Folic Acid Attenuated Learning and Memory Impairment via Inhibition of Oxidative Damage and Acetylcholinesterase Activity in Hypothyroid Rats

Folic Acid Attenuated Learning and Memory Impairment via Inhibition of Oxidative Damage and Acetylcholinesterase Activity in Hypothyroid Rats | 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 Folic Acid Attenuated Learning and Memory Impairment via Inhibition of Oxidative Damage and Acetylcholinesterase Activity in Hypothyroid Rats Sabiheh Amirahmadi, Mahmoud Hosseini, Somaieh Ahmadabady, Mahsa Akbarain, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-383794/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 2 You are reading this latest preprint version Abstract Hypothyroidism has been associated with cognitive decline. Considering the role that has been suggested for folic acid (FA) in cognitive performance, the present study was designed to investigate the effects of FA against hypothyroidism-induced cognitive impairment, oxidative damage and acetylcholinesterase (AChE) activity alterations in propylthiouracil (PTU)-induced hypothyroid rats. In this study, PTU (0.05% in drinking water) and FA (5, 10, and 15 mg/kg, oral gavage) were administered to the rats for a period of 7 weeks. Then, behavioral performance was tested using Morris water maze (MWM) and passive avoidance (PA) tasks. Finally, oxidative stress indicators and AChE activity were assayed in the brain tissues. The impairing effect of hypothyroidism on cognitive performance was markedly alleviated by FA especially at the higher doses. In the MWM test, FA reduced escape latency and travelled distance, compared to the non-treated hypothyroid group. In the PA test, the latency to enter the dark chamber was significantly enhanced by FA as compared to the non-treated hypothyroid group (p < 0.05-p < 0.001). Besides, FA attenuated AChE activity and malondialdehyde level but increased superoxidase dismutase enzyme activity and total thiol content (p < 0.05-p < 0.001). In conclusion, FA could improve learning and memory ability in hypothyroid rats. The observed protective effects may be mediated through regulation of oxidative stress and AChE activity. Molecular Biology Neurology Cellular & Molecular Neuroscience Cholinergic dysfunction Folic acid Hypothyroidism Learning and memory deficit Oxidative injury Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Thyroid hormones (THs) including thyroxine (T4) and triiodothyronine (T3) are of great importance for neuronal developmental stages and play a key role in neurons maturation, migration, differentiation, and signaling. THs-related disorders during development and adulthood can lead to serious clinical problems including cognitive dysfunction (Bernal 2007 ). Since concentrations of THs decrease with increasing age, hypothyroidism is common among elderlies and can lead to symptoms of severe cognitive impairment, including deterioration of learning and memory, perception, reasoning, problem solving, decision making and language by affecting energy consumption processes such as glucose metabolism, which is necessary for neurotransmission (Bégin et al. 2008 ; Annerbo and Lökk 2013 ). THs receptors are abundantly found in the hippocampus. Therefore, THs deficiency can lead to structural and physiological changes as well as a reduction in hippocampal cells growth and their numbers (Cooke et al. 2014 ). In addition, THs deficiency may lead to an imbalance between oxidants and antioxidants, which can result in an increased level of reactive oxygen species (ROS) (Masullo et al. 2018 ). Besides increased production of free radicals, hypothyroidism may lead to a decrease in the capacity of antioxidant defense system (Chakrabarti et al. 2016 ). Studies show that there is a strong association between THs activity and the function of neurotransmitter systems especially the cholinergic system (Fu et al. 2014 ; Wang et al. 2015 ). Clinical and experimental reports suggest that THs replacement therapy can partially reverse negative effects of hypothyroidism on learning and memory, but a full recovery of T3 content in target tissues, especially in the brain, is far from reach and therefore, more appropriate treatments are required (Chaalal et al. 2019 ). It has been reported that antioxidant agents are able to decrease the side effects of hypothyroidism on learning and memory (Asiaei et al. 2017 ; Beheshti et al. 2017 ; Baghcheghi et al. 2018a ; Khordad et al. 2018 ; Baghcheghi et al. 2020 ). In addition, some of the vitamins have been shown to have beneficial effects on hypothyroidism-related cognitive dysfunctions (Beheshti et al. 2017 ; Baghcheghi et al. 2018a ; Baghcheghi et al. 2020 ). Folic acid (FA) is a member of vitamin B family that plays an important role in cognitive activity by increasing the level of vitamins B12 and B6. Deficiency in folate and vitamin B12 may increase the risk of dementia and memory impairment (Ma et al. 2016 ). Clinical evidence showed that approximately two-thirds of patients with anemia or folate and vitamin B12 deficiency have cognitive impairment (Reynolds 2002 ). Homocysteine is a sulfur amino acid and its blood level controlled by FA, and vitamins B12 and B6 (Modaghegh et al. 2016). However, a direct relationship exists between increased homocysteine levels and cognitive impairment (Garcia and Zanibbi 2004). FA deficiency increases homocysteine levels, resulting in DNA damage and apoptosis in the hippocampal neurons. Therefore, FA administration can improve cognitive function by controlling homocysteine levels and acting as an effective antioxidant (Singh et al. 2011 ). Moreover, studies show that FA improves both short-term and long-term memory (Shooshtari et al. 2012 ). Administration of FA in hypothyroid rats improved oxidative stress and hypothalamic monoamines (Ibrahim et al. 2012 ). In addition, folate is metabolically bound to choline and is involved in the synthesis and release of acetylcholine (Crivello et al. 2010 ). Folate deprivation genetically or in the diet, reduces acetylcholine levels and can affect cognitive activity (Chan et al. 2008 ). Given that hypothyroidism is one of the most common diseases that leads to cognitive impairment and since folate deficiency is one of the most important dietary health problems worldwide, this study evaluated the effect of FA on learning and memory, oxidative stress indicators and acetylcholine esterase (AChE) activity in hypothyroid rats. Materials And Methods Chemicals 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), acetylthiocholine iodide and propylthiouracil (PTU) were purchased from Sigma (St. Louis, USA). 5,5´-Dithiobis-2-nitrobenzoic acid (DTNB), 2-thiobarbituric acid (TBA), hydrochloric acid (HCl), trichloroacetic acid (TCA), ethylenediamine tetra acetic acid disodium salt (Na2EDTA), tris (hydroxymethyl) aminomethane (Trizma base), phosphate-buffered saline (PBS), and dimethyl sulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany). Animals and experimental design In the present study, 50 male juvenile (21-22 days old) Wistar rats weighing 50-55 g were used. Animals were kept and treated under standard conditions (with 12h light: dark cycle at 24 ± 2 °C) and they had free access to food and water. