (MWM) as described previously [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To evaluate the cognitive learning function, rats were subjected to training on four consecutive days to locate a hidden escape platform in a water-filled (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C) circular pool (150 cm in diameter, 60 cm in depth), with four trials per day. A memory test was performed on day 5 (probe test), with the removed platform removed from the maze. The movements of the rats were analyzed and processed by The Ethovision XT 11.5 video tracking software (Noldus Information Technology, Wageningen, Netherlands). Four parameters, including time delay in finding the platform (latency), traveled distance, the subjects' swimming speed, and time spent in the target quadrant, were evaluated on training days and the probe day, respectively (the first two parameters on training days and the last two parameters on the probe day).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eThe results of our study were presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and analyzed by Graph pad prism software (GraphPad, version 10.1, La Jolla, CA, USA). One-way and two-way ANOVA and Tukey's post hoc tests were used for statistical testing and comparisons for seizure parameters and MWM test results, respectively. An unpaired t-test was used to compare spent time, traveled distance, and probe in the control, Ex, H lev, and K groups. The \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eComparative analysis of seizure parameters\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e, one-way ANOVA analysis with Tukey's post hoc test revealed that the number of days required for full kindling of the amygdala in animals did not differ significantly among the different interventional groups. There was only an apparent and relative difference between the group subjected to preventive physical exercise (Ex-K, pre-kindling exercise) and the other groups. Still, this observed difference was not substantially meaningful. In addition, the threshold current intensity for the development of AD did not significantly differ among the various groups and was indeed 80\u0026ndash;160 \u0026micro;A for all fully kindled rats in all groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn the context of seizure parameters recording findings, two-way ANOVA analysis with Tukey's post hoc tests results demonstrated that there was a logical correlation between the all 10 experimental groups in the observation of total seizure stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), the maximum seizure stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), maximum seizure duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), total seizure duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), total AD duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), and maximum AD duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Notably, only Ex-K group, which was submitted to preventive physical training before kindling, exhibited a marked difference with the K group on all days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e, one-way ANOVA analysis with Tukey's post hoc test results indicated that there was a logical and positive correlation among all 10 experimental groups regarding the maximum duration of stage 5 of the seizure (Max S5D: Stage 5 Duration) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). There was also a significant correlation among all 10 experimental groups in the context of the mean delay time or latency from electrical stimulation to the onset of stage 1 of the seizure (stage 1 latency, S\u003csub\u003e1\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), stage 2 of the seizure (stage 2 latency, S\u003csub\u003e2\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), stage 3 of the seizure (stage 3 latency, S\u003csub\u003e3\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), stage 4 of the seizure (stage 4 latency, S\u003csub\u003e4\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), and stage 5 of the seizure (stage 5 latency, S\u003csub\u003e5\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Notably, only the Ex-K group, which was submitted to preventive physical exercise before kindling, exhibited a marked difference from the K group on all days.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe effects of treatments on seizure intensity following the kindling process (post-kindling alterations)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs mentioned in the experimental design section, to evaluate the potential anticonvulsant effect of the treatments, rats underwent the rekindling process (six times at a 20-minute interval) on the day after the last intervention, and then various seizure parameters were investigated. In this regard, two-way ANOVA analysis with Tukey's post hoc test results showed that in all therapeutic interventional groups including physical exercise (Ex), low dose of levetiracetam (L lev), high dose of levetiracetam (H lev), and their combination (Ex-L lev and Ex-H lev), there was a substantial reduction in parameters, including seizure stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), seizure duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), dADD (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), the mean delay time or latency from electrical stimulation to the onset of stage1 of seizure (stage 1 latency, S\u003csub\u003e1\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), stage 2 of seizure (stage 2 latency, S\u003csub\u003e2\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and stage 3 of seizure (stage 3 latency, S\u003csub\u003e3\u003c/sub\u003eL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eF) on the challenging (rekindling) day compared to same parameters during the main kindling process. All groups were significantly different from the kindling group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpatial learning and memory\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs is evident from Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the one-way ANOVA analysis with Tukey's post hoc test results indicated that the speed of experimental animals did not differ significantly among all groups on the training days and probe day, indicating no difference in the level of gross motor activity among the different groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe t-test analysis showed that the mean escape latency time of animals at consecutive 4-training days of MWM assay was not significantly different in the high-dose levetiracetam (H Lev) and physical exercise (Ex) groups compared to the control group (Co) (Fig.s 6A and 6B, respectively). On the other hand, and as a notable finding, there was a remarkable difference in control and preventive physical exercise groups compared to the kindling group (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe t-test analysis showed that the mean travel distance of animals at consecutive 4-training days of MWM assay was not significantly different in the high-dose levetiracetam (H Lev) and physical exercise (Ex) groups compared to the control group (Co) (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, respectively). On the other hand, and as a notable finding, there was a remarkable difference in control and preventive physical exercise groups compared to kindling groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, respectively).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the t-test analysis showed that the time spent in the target quadrant (Q1) of animals on the probe trial day of the MWM assay was not significantly different in the high-dose levetiracetam (H Lev) and physical exercise (Ex) groups compared to the control group (Co) (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, respectively). On the other hand, and as a notable finding, there was a remarkable difference in control and preventive physical exercise groups compared to kindling groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, respectively). The results indicated that animals in the kindling group (K) spent less time in the target quadrant (Q1) than the control group, and animals subjected to preventive physical exercise before kindling (Ex-K) spent more time in the target quadrant (Q1) than the kindling (K) group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e9\u003c/span\u003e, two-way ANOVA analysis with Tukey's post hoc test results demonstrated that in physical exercise (Ex), low dose of levetiracetam (L lev), high dose of levetiracetam (H lev), and their combination (Ex-L lev and Ex-H lev) groups, there was a significant reduction in the mean escaped latency time (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e9\u003c/span\u003eA) and the mean the distance (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) of animals at consecutive 4-training days as well as a considerable increase in time spent in target quadrant (Q1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig18\" class=\"InternalRef\"\u003e9\u003c/span\u003eC) of animals at probe trial day compared to kindling (K) group as reported by MWM assay.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGiven the relatively high prevalence of epilepsy, noticeable investigations are being conducted on its etiologic factors and safe novel approaches to prevent and/or treat the disease. To date, a growing number of preclinical studies and clinical trials have demonstrated that physical exercise exhibits beneficial and valuable therapeutic properties against epileptic seizures, as well as behavioral and cognitive complications, through various mechanisms [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this regard, Aguiar et al. reported that a mild-intensity physical exercise program attenuated aging-associated impairments in long-term memory and spatial learning, as assessed by the Morris Water Maze (MWM) assay. Notably, the reported cognitive-improving features of mild-intensity exercise were mainly mediated by the activation of the AKT and CREB pro-survival signaling pathway, which resulted in a marked elevation of the mRNA expression and protein levels of BDNF in the hippocampus of aging animals [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn a study conducted by Gomes et al., physical exercise significantly diminished seizure frequency and reversed impaired long-term memory in adulthood following early-life status epilepticus induced by pilocarpine in rats, according to water maze task observations. Moreover, physical exercise enhanced cell proliferation, promoted anti-apoptotic responses, and normalized BDNF levels in the brains of rats, leading to improved hippocampal neuroplasticity. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. These results are in line with our study that revealed there was a significant reduction in the mean escaped latency time, and the mean distance traveled of animals at consecutive 4-training days as well as a considerable increase in time spent in the target quadrant (Q1) of animals at the probe trial day in rats subjected to regular physical exercise compared to kindling (K) group as reported by MWM assay. In another study, exposure to immediate and 60-day delayed exercise (daily 15 min swimming for 30 days) enhanced dendritic branch points and intersections (DDBPI) and neuron numbers (NN) in the basolateral complex of the amygdala, CA3, CA1, and various regions of limbic system and motor cortex in kainic acid-induced TLE rat model that highlights the helpful effect of regular exercise, even delayed exposure, in improvement of neural plasticity in regions controlling the motor coordination and emotional regulation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, levetiracetam was chosen due to several reasons, including its broad therapeutic index, unique mode of action, acceptable pharmacokinetic feature, fewer drug-drug interactions in comparison to other antiepileptic drugs, lower adverse effects, and proven efficacy in different kindling models, especially against amygdala kindling [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, considering the positive impact of physical exercise on epilepsy, two selected doses of levetiracetam (27 mg/kg BW as a low dose and 54 mg/kg BW as a high dose) alone and in combination with a physical exercise program were used in the current study to compare and investigate their potential effect on kindling-induced epilepsy.