Comparison the effect of atorvastatin and forskolin on memory after global cerebral ischemia in male rats

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For this purpose, we used atorvastatin and forskolin after ischemia to improve neuronal injury and memory problems then compared the effect of these treatments to find the more effective strategy. Methods: Adult male rats (n=70) were divided into 7groups (n=10). Group 1: control; group 2 : sham; group 3: CI (global cerebral ischemia only); group 4: CI+ forskolin; group 5: CI+ atorvastatin; group 6: CI+vehicle1; group 7: CI+vehicle2. Ischemia was induced by occluding the bilateral common carotid artery. Atorvastatin and forskolin were injected intraperitoneally 4, 24, and 42 h after ischemia in group 5 and group6. Memory was evaluated with a water maze test. Data were analyzed by one-way ANOVA. Results : The level of significance was 5% (p ≤ 0.05). Atorvastatin and forskolin treatment significantly reduced neuronal injury and improved memory in comparison with the ischemia group. Rats in the forskolin treated group had better performance in the Morris water maze test. Conclusion : Our findings showed that forskolin is more effective compared to atorvastatin after global cerebral ischemia. Ischemia Atorvastatin Forskolin Memory Water maze Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ischemia stroke is one of the main causes of disability and death in the world(Harukuni and Bhardwaj 2006 ; Mello, Benetti et al. 2009 ). One of the disorders that can be observed in ischemic survivors is learning and memory disorders(Kiyota, Miyamoto et al. 1991 ; Cechetti, Worm et al. 2012 ). Global ischemia-induced by occluding bilateral common carotid artery for 20 minutes causes damage to vulnerable cells(Ábrahám and Lázár 2000 ; Collino, Aragno et al. 2006 ). CA1 region of hippocampus is one of the sensitive regions that can be injured by ischemia (Hartman, Lee et al. 2005 ; Lana, Ugolini et al. 2020). Hippocampus is a sensitive part of the brain that has an important role in memory and learning and is injured by ischemia. Indeed ischemia damages hippocampus neurons and produces memory deficits in a variety of behavioral tasks (Houslay 2001 ; Hartman, Lee et al. 2005 ). One of the important functions of the brain is memory and any impairment of that influence of our life. Pyramidal neurons on the CA1 part of the hippocampus have an important role in memory and learning (Kiyota, Miyamoto et al. 1991 ; Engin, Sigal et al. 2020 ). Studies have shown that cyclic AMP (CAMP) signaling plays a role in the mediation of ischemia-induced neuronal damage and memory problems(Bendel, Bueters et al. 2005 ; Mehan, Dudi et al. 2018). Furthermore, Cyclic AMP (CAMP) plays important role in cell communication. Ischemia produces neuronal Damage in the hippocampus and impairs hippocampal-dependent memory(Baytan, Alkanat et al. 2008 ; Kadam, Perveen et al. 2021). Statins, inhibitors of cholesterol synthesis, are used to prevent cardiovascular complications and these drugs influence some cognitive functions(Zhang, Dong et al. 2020 ). The effect of the most common statins such as simvastatin and atorvastatin have been studied experimentally in different models of memory impairment (Ando, Kametani et al. 1987 ; Georgieva-Kotetarova and Kostadinova 2013 ; Fahim, Wadie et al. 2019 ). Studies showed that learning ability and memory improve after treatment with statins conducted after ischemia injury (Kumar, Kumari et al. ; Patole, Lamdhade et al. 2019; Solmaz, Atasoy et al. 2020 ). Atorvastatin improves cognitive disorders by reduction of the oxidative stress damage of the cortex and the hippocampus in adult rats (Solmaz, Atasoy et al. 2020 ; Davis and Vemuganti 2021 ). Forskolin is a plant that has traditionally been used to treat convulsions and asthma in India, Burma, and Thailand (Barad, Bourtchouladze et al. 1998; Reddymalla, Pureti et al. 2021). Studies have been shown forskolin active adenylate cyclase (AC), which increases cyclic adenosine monophosphate (cAMP) levels, an important signal carrier for cell communication (Reneerkens, Rutten et al. 2009 ; Muntean, Marwari et al. 2022). Indeed, increasing cAMP levels was reported to improve long-term memory (Reneerkens, Rutten et al. 2009 ; Mehan, Parveen et al. 2017 ). Also, forskolin has a neuroprotective effect and improves cognition (Kumar and Singh 2017 ; Mehan, Parveen et al. 2017 ; Gorabi, Kiaie et al. 2019). This study investigated the effect of atorvastatin and forskolin on memory and compared the effect of them to investigate which one is more effective on memory problems after global cerebral ischemia. Material and methods Animal care All the experiments were performed by the guidelines for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The experimental protocol was approved by the institutional animal care and the committee of the Tehran University of Medical Sciences (Tehran, Iran). Experimental Groups We prepared male rats (n = 70) weighing 200-250 gr. Rats were randomly divided into 7 groups: Group 1 : control (n = 10) Group 2: sham group (n = 10); rats have endured the same surgical procedures only. Group 3: Cerebral ischemia (CI followed by occluding the bilateral carotid arteries for 20 min). Group 4: CI + forskolin; surgery was performed under general anesthesia and rats were subjected to 20 min of global ischemia. Rats received intraperitoneal injection of 20 mg/kg forskolin 4, 24, and 42 h after ischemia. Group 5: CI + atorvastatin. Rats were subjected to 20 min of global ischemia and, received 4, 24, and 42 h after intraperitoneal injections of 20 mg/kg atorvastatin. Group 6: CI + vehicle1; surgery was performed under general anesthesia, and rats were subjected to 20 min of global ischemia and then, 4, 24, and 42 h after ischemia rats received intraperitoneal injection of DMSO. Group 7: CI + vehicle2; rats were subjected to 20 min of global ischemia and then 4and24 and 42 h after ischemia rats received intraperitoneal injections of saline. Operative Protocol Surgery was performed under general anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg) administrated as intraperitoneal injections and rats were subjected to global cerebral ischemia followed by the occluding bilateral common carotid artery for 20 minutes. Drug and Dose preparation : Atorvastatin (Sigma Co.) that was dissolved in saline and forskolin that was dissolved in DMSO was administered intraperitoneally at the dose of 20 mg/kg 4, 24, and 42 h after CI. Rats in the treatment