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by Ethics Committee on Animal Research of Mashhad University of Medical Sciences (Approval No. IR.MUMS.MEDICAL.REC.1399.639). The animals were randomly divided into 5 groups including: 1) Control group in which the rats received normal drinking water and vehicle instead of FA, 2) Hypothyroid group in which PTU was added to the drinking water and the rats received vehicle instead of FA. Groups 3, 4 and 5 included Hypothyroid - FA 5, Hypothyroid - FA 10 and Hypothyroid - FA 15 groups which received PTU in their drinking water and were respectively treated with 5, 10 or 15 mg/kg FA, by gavage (Singh et al. 2011; Shooshtari et al. 2012). PTU was daily added to the drinking water and treatment by different doses of FA was carried out orally once a day for 7 weeks. Then, behavioral tests were then done and finally, the blood samples were collected and the animals' brains were removed under deep anesthesia induced using ketamine and xylazine. The serum samples separated from the blood, and hippocampus and cortex samples separated from the brains were kept at -80 ° C until biochemical tests. Morris water maze test (MWM) A water maze test was used to assess spatial learning and memory. The test uses a circular black pool (136 cm in diameter, 60 cm high and 30 cm deep) filled with water (22–24°C) that has an escape platform (10 cm in diameter and 28 cm high). The pool was divided into the following four zones/quadrants: north, south, east and west. The platform was located in the center of the southwest quadrant. To help the animals’ navigation, visual cues were placed around the apparatus. The experiments were performed on 5 consecutive days, each day four trials. In each trial, the animal was randomly located into the water tank and allowed to find the platform. After finding the platform, it was allowed to stay on the platform for 15 seconds. If the animal was not able to find the platform during 60 seconds, it was located on the platform by the experimenter and allowed to stay on it for 15 seconds. The animals rested outside the apparatus for 20 seconds between each trail. The time latency to find the platform and the length of the swimming path were recorded by a video tracking system. On the sixth day, the probe test was performed in such a way that the platform was removed and the rat was allowed to swim for 60 seconds. The time spent and distance traveled in the target quadrant were recorded (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b). Passive avoidance test The passive avoidance (PA) test helps to study non-spatial memory. In this test, an apparatus containing two chambers, one dark and one light, which are separated by a small guillotine door, was used. The floor of the dark chamber is covered with steel bars at a distance of 1 cm. An electric shock is applied to these bars by a stimulator. The experiment was performed in three phases: 1) habituation phase where the animals were placed in the apparatus for two consecutive days (each day for 5 minutes) and allowed to move freely between the two chambers; 2) training phase where the animals were placed in the light chamber and 20 seconds later, the guillotine door was opened. As soon as the animal entered the dark room, the door was closed and an electric shock (2 mA for two seconds) was applied to the animal's feet; and 3) retention phase, done 3, 24, 48 and 72 hours after the training phase, where the animals were placed in the light chamber, the guillotine door was opened, and the time latency in entering the dark compartment, the time spent in the light and dark compartments and the frequency of entering into the dark compartment were recorded (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b). Biochemical tests The blood samples were centrifuged at 500 g for 10 min and the obtained serums were used for measuring the levels of thyroxin. Serum thyroxin level was measured using a radioimmunoassay method in Navid Medical laboratory, Mashhad, Iran. The homogenates of cerebral cortex and hippocampus (10 % w/v) were prepared in ice-cold PBS (0.1 M, pH 7.4). The homogenates were then centrifuged at 4 °C, at 10000×g to separate the supernatants for estimation of malondialdehyde (MDA) and total thiol concentration as well as superoxide dismutase (SOD) and AChE activities. Measurement of MDA and total thiol concentration As a marker of lipid peroxidation, MDA was measured in the hippocampus and cortex. Briefly, one milliliter of each sample was added to 2 ml of TBA/TCA/HCl reagent and the reaction mixtures were incubated in a boiling bath for 45 min. After cooling, the whole solutions were centrifuged at 1000 g for 10 min. Finally, the supernatants were collected and the absorbance of the pink chromogen was measured at 535 nm using a spectrophotometer (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b). The MDA concentration was calculated by the following equation: C (M) = Absorbance/ (1.56 × 10 5 ) To measure thiol content, DTNB was used. In this assay, 50 μl of the homogenates was added to 1 ml of tris-EDTA buffer (pH 8.6) and the first absorbance (A1) was recorded at 412 nm using a spectrophotometer. Afterwards, 20 μl DTNB solution (10 mM in methanol) was added to each sample and the second absorbance (A2) was recorded at the same wavelength (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b). Total thiol concentration (mM) was calculated by the following equation: C (mM) = (A2-A1-B) × 1.07/0.05 × 13.6 Estimation of SOD Activity SOD activity in the cerebral cortex and hippocampus was assessed based on the ability of the enzyme to inhibit autoxidation of pyrogallol (Madesh and Balasubramanian 1998). Based on the method of Madesh et al., each sample (10 μl supernatant from homogenate) was mixed with MTT and pyrogallol solution and then incubated at room temperature. After 5 min, DMSO was added to solubilize the resultant color. The optical absorbance was measured at 570 nm and the activity of SOD was expressed as unit per gram of tissue (Madesh and Balasubramanian 1998). Estimation of AChE Activity The AChE activity in the supernatants was determined by the method of Ellman using acetylthiocholine iodide as a substrate. Briefly, each sample (50 μl) was added into a solution (containing PBS (pH 8), 0.1 ml DTNB (10 mM), and 0.02 ml acetylthiocholine (75 mM). The changes in absorbance of the samples were spectrophotometrically recorded at 412 nm within 10 min and AChE activity was estimated as μmol/g tissue/min (Ellman et al. 1961). Statistical Analysis Statistical analysis was performed using the SPSS 11.5 software and normality of the data was checked by the Kolmogorov–Smirnov test. Data about learning phase in MWM test was analyzed by repeated measures analysis of variance (ANOVA) followed by Tukey’s post hoc test. Other data were analyzed by one-way ANOVA followed by Tukey’s post hoc test. All data are expressed as means ± SEM and a p<0.05 was considered statistically significant. Results Morris water maze The time latency and the distance traveled to reach the platform during the 5-day training in the MWM test in hypothyroid group were significantly increased compared to the control group (P<0.001 for the latency and P<0.05-P<0.001 for the distance). The time latency was significantly reduced in all FA-treated groups compared to the hypothyroid group (P<0.05 to P<0.001) (Fig. 1a). Moreover, the distance traveled to reach the platform in hypothyroid rats with different doses of FA was lower than the hypothyroid group (P<0.05 to P<0.01) (Fig. 1b). The animals in hypothyroid group showed significant decreases in the time spent and distance traveled in target quadrant on the probe day compared to the control group (P<0.001 for both). The hypothyroid animals treated with FA (5, 10 or 15 mg) better remembered the location of the platform and spent longer time and traveled longer distance in the target area of MWM than the hypothyroid group (P<0.001 for all). The animals of the hypothyroid-FA 15 group spent longer time and traveled longer distance in the target area of MWM than the hypothyroid-FA 10 group (P<0.05 and P<0.01, respectively) and traveled a longer distance in the target area than the hypothyroid-FA 5 group (P<0.05). The traveling time in the target area of MWM showed no significant difference between FA-treated hypothyroid rats and the control group. The distance traveled in the target area in the hypothyroid-FA 5 and hypothyroid-FA 10 groups was shorter than that of the control group (P<0.01 for both) but there was no significant difference between hypothyroid-FA 15 and the control group in this regard (Fig. 2). Passive avoidance Based on the results presented in Fig. 3 and 4, in the hypothyroid group, the latency to enter and the time spent in the light chamber were decreased