\u003c/p\u003e\u003cp\u003eThe protective role of levetiracetam against the induction of partial seizure by KA, pilocarpine, and PTZ, as well as its inhibitory effect against kindling (both electrically and chemically), was established in previous animal studies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur results indicated that the development of kindling was partially delayed in animals subjected to pre-kindling physical exercise, highlighting the promising and beneficial role of exercise in preventing epilepsy. Indeed, it should be emphasized that the various parameters of the exercise program, including the intensity, frequency, and duration (acute or chronic), as well as the patient's quality of life, are the main contributors to exercise-induced health effects on patients suffering from epileptic seizures which are in line with previous studies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe findings of the present study showed that there was a logical and positive correlation among all 10 experimental groups in regards to modifying the seizure parameters, including the maximum duration of stage 5 of the seizure (Max S5D: Stage 5 Duration), the mean delay time or latency from electrical stimulation to the onset of stages 1, 2, 3, 4 and 5 of the seizure (S\u003csub\u003e1\u003c/sub\u003eL, S\u003csub\u003e2\u003c/sub\u003eL, S\u003csub\u003e3\u003c/sub\u003eL, S\u003csub\u003e4\u003c/sub\u003eL, and S\u003csub\u003e5\u003c/sub\u003eL). Notably, only the Ex-K group, which was subjected to preventive physical exercise before kindling, exhibited a marked difference from the K group on all days. Therefore, considering the abovementioned findings, there was no significant difference in the recorded seizure parameters among the various experimental groups, and it can be inferred that the degree of nervous system excitability and the susceptibility to the development of epileptic seizures in the abovementioned subgroups did not differ remarkably from each other at the onset of the experiment.\u003c/p\u003e\u003cp\u003eMoreover, our results demonstrated that there was a statistically significant correlation among the treatment interventional groups (Ex, L-lev, H-lev, Ex-L lev, and Ex-H lev) in the reduction of total seizure stages, the maximum seizure stages, maximum seizure duration, total seizure duration, total AD duration, and maximum AD duration. Notably, only the Ex-K group, which was subjected to preventive physical exercise before kindling, exhibited a marked difference from the K group on all days. These findings highlight the potential role of physical exercise as a preventive treatment or prophylactic agent in reducing seizure duration and shortening seizure stages. These findings also suggest that if physical exercise is used as a preventative measure, it may be able to reduce ADD in seizures.\u003c/p\u003e\u003cp\u003eMore specifically, the results of our study demonstrated a significant difference between groups subjected to exercise and receiving low- and high-doses of levetiracetam, as well as their combination, compared to the control group in seizure duration, ADD, S\u003csub\u003e1\u003c/sub\u003eL, S\u003csub\u003e2\u003c/sub\u003eL, and S\u003csub\u003e3\u003c/sub\u003eL parameters of seizure. These data indicate a consequential role of all therapeutic interventions (Ex, L-lev, H-lev, Ex-L lev, and Ex-H lev) in reducing seizure duration and ADD in seizure as well as in increasing S\u003csub\u003e1\u003c/sub\u003eL, S\u003csub\u003e2\u003c/sub\u003eL, and S\u003csub\u003e3\u003c/sub\u003eL. Given that there was a notable difference in the groups that received combinational therapy (Ex-L lev and Ex-H lev) compared to the groups that received monotherapy (Ex, L-lev and H-lev), which indicates synergism between physical exercise and levetiracetam, it is conceivable that exercise as a complementary non-pharmacological treatment option could be able to probably enhance medication adherence and pharmacological approaches (antiepileptic drugs) effectiveness in the management of seizure and dependent comorbidities.\u003c/p\u003e\u003cp\u003eTo date, several studies have been conducted, with results consistent with our current study. For example, Jia et al. reported that a combination of sodium valproate with low-intensity physical exercise alleviated epileptic seizures and related comorbidities like anxiety, depression, and cognitive impairment through down-regulation of TLR4 and NF-κB-dependent neuroinflammatory cytokines, including TNF-α, IL-1β, and IL-6 in the hippocampus of KA-lesioned murine model [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn a similar investigation, it has been revealed that combined therapy of regular swimming exercise plus sodium valproate administration results in prolongation of seizure latency and decline of seizure severity and frequency, confirmed by reduction of epileptiform discharge numbers as well as the numbers of total spike using EEG recording and visual observation methods in PTZ-kindled rat models. Additionally, learning and memory skills were not influenced in groups receiving combination therapy, as indicated by the passive avoidance test [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBarzroodi Pour et al. reported that the addition of regular exercise to the administration of carbamazepine (CBZ) resulted in a significant reduction of seizure severity as well as a remarkable increase in the mean latency in PTZ-kindled rats. As a notable finding, they