groups were fed atorvastatin and forskolin 4, 24, and 42 h after global cerebral ischemia. Nissl staining After 7 day’s treatment of rats with atorvastatin and forskolin rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and then sacrificed. Ketamine was used as an anesthetic drug and xylazine as a sedative one to prevent stress in animals. After thoracotomy, at first, all rats were perfused with 120 cc normal saltines and then with 120 ccs paraformaldehyde 4% intracranial via infusion. After fixation, the brains were removed from the skull. The cryosections (30 μm) were prepared from the hippocampal region. Water maze The Morris water maze (MWM) is one of the tests to examine long term spatial memory. The (MWM) consisted of a large black tank (diameter 183 cm) and was filled with water (27° C). There is an escape platform (diameter 10 cm) in the southeast quadrant of the maze and hidden 2 cm below the surface of the water and rats should escape the platform in trial days. During the probe test, the platform could be changed to under the water. For the visible platform task, the platform took 2 cm above the water’s surface. There is a video camera that saved rats performance and interfaced with a computerized tracking system. In this test, we have the trial for 3 days and on the 4rd-day probe, the trial was done. We used this way to evaluate rats performance and memory after CI. Tunnel test The neuronal nuclear injury was evaluated by terminal deoxynucleotidyl transferase-mediated 2’Deoxyuridine 5’-Triphosphate-biotin nick end labeling (TUNEL) staining after ischemia. The paraffin section (3 µm), 10 slides for every rat, were prepared, xylene was used for deparaffinized, ethanol was utilized for dehydrating, slides were washed in phosphate-buffered saline (PBS) for 30 min at room temperature, and incubated with proteinase k for 30 min at +21 to +37 °C to block endogenous peroxidase (POD). Then, the slides were incubated with the TUNEL reaction mixture at 37 °C for 60 min, washed with PBS, and visible by using converter-POD for 30 min at 37 °C and a humidified atmosphere. Slides were washed with PBS; 50-100 µl 3, 3’-diaminobenzidine- tetrahydrochloride-dihydrate (DAB) was added and washed with PBS. Finally, slides were mounted and investigated by a light microscope at x400 magnification. Statistical analysis Statistical analysis was performed by one-way ANOVA (analysis of variance) followed by post hoc Turkey’s test to compare more than two groups. In the case of comparison of two different groups, an unpaired Student t-test was used. All statistical analyses were performed using Graph Pad Prism (version 5.0, Graph Pad Software Inc., San Diego, California) and significance was considered at P < 0.05. Data were expressed as mean ± SEM. Results Forskolin and atorvastatin reduce neuronal damage Hippocampus region in the rats brain was first examined by routine histology to confirm the presence of histologic changes after ischemia. Brain sections in the control group exhibited shiny cell nuclei and intact cell membrane (picture b) (Fig. 1 ), but in the ischemia group cells had irregular appearance with dense pyknotic nuclei (picture a) (Fig. 1 ). Our histological findings revealed that after ischemia the number of normal neurons significantly decreased (Fig. 2 ). The hippocampal CA1 neuron count was 45 ± 5 in the control group (b) and 3 ± 4 in animals subjected to ischemia (a) (Figoure1). Our findings revealed a reduction of necrotic cells in CI + forskolin and CI + atorvastatin groups. There were larger necrotic cells in the hippocampus of rats in the ischemia group (firure2), as compared with the CI + forskolin and CI + atorvastatin groups. There was no difference between the vehicle group and the ischemia group. **p < 0.05 Figure 2 . The effect of forskolin and atorvastatin on CA1 hippocampal neurons. A representative diagram of coronal sections through the hippocampus shows a reduction of necrotic cells following forskolin and atorvastatin treatment. There were larger necrotic cells in the hippocampus of rats in the ischemia group (B), as compared with the forskolin and atorvastatin-treated group. It is no difference between the vehicle group and the ischemia group. **p < 0.05 Tunnel assay We used the TUNEL assay to determine the presence and degree of apoptosis after ischemia. TUNEL-positive cells were present in the ischemia group (picture a) (Fig. 3 ), and none were detected in the control group (picture b). Apoptotic nuclei were evident in the ischemia group; TUNEL-positive cells were irregular appearance with dense pyknotic nuclei (picture a) (figure). Tunnel negative (picture b) cells in the control, CI + atorvastatin, and CI + forskolin have shiny nuclei and intact cell membrane (picture b) (Fig. 3 ). Treatment with atorvastatin and forskolin improved neuronal damage and decreased the number of damaged cells (picture c, d). Our results showed that the number of hippocampal CA1 normal neurons significantly decreased after ischemia (Fig. 4 ), and treatment with atorvastatin and forskolin increased the number of these neurons. Furthermore, using forskolin after ischemia significantly increased the number of normal neurons compared to atorvastatin treated group. Figure 4 . Tunnel investigation of hippocampus exhibiting the number of normal cells with regular appearance decreased after ischemia. Atorvastatin and forskolin treated groups increased the number of normal cells. There is no different between vehicle groups and ischemia group. **p < 0.05 Effect of forskolin and atorvastatin on learning and memory abilities Morris water maze On day 4 of the study, the time of swimming was significantly increased in the ischemia group than in the normal group or treatment groups. The time of swimming was significantly decreased in treatment groups and time of swimming significantly reduced in the forskolin treated group than atorvastatin treated group ( P < 0.05; Fig. 4 ). There were no significant differences in vehicle groups with the ischemia group. On day 4 distance was recorded to assess rat learning and memory abilities. The distance was significantly decreased ischemia group than in normal group ( P < 0.05). There was no significant difference in distance between ischemia and vehicle groups. Distance significantly increased in CI + forskolin, and CI + atorvastatin groups than in ischemia group. Also, rats in forskolin treated group (CI + forskolin) showed more distance of swimming in comparison with the atorvastatin treated group (CI + atorvastatin), ( P < 0.05; Fig. 5 ). Figure 4 . Effect of treatment with forskolin and atorvastatin on learning and memory in the water maze. Most time of swimming is in the ischemia group. Atorvastatin and forskolin improved rats performance and reduce the time of finding the platform. **p < 0.05 Figure 5 . The Effect of forskolin and atorvastatin treatments on learning and memory in the water maze. The distance in the CI group decreased and treatment with atorvastatin and forskolin increased the distance. It is no difference between the vehicle and the ischemia group. **p < 0.05 Discussion Stroke not only causes physical damage but also cognitive problems. About 60 to 70 percent of patients with a stroke develop movement problems, impaired perception, and memory or language problems (De Luca, Torrisi et al. 2018 ; Lugtmeijer, Lammers et al. 2021 ). The hippocampus is one of the brain regions that play an important role in learning and memory that is very vulnerable to ischemia-reperfusion injury (Tian, Zhang et al. 2013 ; Stevenson, Zheng et al. 2018). In this study, we examined forskolin and atorvastatin treatment effect on inhibiting neuronal damage and memory problems after cerebral ischemia and then compared them to determine effective treatment. Initially, we have developed a mouse model for global cerebral ischemia by occluding bilateral common carotid artery for 20 minutes (Wahul, Joshi et al. 2018 ; León-Moreno, Castañeda-Arellano et al. 2020 ). Then, the histological change of CA1 region of hippocampus were evaluated after ischemia-reperfusion. The histological results showed that atorvastatin and forskolin treatment improved the neuronal damage in the hippocampus, thus preventing neuronal injury, which was similar to the previously reported finding (van der Most, Dolga et al. 2009 ; Ivanova, Sitnikova et al. 2019 ). Atorvastatin exhibited protective effects on survival rate and cognitive disorders after ischemia by inhibiting the release of inflammatory cytokines, oxidative stress, and neuronal apoptosis in brain tissue (Georgieva-Kotetarova and Kostadinova 2013 ; Mancini, Martins et al. 2020 ; Tian, Tai et al. 2020 ). Also, studies showed that using forskolin could reduce ROS production, inflammation and, brain injury (Guo, Bai et al. 2018 ; Patole, Lamdhade et al. 2019). Cerebral ischemia injury (CI) often causes neuronal injury and death, by oxidative stress, inflammation and apoptosis (Liu, Wang et al. 2020 ; Wu, Xiong et al. 2020 ). The oxidative stress damages the hippocampus and memory(Gargouri, Yousif et al. 2018 ; Hajipour, Farbood et al. 2020 ). In view of this, we investigated the apoptosis in neurons of hippocampus area. It is well known that necrosis or apoptosis can aggravate ischemic damage. Ischemia-reperfusion injury is associated with inflammatory response and apoptosis. In the present study, the forskolin and atorvastatin treatment significantly reduced the number of Tunnel-positive cells after ischemia. Furthermore, the forskolin treatment reduced significantly apoptotic cells in comparison with atorvastatin treatment group. Forskolin reduce cell death and apoptosis, which exerts its pharmacological effects mainly by increasing intracellular cAMP levels and decreasing Ros production. On the other hand, treatment with atorvastatin down-regulated caspase-3 expression, reduced cell apoptosis, and improved neurological outcome. These results are similar to those which were found in the current study, which suggests the effects of apoptosis reduction and neuroprotection(Wu, Tang et al. 2014 ; Guo, Bai et al. 2018 ). Water maze test have been used to investigate the functional response and the effect of atorvastatin and forskolin on memory and learning in rats endure global cerebral ischemia (CI). Atorvastatin and forskolin treatment reduced damage to neurons in the CA1 region and improved animal performance in the water maze test compared with the CI group (Bingham, Martin et al. 2012 ; Tian, Ding et al. 2019; Yuan, Zhou et al. 2019 ). Our study revealed that damage to the CA1 of the hippocampus induces deficiency in memory as measured by the Morris water maze. Our study was in line with other studies and showed that treatment with atorvastatin and forskolin improves animal memory and their performance in the water maze test (Mehan, Parveen et al. 2017 ; Yoo, Stremlau et al. 2021 ). There is no significant difference in vehicle groups with the CI group. Studies have been shown forskolin cause the activation of adenylate cyclase (Sapio, Gallo et al. 2017 ). The activation of adenylate cyclase increases cyclic adenosine monophosphate levels a key signal carrier for cell communication. CAMP improves the memory and function of rats (Ando, Kametani et al. 1987 ; Bernabeu, Bevilaqua et al. 1997; Kumar and Singh 2017 ). On the other hand, Atorvastatin improves memory function by decreasing oxidative stress and inflammatory activity in the brain (Solmaz, Atasoy et al. 2020 ). Our study showed that treatment with atorvastatin and forskolin reduced neuronal injury and improved memory compared to the cerebral ischemia group, and forskolin was more effective. Indeed, rats in forskolin treatment group have been shown better performance in the Morris water maze test. Our findings were in line with other studies that investigated the neuroprotective effect of atorvastatin and forskolin on memory and learning, showed that atorvastatin and forskolin were efficient in improving memory deficits after brain injury (Laufs, Gertz et al. 2000 ; Yrjänheikki, Koistinaho et al. 2005 ). Ischemic stroke results from occluding of a blood vessel supplying the brain and is causing increasing stress oxidative, inflammatory, neuronal cells death, apoptosis, and memory problems. In the current study, both forskolin and atorvastatin treatment significantly decreased indications of brain damage, neuronal injury on the CA1 region of the hippocampus, apoptosis, and memory impairment. In addition, our findings showed that rats in the forskolin treated group had better performance in the water maze test in comparison with the atorvastatin treated group. Conclusion In this study global cerebral ischemia had demonstrated significant impairment in learning and memory. 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"Pleiotropic use of Statins as non-lipid-lowering drugs." International Journal of Biological Sciences 16 (14): 2704. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 27 Jun, 2024 Submission checks completed at journal 23 Jun, 2024 First submitted to journal 23 Jun, 2024 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4624330","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319923296,"identity":"62821d31-9346-4f1d-8ae2-c8521a0dc3e7","order_by":0,"name":"Alieh Bashghareh","email":"","orcid":"","institution":"Shahroud University of medical science","correspondingAuthor":false,"prefix":"","firstName":"Alieh","middleName":"","lastName":"Bashghareh","suffix":""},{"id":319923297,"identity":"b512be51-d577-493f-84ef-d8ea650c9e1c","order_by":1,"name":"Peyman Modarresi","email":"","orcid":"","institution":"East Azerbaijan Province, Azad University","correspondingAuthor":false,"prefix":"","firstName":"Peyman","middleName":"","lastName":"Modarresi","suffix":""},{"id":319923298,"identity":"568931d5-c989-4962-b5f6-838e744d5306","order_by":2,"name":"Samaneh Mahdavian","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Samaneh","middleName":"","lastName":"Mahdavian","suffix":""},{"id":319923299,"identity":"72554b5a-74ce-482d-a76b-188d6365a3ba","order_by":3,"name":"Azim Hedayatpour","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACAzCCAMYHcOEEwlrA2pgNDpCqhU3iAB6VcGDO3rzxM0/Nnzx+idxn1R/bttkzsB9+wPBwD24tlj3HiqV5jhkUS85IN7txsO12YgNPmgFDwjM8DruRYyDNw2aQuOFGGhtIC9AXOUC/4HGiwf03xr95/kG0FAC12DPwvyGg5QaPmTRvG0QLA1ALY4MEIVvOpJVZzu0zLpbsecYscebc7cQ2iWcGB/BqOX5484033+Ty+NnTGD9UlN225+dPfvjwBx4tIMDEgxx3bEBMQAMwofzAH92jYBSMglEw0gEA+rRU16FGK0QAAAAASUVORK5CYII=","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Azim","middleName":"","lastName":"Hedayatpour","suffix":""}],"badges":[],"createdAt":"2024-06-23 08:08:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4624330/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4624330/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60340423,"identity":"0360b68b-cdcd-44fc-b811-dc9e5a9f86ec","added_by":"auto","created_at":"2024-07-15 18:25:48","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83474,"visible":true,"origin":"","legend":"\u003cp\u003eHistological change of hippocampus region of brain after ischemia. In the control group, glossy cell nuclei and cell membranes are normal (picture b), and Damaged cells have irregular appearance with dense pyknotic nuclei (picture a).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/733286724bf553a3b7f2d14c.jpg"},{"id":60340419,"identity":"be9c4142-09b2-4808-9d8a-856b5c2aced9","added_by":"auto","created_at":"2024-07-15 18:25:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":42921,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of forskolin and atorvastatin on CA1 hippocampal neurons. A representative diagram of coronal sections through the hippocampus shows a reduction of necrotic cells following forskolin and atorvastatin treatment. There were larger necrotic cells in the hippocampus of rats in the ischemia group (B), as compared with the forskolin and atorvastatin-treated group. It is no difference between the vehicle group and the ischemia group. **p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/6147a22570ceaaa2cb122007.jpg"},{"id":60340422,"identity":"12b1d049-b397-42b8-940a-2d4c4af30a76","added_by":"auto","created_at":"2024-07-15 18:25:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87058,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of apoptotic changes in hippocampal neurons by tunnel test. In ischemia (a), neurons in the CA1 region of the hippocampus are TUNEL positive. Normal cells (b) have a regular appearance, and apoptotic cells (a) are dark and distinct. The number of damaged cells decreased in the treatment groups (Figure c, d).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/a7d11e15ec27f1dc0a92738d.jpg"},{"id":60340424,"identity":"9b6687da-c2aa-49be-a9ce-e0178f37a80e","added_by":"auto","created_at":"2024-07-15 18:25:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":42990,"visible":true,"origin":"","legend":"\u003cp\u003eTunnel investigation of hippocampus exhibiting the number of normal cells with regular appearance decreased after ischemia. Atorvastatin and forskolin treated groups increased the number of normal cells.There is no different between vehicle groups and ischemia group. **p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/b3186fa7e26c8af2b0baec13.jpg"},{"id":60340420,"identity":"a9e43973-f998-426f-8f64-0a4d37963a06","added_by":"auto","created_at":"2024-07-15 18:25:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 4\u003c/strong\u003e. Effect of treatment with forskolin and atorvastatin on learning and memory in the water maze. Most time of swimming is in the ischemia group. Atorvastatin and forskolin improved rats performance and reduce the time of finding the platform. **p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/be1deb2ff797fbe9923d2573.jpg"},{"id":60340418,"identity":"c0028564-68cf-4b49-8ee2-ec261f888ee9","added_by":"auto","created_at":"2024-07-15 18:25:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5\u003c/strong\u003e. The Effect of forskolin and atorvastatin treatments on learning and memory in the water maze. The distance in the CI group decreased and treatment with atorvastatin and forskolin increased the distance. It is no difference between the vehicle and the ischemia group. **p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/e15d6e6cf3b60fa5a14790dc.jpg"},{"id":60340442,"identity":"8fc05df4-80cc-4a3e-a212-d95b8096f65e","added_by":"auto","created_at":"2024-07-15 18:25:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":855215,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4624330/v1/53641f86-c86e-4866-bcba-8859ffa48847.