while the time spent and frequency of entry to the dark chamber were significantly increased compared with the control group, 3, 24, 48 and 72 h after receiving the electric shock (P<0.01 to P<0.001). The latency to enter the dark chamber in all FA-treated hypothyroid groups was higher than that of the control group, 3 h after the delivery of the electric shock (P<0.01, P<0.001 and P<0.001 for FA 5, 10 and 15, respectively). The latency to enter the dark chamber in the hypothyroid-FA 10 and hypothyroid-FA 15 groups was higher than the hypothyroid group 24, 48 and 72 h after the shock (P<0.05 to P0.05). The latency to enter the dark chamber in the hypothyroid-FA 15 group was significantly higher compared to the hypothyroid-FA 5 group, 3, 24, 48 and 72 h post-shock (P<0.01, P<0.001, P<0.001 and P<0.05, respectively). As shown in Fig. 3a, the latency in the hypothyroid-FA 15 group was higher compared to the hypothyroid-FA 10 group, 24 h after the shock (P<0.05). All FA-treated hypothyroid rats spent shorter times in the dark chamber than the hypothyroid group, 3 h post shock time (P<0.001 for the three groups) but there was no significant difference in this parameter among the three FA-treated groups. The rats of the hypothyroid-FA 10 and hypothyroid-FA 15 groups also spent shorter times in the dark chamber than the hypothyroid group, 24, 48 and 72 h post-shock (P<0.01 to P<0.001) but there was no significant difference between the hypothyroid-FA 5 and the hypothyroid group at these time-points (Fig. 3b). The rats of hypothyroid-FA 15 group spent shorter time in the dark chamber than the hypothyroid-FA 5 group, 24, 48 and 72 h post-shock (P<0.05 to P<0.001). In addition, the rats of the hypothyroid-FA 10 group spent shorter time in the dark chamber than the hypothyroid-FA 5 group, 24 and 72 h after the shock (P<0.05 for both times). The results also showed that the FA-treated hypothyroid animals spent longer times in the light chamber than the hypothyroid group 3 h post-shock (P<0.001 for all groups) but there was no significant difference among FA-treated hypothyroid groups in this regard. Moreover, the rats of hypothyroid-FA 10 and hypothyroid-FA 15 groups spent longer times in the light chamber than the hypothyroid group, 24, 48 and 72 h post-shock (P<0.01 to P<0.001) but there was no significant difference between hypothyroid-FA 5 and hypothyroid groups (Fig. 4a). The time spent in the dark chamber in the hypothyroid-FA 10 and hypothyroid-FA 15 groups was longer than the hypothyroid-FA 5 group, 24, 48 and 72 h post-shock (P<0.05 to P<0.001). The results of the number of dark chamber entries (Fig. 4b) showed that the frequency of entries in the hypothyroid-FA 5 group was higher than the control group (P<0.01 to P<0.001) but there was no significant difference between the hypothyroid-FA 5 and control groups. The rats of the hypothyroid-FA 10 and hypothyroid-FA 15 groups had lower number of entries into the dark chamber than the hypothyroid group at all time-points after the shock (P<0.05 to P<0.001). The number of entries in the hypothyroid-FA 10 and hypothyroid-FA 15 groups was lower than the hypothyroid-FA group (P<0.05 to P<0.001). MDA and thiol concentrations in cortical and hippocampal tissues Hypothyroidism induced by PTU increased MDA but decreased total thiol levels in both cortex and hippocampus (P<0.001 for all) (Fig. 5 and 6). FA at all doses reduced the MDA concentration while increased thiol content in the hippocampus (P<0.05 to P<0.001). Also, FA 10 and 15 mg/kg decreased MDA while increased thiol content in the cortex of hypothyroid rats compared to the hypothyroid group (P<0.05 to P<0.001) but there was no significant difference between hypothyroid-FA 5 and hypothyroid groups in the cortical levels of MDA and thiol. There was no significant difference among the three FA-treated groups in the hippocampal levels of MDA and thiol but in the cortex of the hypothyroid-FA 15 group, MDA concentration was lower (P<0.01) while thiol content was higher (P<0.05) than the hypothyroid-FA 5 group (Fig. 5 and 6). SOD activity in hippocampal and cortical tissues Figure 7 shows that hippocampal and cortical SOD activity significantly decreased following hypothyroidism induced by PTU administration (P<0.001 for both tissues). SOD activity in hypothyroid-FA 10 and hypothyroid-FA 15 groups significantly increased compared to the hypothyroid group (P<0.01 and P<0.001) and it was higher in the hypothyroid-FA 15 group than the hypothyroid-FA 5 group (P<0.001 in the hippocampus and P<0.01 in the cortex). There was no significant difference between the hypothyroid-FA 5 and the hypothyroid group in both hippocampus and cortex SOD activity (Figure 7). Both hippocampal and cortical SOD activity in all FA-treated hypothyroid groups were still lower than that of the control group (P<0.05 to P<0.001). AChE activity in hippocampal and cortical tissues Fig. 8 shows that hippocampal and cortical AChE activity significantly increased following hypothyroidism induced by PTU (P<0.001 for both cortex and hippocampus tissues). Administration of 10 and 15 mg/kg of FA reduced AChE activity in the hippocampus and cortex compared with the hypothyroid group (P<0.05 to P<0.001) but there was no significant difference between the hypothyroid-FA 5 and the hypothyroid group. AChE activity in the cortex of both hypothyroid-FA 10 and hypothyroid-FA 15 groups was lower than that of the hypothyroid-FA 5 group (P<0.01 for both) but there was no significant difference in the hippocampal AChE activity among FA-treated hypothyroid groups. The results also showed that AChE activity in the hippocampus of all FA-treated hypothyroid groups and in the cortex of hypothyroid-FA 5 group was higher than that of the control group (P<0.05 to P<0.001). Thyroxin level in the serum The results showed that serum thyroxin level in the hypothyroid group was significantly lower than the control group (P<0.001). The results also showed that FA was not able to reverse serum thyroxin level as there was no significant difference between FA-treated hypothyroid groups and the hypothyroid group. Serum thyroxin level in all FA-treated hypothyroid groups was lower than the control group (P<0.001 for all cases). Discussion In the present study, PTU administration induced a hypothyroidism state as reflected by a low serum level of thyroxin in the hypothyroid group compared to the control group. PTU as a well-known drug used for hyperthyroidism treatment, has been frequently used to produce hypothyroidism in rodents (Asiaei et al. 2017 ; Beheshti et al. 2017 ; Baghcheghi et al. 2018a ; Baghcheghi et al. 2018b ; Baghcheghi et al. 2020 ). Hypothyroidism, especially during developmental and growth periods has been shown to negatively affect the central nervous system (Asiaei et al. 2017 ; Beheshti et al. 2017 ; Baghcheghi et al. 2018a , Baghcheghi et al. 2018b ; Baghcheghi et al. 2020 ). Studies in humans and animals have shown that hypothyroidism during developmental period impairs cognitive functions such as attention, learning and memory (Hosseini et al. 2010 ; Beheshti et al. 2017 ; Baghcheghi et al. 2018a ; Baghcheghi et al. 2018b ). The results of the current study showed that PTU-induced hypothyroidism was accompanied with learning and memory impairment as confirmed by both MWM and PA tests. Also, we found that the rats of the hypothyroid group spent longer time to reach the platform during the five-day learning period in the MWM test. The rats of the hypothyroid group also traveled longer distance to reach the hidden platform than the control group. Interestingly, the rats of the hypothyroid group could not remember the location of the platform and spent less time and traveled shorter distance in the target area in the probe trial in the MWM. The results of PA test also showed that the rats of the hypothyroid group had a shorter delay but a higher frequency in entering the dark compartment, and spent longer time there, than the control group. These results are consistent with the previous