also demonstrated that the distribution of glutamate decarboxylase (GAD), an enzyme responsible for the conversion of glutamate into GABA, was elevated in both the CA3 and CA1 regions of the hippocampus and the cortical area in groups subjected to the combination therapy of exercise and CBZ. Based on our results and supporting evidence, it can be inferred that regular exercise has the potential to enhance the efficacy of antiepileptic medications and reduce the required doses of these medications, as well as minimize associated adverse outcomes. The improvement of GABA and the downstream signaling pathway may also contribute to the synergistic effect of exercise and antiepileptic medications [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCognitive impairments are prominent manifestations in patients who have epilepsy, while limited successful and effective medicines are available at present [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. As another significant finding of our study, preventive physical exercise attenuated the spatial learning and memory deficits following amygdala electrical kindling-induced epileptic seizures. The results of the present study demonstrated that in all therapeutic interventional groups including Ex, L lev, H lev, and their combination (Ex-L lev and Ex-H lev), there was a significant reduction in the mean escaped latency time and the mean distance traveled of animals at consecutive 4-training days as well as a considerable increase in time spent in target quadrant (Q1) of animals at probe trial day compared to kindling (K) group as reported by MWM assay. More specifically, there was a notable difference between the groups that received combinational therapy (Ex-L lev and Ex-H lev) and the groups that received monotherapy (Ex, L-lev, and H-lev), indicating the synergistic effect of physical exercise and levetiracetam. Notably, these findings align with reports from previous investigations [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn line with our study, Gorantla et al. reported that both immediate and delayed exposures to swimming exercise enhanced the percent bias, correct responses, and number of changes based on T-maze testing observations in the kainate-induced TLE model of rats. These findings suggest that even delayed exposure to exercise was beneficial in improving seizure-associated cognitive and behavioral dysfunctions [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn another investigation, cognitive function was enhanced in PTZ-induced epileptic rats subjected to both long-term forced swimming and endurance running, as confirmed by improved performance in the passive avoidance test and object recognition test. Moreover, the decreased levels of synaptic proteins and impaired function of GABAergic neurons were ameliorated in rats subjected to exercise. Furthermore, antagonizing CA3 GABAergic neurons hindered the reversal effects of physical activity on seizures and related cognitive skill dysfunction. These data suggest that long-term and regular exercise can improve cognitive skills by normalizing GABAergic function and synaptic plasticity in the CA3 area of the hippocampus [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA study conducted by Almeida et al. demonstrated that seizure-associated memory deficits are mitigated by a 4-week resistance exercise program, as indicated by inhibitory avoidance test records. They reported that IGF-1 levels increased in pilocarpine-lesioned epileptic rats, and resistance exercise restored them to control levels. On the other hand, reductions in BDNF levels, as well as hypoactivation of ERK and mTOR, were observed in epileptic rats, and these values were restored to control levels by resistance exercise [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn a study evaluating the effect of treadmill exercise on behavioral rehabilitation in pilocarpine-induced epileptic rat models, cognitive tests (Y-maze, Open-field, and Novel object recognition assays) confirmed the enhancement of spatial and object memory following endurance exercise, and electrophysiology-based studies indicated the conservation of hippocampal neuronal plasticity as a consequence of physical activity. Investigations of the potential underlying mechanisms of these observed effects affirmed that exercise protects parvalbumin-expressing interneurons in the hippocampus, probably through the quenching of neuroinflammation and upgrading the integrity of the BBB [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCollectively, the aforementioned findings suggest that regular physical exercise may contribute to neuroprotection and attenuate the progression of kindling.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFinally, based on the findings of the current study, exercise appears to have an anticonvulsant effect in combination with levetiracetam, a well-proven and highly effective medication for treating various types of epilepsy. This effect was confirmed across all seizure parameters examined in our investigation. Moreover, exercise can improve kindling-induced memory and spatial learning deficits as effectively as levetiracetam. On the other hand, no significant difference was observed between the therapeutic effects of low and high doses of levetiracetam, so it can be inferred that the full therapeutic effect can be achieved even with a low dose, and this effect does not accelerate upon increasing the dose; however, the combination of exercise with low- and high-doses of levetiracetam enhances the therapeutic efficacy in all seizure parameters and improves memory and spatial learning cognitive functions. These beneficial effects should be taken into consideration for the potential clinical translation of physical exercise as a non-pharmacological intervention in combination with conventional antiepileptic medications for more efficacious control or treatment of seizures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cp\u003eAll experiment practices were approved by the Research Ethics Committees of Biosafety \u0026amp; laboratory, Tehran University of Medical Sciences \u0026amp; Health Services (Ethical code: IR.TUMS.TIPS.REC.1397.015).