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison the effect of atorvastatin and forskolin on memory after global cerebral ischemia in male rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIschemia stroke is one of the main causes of disability and death in the world(Harukuni and Bhardwaj \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mello, Benetti et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). One of the disorders that can be observed in ischemic survivors is learning and memory disorders(Kiyota, Miyamoto et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Cechetti, Worm et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Global ischemia-induced by occluding bilateral common carotid artery for 20 minutes causes damage to vulnerable cells(\u0026Aacute;brah\u0026aacute;m and L\u0026aacute;z\u0026aacute;r \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Collino, Aragno et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). CA1 region of hippocampus is one of the sensitive regions that can be injured by ischemia (Hartman, Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Lana, Ugolini et al. 2020). Hippocampus is a sensitive part of the brain that has an important role in memory and learning and is injured by ischemia. Indeed ischemia damages hippocampus neurons and produces memory deficits in a variety of behavioral tasks (Houslay \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hartman, Lee et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). One of the important functions of the brain is memory and any impairment of that influence of our life. Pyramidal neurons on the CA1 part of the hippocampus have an important role in memory and learning (Kiyota, Miyamoto et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Engin, Sigal et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies have shown that cyclic AMP (CAMP) signaling plays a role in the mediation of ischemia-induced neuronal damage and memory problems(Bendel, Bueters et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mehan, Dudi et al. 2018). Furthermore, Cyclic AMP (CAMP) plays important role in cell communication. Ischemia produces neuronal Damage in the hippocampus and impairs hippocampal-dependent memory(Baytan, Alkanat et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kadam, Perveen et al. 2021). Statins, inhibitors of cholesterol synthesis, are used to prevent cardiovascular complications and these drugs influence some cognitive functions(Zhang, Dong et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The effect of the most common statins such as simvastatin and atorvastatin have been studied experimentally in different models of memory impairment (Ando, Kametani et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Georgieva-Kotetarova and Kostadinova \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fahim, Wadie et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies showed that learning ability and memory improve after treatment with statins conducted after ischemia injury (Kumar, Kumari et al. ; Patole, Lamdhade et al. 2019; Solmaz, Atasoy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Atorvastatin improves cognitive disorders by reduction of the oxidative stress damage of the cortex and the hippocampus in adult rats (Solmaz, Atasoy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Davis and Vemuganti \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Forskolin is a plant that has traditionally been used to treat convulsions and asthma in India, Burma, and Thailand (Barad, Bourtchouladze et al. 1998; Reddymalla, Pureti et al. 2021). Studies have been shown forskolin active adenylate cyclase (AC), which increases cyclic adenosine monophosphate (cAMP) levels, an important signal carrier for cell communication (Reneerkens, Rutten et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Muntean, Marwari et al. 2022). Indeed, increasing cAMP levels was reported to improve long-term memory (Reneerkens, Rutten et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Mehan, Parveen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Also, forskolin has a neuroprotective effect and improves cognition (Kumar and Singh \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Mehan, Parveen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gorabi, Kiaie et al. 2019). This study investigated the effect of atorvastatin and forskolin on memory and compared the effect of them to investigate which one is more effective on memory problems after global cerebral ischemia.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal care\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experiments were performed by the guidelines for the care and use of laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The experimental protocol was approved by the institutional animal care and the committee of the Tehran University of Medical Sciences (Tehran, Iran).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Groups\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe prepared male rats (n = 70) weighing 200-250 gr. Rats were randomly divided into 7 groups:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 1\u003c/strong\u003e: control (n = 10)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 2:\u0026nbsp;\u003c/strong\u003esham group (n = 10); rats have endured the same surgical procedures only.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 3:\u0026nbsp;\u003c/strong\u003eCerebral ischemia (CI followed by occluding the bilateral carotid arteries for 20 min).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eGroup 4:\u0026nbsp;\u003c/strong\u003eCI + forskolin; surgery was performed under general anesthesia and rats were subjected to 20 min of global ischemia. Rats received intraperitoneal injection of 20 mg/kg forskolin 4, 24, and 42 h after ischemia.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eGroup 5:\u0026nbsp;\u003c/strong\u003eCI + atorvastatin. Rats were subjected to 20 min of global ischemia and, received 4, 24, and 42 h after intraperitoneal injections of 20 mg/kg atorvastatin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 6:\u0026nbsp;\u003c/strong\u003eCI + vehicle1; surgery was performed under general anesthesia, and rats were subjected to 20 min of global ischemia and then, 4, 24, and 42 h after ischemia rats received intraperitoneal injection of DMSO.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup 7:\u0026nbsp;\u003c/strong\u003eCI + vehicle2; rats were subjected to 20 min of global ischemia and then 4and24 and 42 h after ischemia rats received intraperitoneal injections of saline.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOperative Protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurgery was performed under general anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg) administrated as intraperitoneal injections and rats were subjected to global cerebral ischemia followed by the occluding bilateral common carotid artery for 20 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eDrug and Dose preparation\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eAtorvastatin (Sigma Co.) that was dissolved in saline and forskolin that was dissolved in DMSO was administered intraperitoneally at the dose of 20 mg/kg 4, 24, and 42 h after CI. Rats in the treatment groups were fed atorvastatin and forskolin 4, 24, and 42 h after global cerebral ischemia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNissl staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 7 day’s treatment of rats with atorvastatin and forskolin rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and then sacrificed. Ketamine was used as an anesthetic drug and xylazine as a sedative one to prevent stress in animals. After thoracotomy, at first, all rats were perfused with 120 cc normal saltines and then with 120 ccs paraformaldehyde 4% intracranial \u003cem\u003evia\u0026nbsp;\u003c/em\u003einfusion. After fixation, the brains were removed from the skull. The cryosections (30 μm) were prepared from the hippocampal region.