studies which showed that hypothyroidism during lactation, infancy or developmental periods causes cognitive impairments such as learning and memory dysfunction (Hosseini et al. 2010 ; Beydoun et al. 2013 ; Farrokhi et al. 2014 ). Thyroid hormones are known to be important for non-hippocampal and hippocampal-related learning and memory, synaptic flexibility, and neurogenesis (Cooke et al. 2014 ; Asiaei et al. 2017 ; Baghcheghi et al. 2020 ). The exact mechanism(s) responsible for adverse effects of hypothyroidism on learning and memory have not been well elucidated. It is suggested that hypothyroidism causes cerebral atrophy and cerebral neuro-inflammation as it is also accompanied with amyloid beta (Aβ) production, tau hyperphosphorylation, and impairment of signaling pathways responsible for hippocampal-dependent spatial memory (Beydoun et al. 2013 ; Chaalal et al. 2019 ). An imbalance between the production of peroxidants and antioxidants and production of high levels of ROS and RNS (reactive nitrogen species) are also suggested to have a role in negative impacts of hyperthyroidism on learning and memory (Venditti and Di 2006). The results of the current study also showed that hypothyroidism-associated learning and memory was accompanied with a decrease in thiol content and SOD activity but an increase in MDA level in both hippocampus and cortex tissues. These findings confirmed the occurrence of an oxidative stress state in the brain of hypothyroid rats which may have a role in learning and memory impairing effects of hypothyroidism seen in the present study. The results of previous researches have shown that MDA levels are reduced in patients with controlled hypothyroidism due to antioxidant mechanisms mediated by thyroid hormones (Villanueva et al. 2013 ). It has also been previously reported that hypothyroidism is accompanied with a decrease in thiol content and SOD and CAT activities in the brain. Interestingly, AChE activity in the hippocampus and cortex of hypothyroid rats was significantly decreased compared to the control ones. Acetylcholine is one of the major neurotransmitters involved in cognitive function. It has been previously reported that hypothyroidism is associated with cholinergic system dysfunction (Smith et al. 2002 ). It was also shown that thyroxin increased choline acetyltransferase activity and acetylcholine level in the brain and consequently, improved learning and memory (Fu et al. 2014 ). Considering the results of the present study and the mentioned evidence, negative effects of hypothyroidism may at least in part be due to its effects on the cholinergic system. Supplementation with THs is widely done to treat hypothyroidism. Levothyroxine has been reported to reduce hippocampal cognitive impairment in hypothyroid mice (Smith et al. 2002 ; Fu et al. 2014 ). Recently, vitamins C and E and anti-oxidant natural products were suggested to reduce the adverse effects of hypothyroidism on brain functions including learning and memory (Beheshti et al. 2017 ; Baghcheghi et al. 2018a ; Baghcheghi et al. 2018b ; Baghcheghi et al. 2020 ). In the current research, treatment by three doses of FA (i.e. 5, 10 and 15 mg/kg) improved learning and memory of the hypothyroid rats. The results showed that the hypothyroid rats treated with different doses of FA spent less time and traveled shorter distances to reach the platform during the 5-day learning period in the MWM. They also better remembered the location of the platform and spent more time and traveled longer distances in the target area of the MWM in the probe trial. The results of PA test also showed that treatment by different doses of FA prolonged the latency to enter the dark compartment and increased the total time spent in the light compartment while decreased the time in the dark chamber and reduced the number of entries to the dark compartment. To the best of our knowledge, the effects of FA on hypothyroidism-associated learning and memory impairment were not previously reported. Previous studies have shown that administration of vitamin B improves cognitive function in people with low FA levels by decreasing homocysteine (Shooshtari et al. 2012 ). FA deficiency has been reported to be followed by increases in homocysteine and cognition and learning and memory impairments (Dam et al. 2017 ). Our results also showed that FA attenuated MDA while increased thiol and SOD in both hippocampus and cortex tissues. It has been previously reported that FA improves the level of glutathione (GSH), but causes a significant reduction in brain MDA levels, indicating suppression of lipid peroxidation (Singh et al. 2011 ). FA administration in patients with Alzheimer's disease could improve cognition and reduce inflammatory factors (Chen et al. 2016 ; Calderón et al. 2020). FA deficiency increases the risk of other neurological disorders including stroke, through induction of oxidative DNA damage associated with morphological damage and increased cell autophagy function (Zhao et al. 2016 ). Short-term administration of FA for 7 weeks in patients with metformin-treated type 2 diabetes was able to significantly decrease serum MDA levels (Aghamohammadi et al. 2011 ). Moreover, FA with or without vitamin B12 given for 30 days could prevent mitochondrial dysfunction and DNA damage caused by short-term treatment with arsenic trioxide in rats (Majumdar et al. 2009 ). FA treatment in stressed rats was able to reduce depressive-like behaviors and brain oxidative damage, and ameliorate hippocampal antioxidant imbalance (Budni et al. 2013; Réus et al. 2018 ; Menegas et al. 2020 ). To better understand the responsible mechanism(s), AChE activity was also evaluated in the brain. The results showed that FA decreases AChE activity in the hippocampus and cortex. Considering these results, it seems that learning and memory improving effects of FA seen in the present study are at least in part due to its attenuating effect on AChE activity. It has also been reported that FA deficiency is associated with cholinergic system dysfunction and increased AChE activity (Crivello et al. 2010 ). Nevertheless, FA was not able to restore the thyroxin concentration to normal levels. Thus, it seems that FA was not able to protect the thyroid grain from damaging effects of PTU. To better understand the effects of FA on serum thyroxin level, further investigations using other animal models including thyroidectomy model need to be done. Conclusion FA could improve learning and memory ability in hypothyroid rats. The observed protective effects may have been achieved by suppression of oxidative stress and regulation of AChE activity. Declarations Conflict of interest The authors declare no conflict of interest. Ethical approval All procedures performed in this studies including animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Ethical Committee of the Animal Research of Mashhad University of Medical Sciences (Ethical code: IR.MUMS.MEDICAL.REC.1399.639 ). Data availability statement The authors confirm that all data generated or analyzed during this study are included in this published article. Author contributions Mahmoud Hosseini conducted conception and design of the project, Sabiheh Amirahmadi, Somaieh Ahmadabady, Mahsa Akbarain, Kataneh Abrari, Arezoo Rajbian, and Farzaneh Vafaee performed the experiments. Mahmoud Hosseini, Arezoo Rajbian, and Farzaneh Vafaee prepared a draft of the manuscript. Mahmoud Hosseini, Arezoo Rajbian, Farzaneh Vafaee performed statistical analysis. Mahmoud Hosseini, Farzaneh Vafaee, and Arezoo Rajbian provided final revision of the manuscript. All authors contributed to manuscript preparation and approved the submitted version. Funding This study was funded by Vice Chancellor for Research and Technology, Mashhad University of Medical Sciences with the following grant number: 991073 Acknowledgments The authors appreciate the Vice Chancellor for Research and Technology, Mashhad University of Medical Sciences for financial support (NO: 991073). References Aghamohammadi V, Gargari BP, Aliasgharzadeh A (2011) Effect of folic acid supplementation on homocysteine, serum total antioxidant capacity, and malondialdehyde in patients with type 2 diabetes mellitus. J Am Coll Nutr 30(3):210–215. https://doi.org/10.1080/07315724.2011.10719962 Annerbo S, Lökk J (2013) A clinical review of the association of thyroid stimulating hormone and cognitive impairment. 