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis research was supported by a grant from the Tehran University of Medical Sciences, Tehran, Iran.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAbbas Kebriaeezadeh suppervising research and electrical kindling design Mohammad Sharizadeh supervising and reseach design for MAZ and memory study Ghoran Taghizadeh research design for physical activity testReza Zafari researcher Hassan Gheibi Co researcherMamoud Rezaei co researcher\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eTehran University of Medical Sciences\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMilligan TA. Epilepsy: a clinical overview. Am J Med (2021) 134(7):840\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlair RDG. Temporal lobe epilepsy semiology. Epilepsy Res Treat (2012) 2012(1):751510.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHenning O et al. Temporal lobe epilepsy. Tidsskr Den Nor legeforening (2023) 143.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi Z, Cao W, Sun H, Wang X, Li S, Ran X, Zhang H. Potential clinical and biochemical markers for the prediction of drug-resistant epilepsy: a literature review. Neurobiol Dis (2022)174:105872.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLoPinto-Khoury C. Long-term effects of antiseizure medications. Semin Neurol (2022) 42(05):583\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBesag FMC, Vasey MJ. Neurocognitive effects of antiseizure medications in children and adolescents with epilepsy. 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Neural Regen Res (2024) 19(3):657\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"Epilepsy, Physical exercise, amygdala electrical Kindling, Levetiracetam, Learning, Memory","lastPublishedDoi":"10.21203/rs.3.rs-7215509/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7215509/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eA recent body of evidence has suggested regular exercise as a promising complementary therapeutic strategy in the management of epilepsy and its related cognitive impairments.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e\u003cp\u003eTo put it to the test, our study aimed to comparatively examine the effects of physical exercise, low and high doses of levetiracetam, or the combination of both on amygdala electrical kindling-induced epilepsy in rats, as well as the consequent learning and memory impairments.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMale Wistar rats were randomly divided into ten groups (n\u0026thinsp;=\u0026thinsp;7 per group) as the following: (I) Control (without kindling and exercise), (II) Lev (receiving a high dose of levetiracetam without kindling and exercise), (III) Ex (subjected to exercise without kindling), (IV) Ex-K (subjected to preventive exercise before kindling), (V) K (subjected to kindling without any intervention), (VI) K-Ex (subjected to exercise after kindling), (VII) K-L lev (receiving a low dose of levetiracetam after kindling), (VIII) K-H lev (receiving a high dose of levetiracetam after kindling), (IX) K-Ex-L lev (subjected to exercise and receiving a low dose of levetiracetam after kindling), and (X) K-Ex-H lev (subjected to exercise and receiving a high dose of levetiracetam after kindling). After the kindling procedure and interventions, the seizure parameters, including dADD, S\u003csub\u003e1\u003c/sub\u003eL, S\u003csub\u003e2\u003c/sub\u003eL, S\u003csub\u003e3\u003c/sub\u003eL, S\u003csub\u003e4\u003c/sub\u003eL, S\u003csub\u003e5\u003c/sub\u003eL, Max S5D, and Max ADD, were recorded, and seizure-related behavioral changes were evaluated using the MWM test.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eOur findings showed that in all therapeutic interventional groups, including Ex, L lev, H lev, and their combination (Ex-L lev and Ex-H lev), there was a substantial reduction in parameters, including seizure stages, seizure duration, and dADD. In contrast, there was a significant increase in the mean delay time or latency from electrical stimulation to the onset of stages 1, 2, and 3 of seizure (S\u003csub\u003e1\u003c/sub\u003eL, S2L, and S3L), and all groups were significantly different from the kindling group. Moreover, the kindling-induced spatial memory and learning deficit was remarkably ameliorated by preventive exercise, Ex, L lev, H lev, and their combination.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur study reveals that, in conjunction with levetiracetam, regular exercise can ameliorate the intensity and frequency of amygdala electrical kindling-induced epileptic seizures, as well as the consequent spatial memory and learning impairments.\u003c/p\u003e","manuscriptTitle":"The effect of physical activity on epileptic seizures and consequent learning and memory impairment in electrical amygdala kindling model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-05 20:19:27","doi":"10.21203/rs.3.rs-7215509/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":"58615d64-25e1-481e-b951-987582ab9669","owner":[],"postedDate":"September 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T15:59:43+00:00","versionOfRecord":{"articleIdentity":"rs-7215509","link":"https://doi.org/10.1007/s40199-025-00583-w","journal":{"identity":"daru-journal-of-pharmaceutical-sciences","isVorOnly":false,"title":"DARU Journal of Pharmaceutical Sciences"},"publishedOn":"2026-01-27 15:57:45","publishedOnDateReadable":"January 27th, 2026"},"versionCreatedAt":"2025-09-05 20:19:27","video":"","vorDoi":"10.1007/s40199-025-00583-w","vorDoiUrl":"https://doi.org/10.1007/s40199-025-00583-w","workflowStages":[]},"version":"v1","identity":"rs-7215509","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7215509","identity":"rs-7215509","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}