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWater maze\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Morris water maze (MWM) is one of the tests to examine long term spatial memory. The (MWM) consisted of a large black tank (diameter 183 cm) and was filled with water (27° C). There is an escape platform (diameter 10 cm) in the southeast quadrant of the maze and hidden 2 cm below the surface of the water and rats should escape the platform in trial days. During the probe test, the platform could be changed to under the water. For the visible platform task, the platform took 2 cm above the water’s surface. There is a video camera that saved rats performance and interfaced with a computerized tracking system. In this test, we have the trial for 3 days and on the 4rd-day probe, the trial was done. We used this way to evaluate rats performance and memory after CI.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eTunnel test\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe neuronal nuclear injury was evaluated by terminal deoxynucleotidyl transferase-mediated 2’Deoxyuridine 5’-Triphosphate-biotin nick end labeling (TUNEL) staining after ischemia. The paraffin section (3 µm), 10 slides for every rat, were prepared, xylene was used for deparaffinized, ethanol was utilized for dehydrating, slides were washed in phosphate-buffered saline (PBS) for 30 min at room temperature, and incubated with proteinase k for 30 min at +21 to +37 °C to block endogenous peroxidase (POD). Then, the slides were incubated with the TUNEL reaction mixture at 37 °C for 60 min, washed with PBS, and visible by using converter-POD for 30 min at 37 °C and a humidified atmosphere. Slides were washed with PBS; 50-100 µl 3, 3’-diaminobenzidine- tetrahydrochloride-dihydrate (DAB) was added and washed with PBS. Finally, slides were mounted and investigated by a light microscope at x400 magnification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed by one-way ANOVA (analysis of variance) followed by post hoc Turkey’s test to compare more than two groups. In the case of comparison of two different groups, an unpaired Student t-test was used. All statistical analyses were performed using Graph Pad Prism (version 5.0, Graph Pad Software Inc., San Diego, California) and significance was considered at P \u0026lt; 0.05. Data were expressed as mean ± SEM.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eForskolin and atorvastatin reduce neuronal damage\u003c/h2\u003e \u003cp\u003eHippocampus region in the rats brain was first examined by routine histology to confirm the presence of histologic changes after ischemia. Brain sections in the control group exhibited shiny cell nuclei and intact cell membrane (picture b) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), but in the ischemia group cells had irregular appearance with dense pyknotic nuclei (picture a) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our histological findings revealed that after ischemia the number of normal neurons significantly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The hippocampal CA1 neuron count was 45\u0026thinsp;\u0026plusmn;\u0026thinsp;5 in the control group (b) and 3\u0026thinsp;\u0026plusmn;\u0026thinsp;4 in animals subjected to ischemia (a) (Figoure1). Our findings revealed a reduction of necrotic cells in CI\u0026thinsp;+\u0026thinsp;forskolin and CI\u0026thinsp;+\u0026thinsp;atorvastatin groups. There were larger necrotic cells in the hippocampus of rats in the ischemia group (firure2), as compared with the CI\u0026thinsp;+\u0026thinsp;forskolin and CI\u0026thinsp;+\u0026thinsp;atorvastatin groups. There was no difference between the vehicle group and the ischemia group. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The effect of forskolin and atorvastatin on CA1 hippocampal neurons. A representative diagram of coronal sections through the hippocampus shows a reduction of necrotic cells following forskolin and atorvastatin treatment. There were larger necrotic cells in the hippocampus of rats in the ischemia group (B), as compared with the forskolin and atorvastatin-treated group. It is no difference between the vehicle group and the ischemia group. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTunnel assay\u003c/h2\u003e \u003cp\u003eWe used the TUNEL assay to determine the presence and degree of apoptosis after ischemia. TUNEL-positive cells were present in the ischemia group (picture a) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e), and none were detected in the control group (picture b). Apoptotic nuclei were evident in the ischemia group; TUNEL-positive cells were irregular appearance with dense pyknotic nuclei (picture a) (figure). Tunnel negative (picture b) cells in the control, CI\u0026thinsp;+\u0026thinsp;atorvastatin, and CI\u0026thinsp;+\u0026thinsp;forskolin have shiny nuclei and intact cell membrane (picture b) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Treatment with atorvastatin and forskolin improved neuronal damage and decreased the number of damaged cells (picture c, d). Our results showed that the number of hippocampal CA1 normal neurons significantly decreased after ischemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and treatment with atorvastatin and forskolin increased the number of these neurons. Furthermore, using forskolin after ischemia significantly increased the number of normal neurons compared to atorvastatin treated group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Tunnel investigation of hippocampus exhibiting the number of normal cells with regular appearance decreased after ischemia. Atorvastatin and forskolin treated groups increased the number of normal cells. There is no different between vehicle groups and ischemia group. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of forskolin and atorvastatin on learning and memory abilities\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eMorris water maze\u003c/h2\u003e \u003cp\u003eOn day 4 of the study, the time of swimming was significantly increased in the ischemia group than in the normal group or treatment groups. The time of swimming was significantly decreased in treatment groups and time of swimming significantly reduced in the forskolin treated group than atorvastatin treated group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There were no significant differences in vehicle groups with the ischemia group. On day 4 distance was recorded to assess rat learning and memory abilities. The distance was significantly decreased ischemia group than in normal group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant difference in distance between ischemia and vehicle groups. Distance significantly increased in CI\u0026thinsp;+\u0026thinsp;forskolin, and CI\u0026thinsp;+\u0026thinsp;atorvastatin groups than in ischemia group. Also, rats in forskolin treated group (CI\u0026thinsp;+\u0026thinsp;forskolin) showed more distance of swimming in comparison with the atorvastatin treated group (CI\u0026thinsp;+\u0026thinsp;atorvastatin), (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Effect of treatment with forskolin and atorvastatin on learning and memory in the water maze. Most time of swimming is in the ischemia group. Atorvastatin and forskolin improved rats performance and reduce the time of finding the platform. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The Effect of forskolin and atorvastatin treatments on learning and memory in the water maze. The distance in the CI group decreased and treatment with atorvastatin and forskolin increased the distance. It is no difference between the vehicle and the ischemia group. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStroke not only causes physical damage but also cognitive problems. About 60 to 70 percent of patients with a stroke develop movement problems, impaired perception, and memory or language problems (De Luca, Torrisi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lugtmeijer, Lammers et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The hippocampus is one of the brain regions that play an important role in learning and memory that is very vulnerable to ischemia-reperfusion injury (Tian, Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Stevenson, Zheng et al. 2018). In this study, we examined forskolin and atorvastatin treatment effect on inhibiting neuronal damage and memory problems after cerebral ischemia and then compared them to determine effective treatment. Initially, we have developed a mouse model for global cerebral ischemia by occluding bilateral common carotid artery for 20 minutes (Wahul, Joshi et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Le\u0026oacute;n-Moreno, Casta\u0026ntilde;eda-Arellano et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Then, the histological change of CA1 region of hippocampus were evaluated after ischemia-reperfusion. The histological results showed that atorvastatin and forskolin treatment improved the neuronal damage in the hippocampus, thus preventing neuronal injury, which was similar to the previously reported finding (van der Most, Dolga et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ivanova, Sitnikova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Atorvastatin exhibited protective effects on survival rate and cognitive disorders after ischemia by inhibiting the release of inflammatory cytokines, oxidative stress, and neuronal apoptosis in brain tissue (Georgieva-Kotetarova and Kostadinova \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mancini, Martins et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tian, Tai et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Also, studies showed that using forskolin could reduce ROS production, inflammation and, brain injury (Guo, Bai et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Patole, Lamdhade et al. 2019). Cerebral ischemia injury (CI) often causes neuronal injury and death, by oxidative stress, inflammation and apoptosis (Liu, Wang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wu, Xiong et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The oxidative stress damages the hippocampus and memory(Gargouri, Yousif et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hajipour, Farbood et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In view of this, we investigated the apoptosis in neurons of hippocampus area. It is well known that necrosis or apoptosis can aggravate ischemic damage. Ischemia-reperfusion injury is associated with inflammatory response and apoptosis. In the present study, the forskolin and atorvastatin treatment significantly reduced the number of Tunnel-positive cells after ischemia. Furthermore, the forskolin treatment reduced significantly apoptotic cells in comparison with atorvastatin treatment group.\u003c/p\u003e \u003cp\u003eForskolin reduce cell death and apoptosis, which exerts its pharmacological effects mainly by increasing intracellular cAMP levels and decreasing Ros production. On the other hand, treatment with atorvastatin down-regulated caspase-3 expression, reduced cell apoptosis, and improved neurological outcome. These results are similar to those which were found in the current study, which suggests the effects of apoptosis reduction and neuroprotection(Wu, Tang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Guo, Bai et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Water maze test have been used to investigate the functional response and the effect of atorvastatin and forskolin on memory and learning in rats endure global cerebral ischemia (CI). Atorvastatin and forskolin treatment reduced damage to neurons in the CA1 region and improved animal performance in the water maze test compared with the CI group (Bingham, Martin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tian, Ding et al. 2019; Yuan, Zhou et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our study revealed that damage to the CA1 of the hippocampus induces deficiency in memory as measured by the Morris water maze. Our study was in line with other studies and showed that treatment with atorvastatin and forskolin improves animal memory and their performance in the water maze test (Mehan, Parveen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yoo, Stremlau et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). There is no significant difference in vehicle groups with the CI group.