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Toxicol Ind Health 28(3):253–261. https://doi.org/10.1177/0748233711410913 Khordad E, Alipour F, Beheshti F, Hosseini M, Rajabzadeh AA, Asiaei F, Seghatoleslam M (2018) Vitamin C prevents hypothyroidism associated neuronal damage in the hippocampus of neonatal and juvenile rats: A stereological study. J Chem Neuroanat 93:48–56. https://doi.org/ 10.1016/j.jchemneu.2017.11.011 Ma F, Wu T, Zhao J, Song A, Liu H, Xu W, Huang G (2016) Folic acid supplementation improves cognitive function by reducing the levels of peripheral inflammatory cytokines in elderly Chinese subjects with MCI. Sci Rep 6:37486. https://doi.org/10.1038/srep37486 Madesh M, Balasubramanian KA (1998) Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide. 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Metab Brain Dis 35(2):413–425. https://doi.org/10.1007/s11011-019-00503-3 . Modaghegh MH, Ravari H, Haghighi MZ, Rajabnejad A (2016) Effect of folic acid therapy on homocysteine level in patients with atherosclerosis or Buerger's disease and in healthy individuals: A clinical trial. Electron Physician 8(10):3138–3143. https://doi.org/10.19082/3138 Réus GZ, Maciel AL, Abelaira HM, de Moura AB, de Souza TG, Dos Santos TR, Darabas AC, Parzianello M, Matos D, Abatti M, Vieira AC, Fucillini V, Michels M, Dal-Pizzol F, Quevedo J (2018) ω-3 and folic acid act against depressive-like behavior and oxidative damage in the brain of rats subjected to early- or late-life stress. Nutrition 53:120–133. https://doi.org/10.1016/j.nut.2018.03.006 Reynolds EH (2002) Folic acid, ageing, depression, and dementia. BMJ 324(7352):1512–1515. https://doi.org/10.1136/bmj.324.7352.1512 Shooshtari MK, Moazedi AA, Parham GA (2012) Memory and motor coordination improvement by folic Acid supplementation in healthy adult male rats. Iran J Basic Med Sci 15(6):1173–1179 Singh R, Kanwar SS, Sood PK, Nehru B (2011) Beneficial effects of folic acid on enhancement of memory and antioxidant status in aged rat brain. Cell Mol Neurobiol 31(1):83–91. https://doi.org/10.1007/s10571-010-9557-1 Smith JW, Evans AT, Costall B, Smythe JW (2002) Thyroid hormones, brain function and cognition: a brief review. Neurosci Biobehav Rev 26(1):45–60. https://doi.org/10.1016/s0149-7634(01)00037-9 Venditti P, Di Meo S (2006) Thyroid hormone-induced oxidative stress. Cell Mol Life Sci 63(4):414–434. https://doi.org/10.1007/s00018-005-5457-9 Villanueva I, Alva-Sánchez C, Pacheco-Rosado J (2013) The role of thyroid hormones as inductors of oxidative stress and neurodegeneration. Oxid Med Cell Longev 2013:218145. https://doi.org/10.1155/2013/218145 Wang F, Zeng X, Zhu Y, Ning D, Liu J, Liu C, Jia X, Zhu D (2015) Effects of thyroxine and donepezil on hippocampal acetylcholine content, acetylcholinesterase activity, synaptotagmin-1 and SNAP-25 expression in hypothyroid adult rats. Mol Med Rep 11(2):775–782. https://doi.org/10.3892/mmr.2014.2825 Zhao Y, Huang G, Chen S, Gou Y, Dong Z, Zhang X (2016) Folic acid deficiency increases brain cell injury via autophagy enhancement after focal cerebral ischemia. J Nutr Biochem 38:41–49. https://doi.org/10.1016/j.jnutbio.2016.08.009 Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 31 Mar, 2021 First submitted to journal 31 Mar, 2021 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. 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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-383794","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":19582372,"identity":"a92401af-73d4-4773-bc71-d9d72d1455c8","order_by":0,"name":"Sabiheh Amirahmadi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences Faculty of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sabiheh","middleName":"","lastName":"Amirahmadi","suffix":""},{"id":19582373,"identity":"c54052a8-64a8-4806-9781-ce5e1229fc7b","order_by":1,"name":"Mahmoud Hosseini","email":"","orcid":"","institution":"Mashhad University of Medical Sciences Faculty of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mahmoud","middleName":"","lastName":"Hosseini","suffix":""},{"id":19582374,"identity":"70928e20-6a36-4b86-b58f-6fb29f03fac4","order_by":2,"name":"Somaieh Ahmadabady","email":"","orcid":"","institution":"Mashhad University of Medical Sciences Faculty of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Somaieh","middleName":"","lastName":"Ahmadabady","suffix":""},{"id":19582375,"identity":"a5fb71b1-9080-4b77-a4b0-7d61c50ddb90","order_by":3,"name":"Mahsa Akbarain","email":"","orcid":"","institution":"Mashhad University of Medical Sciences Faculty of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"","lastName":"Akbarain","suffix":""},{"id":19582376,"identity":"b33a301f-ee12-48b2-8c5a-1759d8dd1ebd","order_by":4,"name":"Kataneh Abrari","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kataneh","middleName":"","lastName":"Abrari","suffix":""},{"id":19582377,"identity":"10b43e7d-702f-488d-a165-a7f90d4604f0","order_by":5,"name":"Farzaneh Vafaee","email":"","orcid":"","institution":"Mashhad University of Medical Sciences Faculty of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Farzaneh","middleName":"","lastName":"Vafaee","suffix":""},{"id":19582378,"identity":"21e702de-2bff-4ca7-ad38-12edc265429c","order_by":6,"name":"Arezoo Rajabian","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYHCCBGYGAxDNfAAudACHUnQtbAlEa2FghlA8BsS5yrz9wMPPBQV2efyzz3zdzJtTx8DffoDxcAUeLTJnEpKlZxgkF0ucy912m3fbYQaJMwkMB8/g0SLBkJAgzWPAnNhwhhekBeiLGwwMBxvwaeF/kPybx6A+cf4ZnmdALXUM8gS1SCSkAW05nLjhDA8bUAsw9AhreZBmPcPgeOLGM2xmN+duO8xjeCaxgYDDcpJvF/ypTpx3hvnZjbfb6uTkjh8+/BGfFmB0JKByGRgY8WtgYGA/QEDBKBgFo2AUjHgAAPvFTvoAGG7jAAAAAElFTkSuQmCC","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Arezoo","middleName":"","lastName":"Rajabian","suffix":""}],"badges":[],"createdAt":"2021-04-01 14:40:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-383794/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-383794/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":8562934,"identity":"745a416f-8f93-4d2e-a057-6ab02619f713","added_by":"auto","created_at":"2021-04-28 18:45:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30786,"visible":true,"origin":"","legend":"The results of traveling time and distance during 5 days learning in Morris water maze. The data were expressed as mean ± SEM (n = 10). *P\u003c0.05, ***P\u003c0.001 compared to the control group. +P\u003c0.05, ++P\u003c0.01, +++P\u003c0.001 comparison between Hypothyroid FA 5 and Hypothyroid groups, $P\u003c0.05, $$P\u003c0.01, $$$P\u003c0.001 comparison between Hypothyroid FA 10 and Hypothyroid groups, \u0026P\u003c0.05, \u0026\u0026P\u003c0.01, \u0026\u0026\u0026P\u003c0.001 comparison between Hypothyroid FA 15 and Hypothyroid groups FA: Folic acid.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/f3076b6861bc8cc25c904376.png"},{"id":8563104,"identity":"2105b176-457e-4a31-be1c-186365d29c1c","added_by":"auto","created_at":"2021-04-28 18:48:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37363,"visible":true,"origin":"","legend":"The results of traveling time and distance in the target quadrant during probe test in Morris water maze. The data were expressed as mean ± SEM (n = 10). **P\u003c0.01 and ***P\u003c0.001 compared to the control group. +++P\u003c0.001 compared to Hypothyroid group, $P\u003c0.05 compared to Hypothyroid FA 5 group, \u0026P\u003c0.05 and \u0026\u0026P\u003c0.01compared to Hypothyroid FA 10 group. FA: Folic acid","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/cb25e419bf638695e199a1f0.png"},{"id":8562927,"identity":"72ab266a-a002-4e54-9ec6-3bcbcfcb7516","added_by":"auto","created_at":"2021-04-28 18:45:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52030,"visible":true,"origin":"","legend":"The results of delay time for entering the dark and the total time spent in the dark in passive avoidance test. The data were expressed as mean ± SEM (n = 10). *P\u003c0.05, **P\u003c0.01 and ***P\u003c0.001 compared to the control group. +P\u003c0.05, ++P\u003c0.01 and +++P\u003c0.001 compared to Hypothyroid group, $P\u003c0.05, $$P\u003c0.01 and $$$P\u003c0.001 compared to Hypothyroid FA 5 group, \u0026P\u003c0.05 compared to Hypothyroid FA 10 group. FA: Folic acid","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/ea90c82b8819bfc88c202b94.png"},{"id":8563106,"identity":"cff904a1-4a57-4151-8853-c51b1aabf529","added_by":"auto","created_at":"2021-04-28 18:48:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51264,"visible":true,"origin":"","legend":"The results of total time spent in the light and the number of entering into the dark in passive avoidance test. The data were expressed as mean ± SEM (n = 10). **P\u003c0.01 and ***P\u003c0.001 compared to the control group. +P\u003c0.05, ++P\u003c0.01 and +++P\u003c0.001 compared to Hypothyroid group, $P\u003c0.05, $$P\u003c0.01 and $$$P\u003c0.001 compared to Hypothyroid FA 5 group. FA: Folic acid","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/ce4d06a786fbef4d1f5fa868.png"},{"id":8563206,"identity":"ef64b98c-1614-4f9e-a13f-811fb58b68f6","added_by":"auto","created_at":"2021-04-28 18:51:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":35680,"visible":true,"origin":"","legend":"The results of MDA in the hippocampus (a) and cortex (b). The data were expressed as mean ± SEM (n = 10).*P\u003c0.05, **P\u003c0.01 and ***P\u003c0.001 compared to the control group. ++P\u003c0.01 and +++P\u003c0.001 compared to Hypothyroid group, $$P\u003c0.01 compared to Hypothyroid FA 5 group. FA: Folic acid","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/241cbf2b97409b794439cac5.png"},{"id":8563105,"identity":"5c07e7db-e695-4ebf-b50f-6360620199c6","added_by":"auto","created_at":"2021-04-28 18:48:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":34168,"visible":true,"origin":"","legend":"The results of thiol in the hippocampus (a) and cortex (b). The data were expressed as mean ± SEM (n = 10). ***P\u003c0.001 compared to the control group. +P\u003c0.05 and ++P\u003c0.01 compared to Hypothyroid group, $P\u003c0.05 compared to Hypothyroid FA 5 group. FA: Folic acid","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/62820bd5d6d7541752e3f82a.png"},{"id":8563108,"identity":"7a97867b-04b9-45b3-b4ee-55c008b9ad05","added_by":"auto","created_at":"2021-04-28 18:48:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":32754,"visible":true,"origin":"","legend":"The results of SOD in the hippocampus (a) and cortex (b). The data were expressed as mean ± SEM (n = 10). *P\u003c0.05, **P\u003c0.01 and ***P\u003c0.001 compared to the control group. ++P\u003c0.01 and +++P\u003c0.001 compared to Hypothyroid group, $$P\u003c0.01 and $$$P\u003c0.001 compared to Hypothyroid FA 5 group. FA: Folic acid","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/fb18e4e20caae65f09dfd972.png"},{"id":8563207,"identity":"ef8e3b3c-b471-4e02-bd69-778cdd2f18f1","added_by":"auto","created_at":"2021-04-28 18:51:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":39595,"visible":true,"origin":"","legend":"The results of AChE activity in the hippocampus (a) and cortex (b). *P\u003c0.05, and ***P\u003c0.001 compared to the control group. The data were expressed as mean ± SEM (n = 10). +P\u003c0.05, ++P\u003c0.01 and +++P\u003c0.001 compared to Hypothyroid group, $$P\u003c0.01 compared to Hypothyroid FA 5 group. FA: Folic acid","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/4494bca96f8a3515313aaad5.png"},{"id":8563109,"identity":"24a7f6ea-7296-48f3-abef-dae2d46dffff","added_by":"auto","created_at":"2021-04-28 18:48:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":14531,"visible":true,"origin":"","legend":"The results of thyroxin level in the serum. ***P\u003c0.001 compared to the control group. The data were expressed as mean ± SEM (n = 10). FA: Folic acid","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/e4c49dc64cd8e1be23ddc17b.png"},{"id":13689290,"identity":"a5f47023-a770-446d-9fe9-8ca0f716c3f7","added_by":"auto","created_at":"2021-09-17 12:28:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":642080,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-383794/v1/2aa256bb-33a8-48d2-a3db-a321c5e5ac93.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eFolic Acid Attenuated Learning and Memory Impairment via Inhibition of Oxidative Damage and Acetylcholinesterase Activity in Hypothyroid Rats\u003c/p\u003e","fulltext":[{"header":"Introduction","content":" \u003cp\u003eThyroid hormones (THs) including thyroxine (T4) and triiodothyronine (T3) are of great importance for neuronal developmental stages and play a key role in neurons maturation, migration, differentiation, and signaling. THs-related disorders during development and adulthood can lead to serious clinical problems including cognitive dysfunction (Bernal \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Since concentrations of THs decrease with increasing age, hypothyroidism is common among elderlies and can lead to symptoms of severe cognitive impairment, including deterioration of learning and memory, perception, reasoning, problem solving, decision making and language by affecting energy consumption processes such as glucose metabolism, which is necessary for neurotransmission (B\u0026eacute;gin et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Annerbo and L\u0026ouml;kk \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). THs receptors are abundantly found in the hippocampus. Therefore, THs deficiency can lead to structural and physiological changes as well as a reduction in hippocampal cells growth and their numbers (Cooke et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, THs deficiency may lead to an imbalance between oxidants and antioxidants, which can result in an increased level of reactive oxygen species (ROS) (Masullo et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Besides increased production of free radicals, hypothyroidism may lead to a decrease in the capacity of antioxidant defense system (Chakrabarti et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Studies show that there is a strong association between THs activity and the function of neurotransmitter systems especially the cholinergic system (Fu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Clinical and experimental reports suggest that THs replacement therapy can partially reverse negative effects of hypothyroidism on learning and memory, but a full recovery of T3 content in target tissues, especially in the brain, is far from reach and therefore, more appropriate treatments are required (Chaalal et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been reported that antioxidant agents are able to decrease the side effects of hypothyroidism on learning and memory (Asiaei et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Beheshti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Khordad et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In addition, some of the vitamins have been shown to have beneficial effects on hypothyroidism-related cognitive dysfunctions (Beheshti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFolic acid (FA) is a member of vitamin B family that plays an important role in cognitive activity by increasing the level of vitamins B12 and B6. Deficiency in folate and vitamin B12 may increase the risk of dementia and memory impairment (Ma et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Clinical evidence showed that approximately two-thirds of patients with anemia or folate and vitamin B12 deficiency have cognitive impairment (Reynolds \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Homocysteine is a sulfur amino acid and its blood level controlled by FA, and vitamins B12 and B6 (Modaghegh et al. 2016). However, a direct relationship exists between increased homocysteine levels and cognitive impairment (Garcia and Zanibbi 2004). FA deficiency increases homocysteine levels, resulting in DNA damage and apoptosis in the hippocampal neurons. Therefore, FA administration can improve cognitive function by controlling homocysteine levels and acting as an effective antioxidant (Singh et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, studies show that FA improves both short-term and long-term memory (Shooshtari et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Administration of FA in hypothyroid rats improved oxidative stress and hypothalamic monoamines (Ibrahim et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, folate is metabolically bound to choline and is involved in the synthesis and release of acetylcholine (Crivello et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Folate deprivation genetically or in the diet, reduces acetylcholine levels and can affect cognitive activity (Chan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven that hypothyroidism is one of the most common diseases that leads to cognitive impairment and since folate deficiency is one of the most important dietary health problems worldwide, this study evaluated the effect of FA on learning and memory, oxidative stress indicators and acetylcholine esterase (AChE) activity in hypothyroid rats.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eChemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), acetylthiocholine iodide and propylthiouracil (PTU) were purchased from Sigma (St. Louis, USA).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;5,5\u0026acute;-Dithiobis-2-nitrobenzoic acid (DTNB), 2-thiobarbituric acid (TBA), hydrochloric acid (HCl), trichloroacetic acid (TCA), ethylenediamine tetra acetic acid disodium salt (Na2EDTA), tris (hydroxymethyl) aminomethane (Trizma base), phosphate-buffered saline (PBS), and dimethyl sulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003cstrong\u003eand experimental design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the present study, 50 male juvenile (21-22 days old) Wistar rats weighing 50-55 g were used. Animals were kept and treated under standard conditions (with 12h light: dark cycle at 24 \u0026plusmn; 2 \u0026deg;C) and they had free access to food and water. All experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by Ethics Committee on Animal Research of Mashhad University of Medical Sciences (Approval No. IR.MUMS.MEDICAL.REC.1399.639).\u003c/p\u003e\n\u003cp\u003eThe animals were randomly divided into 5 groups including: 1) Control group in which the rats received normal drinking water and vehicle instead of FA, 2) Hypothyroid group in which PTU was added to the drinking water and the rats received vehicle instead of FA. Groups 3, 4 and 5 included Hypothyroid - FA 5, Hypothyroid - FA 10 and Hypothyroid - FA 15 groups which received PTU in their drinking water and were respectively treated with 5, 10 or 15 mg/kg FA, by gavage (Singh et al. 2011; Shooshtari et al. 2012). PTU was daily added to the drinking water and treatment by different doses of FA was carried out orally once a day for 7 weeks. Then, behavioral tests were then done and finally, the blood samples were collected and the animals' brains were removed under deep anesthesia induced using ketamine and xylazine. The serum samples separated from the blood, and hippocampus and cortex samples separated from the brains were kept at -80 \u0026deg; C until biochemical tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorris water maze test (MWM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA water maze test was used to assess spatial learning and memory. The test uses a circular black pool (136 cm in diameter, 60 cm high and 30 cm deep) filled with water (22\u0026ndash;24\u0026deg;C) that has an escape platform (10 cm in diameter and 28 cm high). The pool was divided into the following four zones/quadrants: north, south, east and west. The platform was located in the center of the southwest quadrant. To help the animals\u0026rsquo; navigation, visual cues were placed around the apparatus. The experiments were performed on 5 consecutive days, each day four trials. In each trial, the animal was randomly located into the water tank and allowed to find the platform. After finding the platform, it was allowed to stay on the platform for 15 seconds. If the animal was not able to find the platform during 60 seconds, it was located on the platform by the experimenter and allowed to stay on it for 15 seconds. The animals rested outside the apparatus for 20 seconds between each trail. The time latency to find the platform and the length of the swimming path were recorded by a video tracking system. On the sixth day, the probe test was performed in such a way that the platform was removed and the rat was allowed to swim for 60 seconds. The time spent and distance traveled in the target quadrant were recorded (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePassive avoidance test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe passive avoidance (PA) test helps to study non-spatial memory. In this test, an apparatus containing two chambers, one dark and one light, which are separated by a small guillotine door, was used. The floor of the dark chamber is covered with steel bars at a distance of 1 cm. An electric shock is applied to these bars by a stimulator. The experiment was performed in three phases: 1) habituation phase where the animals were placed in the apparatus for two consecutive days (each day for 5 minutes) and allowed to move freely between the two chambers; 2) training phase where the animals were placed in the light chamber and 20 seconds later, the guillotine door was opened. As soon as the animal entered the dark room, the door was closed and an electric shock (2 mA for two seconds) was applied to the animal's feet; and 3) retention phase, done 3, 24, 48 and 72 hours after the training phase, where the animals were placed in the light chamber, the guillotine door was opened, and the time latency in entering the dark compartment, the time spent in the light and dark compartments and the frequency of entering into the dark compartment were recorded (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood samples were centrifuged at 500\u0026nbsp;\u003cem\u003eg\u003c/em\u003e\u0026nbsp;for 10 min and the obtained serums were used for measuring the levels of thyroxin. Serum thyroxin level was measured using a radioimmunoassay method in Navid Medical laboratory, Mashhad, Iran. The homogenates of cerebral cortex and hippocampus (10 % w/v) were prepared in ice-cold PBS (0.1 M, pH 7.4). The homogenates were then centrifuged at 4 \u0026deg;C, at 10000\u0026times;g to separate the supernatants for estimation of malondialdehyde (MDA) and total thiol concentration as well as superoxide dismutase (SOD) and AChE activities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of MDA and total thiol concentration \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a marker of lipid peroxidation, MDA was measured in the hippocampus and cortex. Briefly, one milliliter of each sample was added to 2 ml of TBA/TCA/HCl reagent and the reaction mixtures were incubated in a boiling bath for 45 min. After cooling, the whole solutions were centrifuged at 1000 g for 10 min. Finally, the supernatants were collected and the absorbance of the pink chromogen was measured at 535 nm using a spectrophotometer (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b). The MDA concentration was calculated by the following equation:\u003c/p\u003e\n\u003cp\u003eC (M) = Absorbance/ (1.56 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e)\u003c/p\u003e\n\u003cp\u003eTo measure thiol content, DTNB was used. In this assay, 50 \u0026mu;l of the homogenates was added to 1 ml of tris-EDTA buffer (pH 8.6) and the first absorbance (A1) was recorded at 412 nm using a spectrophotometer. Afterwards, 20 \u0026mu;l DTNB solution (10 mM in methanol) was added to each sample and the second absorbance (A2) was recorded at the same wavelength (Beheshti et al. 2017; Baghcheghi et al. 2018a; Baghcheghi et al. 2018b). Total thiol concentration (mM) was calculated by the following equation:\u003c/p\u003e\n\u003cp\u003eC (mM) = (A2-A1-B) \u0026times; 1.07/0.05 \u0026times; 13.6\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstimation of SOD Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOD activity in the cerebral cortex and hippocampus was assessed based on the ability of the enzyme to inhibit autoxidation of pyrogallol (Madesh and Balasubramanian 1998). Based on the method of Madesh et al., each sample (10 \u0026mu;l supernatant from homogenate) was mixed with MTT and pyrogallol solution and then incubated at room temperature. After 5 min, DMSO was added to solubilize the resultant color. The optical absorbance was measured at 570 nm and the activity of SOD was expressed as unit per gram of tissue (Madesh and Balasubramanian 1998).