\u003c/p\u003e \u003cp\u003eStudies have been shown forskolin cause the activation of adenylate cyclase (Sapio, Gallo et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The activation of adenylate cyclase increases cyclic adenosine monophosphate levels a key signal carrier for cell communication. CAMP improves the memory and function of rats (Ando, Kametani et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Bernabeu, Bevilaqua et al. 1997; Kumar and Singh \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, Atorvastatin improves memory function by decreasing oxidative stress and inflammatory activity in the brain (Solmaz, Atasoy et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur study showed that treatment with atorvastatin and forskolin reduced neuronal injury and improved memory compared to the cerebral ischemia group, and forskolin was more effective. Indeed, rats in forskolin treatment group have been shown better performance in the Morris water maze test. Our findings were in line with other studies that investigated the neuroprotective effect of atorvastatin and forskolin on memory and learning, showed that atorvastatin and forskolin were efficient in improving memory deficits after brain injury (Laufs, Gertz et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Yrj\u0026auml;nheikki, Koistinaho et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Ischemic stroke results from occluding of a blood vessel supplying the brain and is causing increasing stress oxidative, inflammatory, neuronal cells death, apoptosis, and memory problems. In the current study, both forskolin and atorvastatin treatment significantly decreased indications of brain damage, neuronal injury on the CA1 region of the hippocampus, apoptosis, and memory impairment. In addition, our findings showed that rats in the forskolin treated group had better performance in the water maze test in comparison with the atorvastatin treated group.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study global cerebral ischemia had demonstrated significant impairment in learning and memory. Treatment with atorvastatin and forskolin improved the global cerebral ischemia-induced impairment of learning and memory. Furthermore, in the forskolin treated group rats had better performance in the water maze test thus, forskolin could be more effective to improve neuronal injury and memory in comparison with atorvastatin.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eModarres and Mahdavian collected dataHedayatpour design the studyBashghareh wrote article\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eWe thank the Tehran University of medical science and the department of anatomy.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Aacute;brah\u0026aacute;m, H. and G. 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(2020). \u0026quot;Pleiotropic use of Statins as non-lipid-lowering drugs.\u0026quot; \u003cu\u003eInternational Journal of Biological Sciences\u003c/u\u003e \u003cstrong\u003e16\u003c/strong\u003e(14): 2704.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-computational-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcns","sideBox":"Learn more about [Journal of Computational Neuroscience](http://link.springer.com/journal/10827)","snPcode":"10827","submissionUrl":"https://submission.nature.com/new-submission/10827/3","title":"Journal of Computational Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ischemia, Atorvastatin, Forskolin, Memory, Water maze","lastPublishedDoi":"10.21203/rs.3.rs-4624330/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4624330/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e This study aimed to improve memory impairment after global cerebral ischemia. For this purpose, we used atorvastatin and forskolin after ischemia to improve neuronal injury and memory problems then compared the effect of these treatments to find the more effective strategy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Adult male rats (n=70) were divided into 7groups (n=10). \u003cstrong\u003eGroup 1: \u003c/strong\u003econtrol; \u003cstrong\u003egroup 2\u003c/strong\u003e: sham; \u003cstrong\u003egroup 3:\u003c/strong\u003e CI (global cerebral ischemia only);\u003cstrong\u003e group 4: \u003c/strong\u003e\u0026nbsp;CI+ forskolin;\u003cstrong\u003e group 5: \u003c/strong\u003e\u0026nbsp;CI+ atorvastatin; \u003cstrong\u003egroup 6:\u003c/strong\u003e CI+vehicle1; \u003cstrong\u003egroup 7:\u003c/strong\u003e CI+vehicle2. Ischemia was induced by occluding the bilateral common carotid artery. Atorvastatin and forskolin were injected intraperitoneally 4, 24, and 42 h after ischemia in group 5 and group6. Memory was evaluated with a water maze test. Data were analyzed by one-way ANOVA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The level of significance was 5% (p ≤ 0.05). Atorvastatin and forskolin treatment significantly reduced neuronal injury and improved memory in comparison with the ischemia group. Rats in the forskolin treated group had better performance in the Morris water maze test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Our findings showed that forskolin is more effective compared to atorvastatin after global cerebral ischemia.\u003c/p\u003e","manuscriptTitle":"Comparison the effect of atorvastatin and forskolin on memory after global cerebral ischemia in male rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-15 18:25:43","doi":"10.21203/rs.3.rs-4624330/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-06-27T18:29:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-23T23:43:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Computational Neuroscience","date":"2024-06-23T08:07:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-computational-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcns","sideBox":"Learn more about [Journal of Computational Neuroscience](http://link.springer.com/journal/10827)","snPcode":"10827","submissionUrl":"https://submission.nature.com/new-submission/10827/3","title":"Journal of Computational Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"44056c40-6845-4f2e-8d9d-902b5b7c8a05","owner":[],"postedDate":"July 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-07-15T18:25:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-15 18:25:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4624330","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4624330","identity":"rs-4624330","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}