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEstimation of AChE\u003c/strong\u003e\u003cstrong\u003e Activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AChE activity in the supernatants was determined by the method of Ellman using acetylthiocholine iodide as a substrate. Briefly, each sample (50 \u0026mu;l) was added into a solution (containing PBS (pH 8), 0.1 ml DTNB (10 mM), and 0.02 ml acetylthiocholine (75 mM). The changes in absorbance of the samples were spectrophotometrically recorded at 412\u0026nbsp;nm within 10 min and AChE activity was estimated as \u0026mu;mol/g tissue/min (Ellman et al. 1961).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using the SPSS 11.5 software and normality of the data was checked by the Kolmogorov\u0026ndash;Smirnov test. Data about learning phase in MWM test was analyzed by repeated measures analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s \u003cem\u003epost hoc\u003c/em\u003e test. Other data were analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s \u003cem\u003epost hoc\u003c/em\u003e test. All data are expressed as means \u0026plusmn; SEM and a p\u0026lt;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorris water maze\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe time latency and the distance traveled to reach the platform during the 5-day training in the MWM test in hypothyroid group were significantly increased compared to the control group (P\u0026lt;0.001 for the latency and P\u0026lt;0.05-P\u0026lt;0.001 for the distance). The time latency was significantly reduced in all FA-treated groups compared to the hypothyroid group (P\u0026lt;0.05 to P\u0026lt;0.001) (Fig. 1a). Moreover, the distance traveled to reach the platform in hypothyroid rats with different doses of FA was lower than the hypothyroid group (P\u0026lt;0.05 to P\u0026lt;0.01) (Fig. 1b).\u003c/p\u003e\n\u003cp\u003eThe animals in hypothyroid group showed significant decreases in the time spent and distance traveled in target quadrant on the probe day compared to the control group (P\u0026lt;0.001 for both). The hypothyroid animals treated with FA (5, 10 or 15 mg) better remembered the location of the platform and spent longer time and traveled longer distance in the target area of MWM than the hypothyroid group (P\u0026lt;0.001 for all). The animals of the hypothyroid-FA 15 group spent longer time and traveled longer distance in the target area of MWM than the hypothyroid-FA 10 group (P\u0026lt;0.05 and P\u0026lt;0.01, respectively) and traveled a longer distance in the target area than the hypothyroid-FA 5 group (P\u0026lt;0.05). The traveling time in the target area of MWM showed no significant difference between FA-treated hypothyroid rats and the control group. The distance traveled in the target area in the hypothyroid-FA 5 and hypothyroid-FA 10 groups was shorter than that of the control group (P\u0026lt;0.01 for both) but there was no significant difference between hypothyroid-FA 15 and the control group in this regard (Fig. 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePassive avoidance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the results presented in Fig. 3 and 4, in the hypothyroid group, the latency to enter and the time spent in the light chamber were decreased while the time spent and frequency of entry to the dark chamber were significantly increased compared with the control group, 3, 24, 48 and 72 h after receiving the electric shock (P\u0026lt;0.01 to P\u0026lt;0.001). The latency to enter the dark chamber in all FA-treated hypothyroid groups was higher than that of the control group, 3 h after the delivery of the electric shock (P\u0026lt;0.01, P\u0026lt;0.001 and P\u0026lt;0.001 for FA 5, 10 and 15, respectively). The latency to enter the dark chamber in the hypothyroid-FA 10 and hypothyroid-FA 15 groups was higher than the hypothyroid group 24, 48 and 72 h after the shock (P\u0026lt;0.05 to P\u0026lt;0.001) but there was no significant difference between the hypothyroid-FA 5 and the hypothyroid groups at these time-points (P\u0026gt;0.05). The latency to enter the dark chamber in the hypothyroid-FA 15 group was significantly higher compared to the hypothyroid-FA 5 group, 3, 24, 48 and 72 h post-shock (P\u0026lt;0.01, P\u0026lt;0.001, P\u0026lt;0.001 and P\u0026lt;0.05, respectively). As shown in Fig. 3a, the latency in the hypothyroid-FA 15 group was higher compared to the hypothyroid-FA 10 group, 24 h after the shock (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eAll FA-treated hypothyroid rats spent shorter times in the dark chamber than the hypothyroid group, 3 h post shock time (P\u0026lt;0.001 for the three groups) but there was no significant difference in this parameter among the three FA-treated groups. The rats of the hypothyroid-FA 10 and hypothyroid-FA 15 groups also spent shorter times in the dark chamber than the hypothyroid group, 24, 48 and 72 h post-shock (P\u0026lt;0.01 to P\u0026lt;0.001) but there was no significant difference between the hypothyroid-FA 5 and the hypothyroid group at these time-points (Fig. 3b). The rats of hypothyroid-FA 15 group spent shorter time in the dark chamber than the hypothyroid-FA 5 group, 24, 48 and 72 h post-shock (P\u0026lt;0.05 to P\u0026lt;0.001). In addition, the rats of the hypothyroid-FA 10 group spent shorter time in the dark chamber than the hypothyroid-FA 5 group, 24 and 72 h after the shock (P\u0026lt;0.05 for both times).\u003c/p\u003e\n\u003cp\u003eThe results also showed that the FA-treated hypothyroid animals spent longer times in the light chamber than the hypothyroid group 3 h post-shock (P\u0026lt;0.001 for all groups) but there was no significant difference among FA-treated hypothyroid groups in this regard. Moreover, the rats of hypothyroid-FA 10 and hypothyroid-FA 15 groups spent longer times in the light chamber than the hypothyroid group, 24, 48 and 72 h post-shock (P\u0026lt;0.01 to P\u0026lt;0.001) but there was no significant difference between hypothyroid-FA 5 and hypothyroid groups (Fig. 4a). The time spent in the dark chamber in the hypothyroid-FA 10 and hypothyroid-FA 15 groups was longer than the hypothyroid-FA 5 group, 24, 48 and 72 h post-shock (P\u0026lt;0.05 to P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eThe results of the number of dark chamber entries (Fig. 4b) showed that the frequency of entries in the hypothyroid-FA 5 group was higher than the control group (P\u0026lt;0.01 to P\u0026lt;0.001) but there was no significant difference between the hypothyroid-FA 5 and control groups. The rats of the hypothyroid-FA 10 and hypothyroid-FA 15 groups had lower number of entries into the dark chamber than the hypothyroid group at all time-points after the shock (P\u0026lt;0.05 to P\u0026lt;0.001). The number of entries in the hypothyroid-FA 10 and hypothyroid-FA 15 groups was lower than the hypothyroid-FA group (P\u0026lt;0.05 to P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMDA and thiol concentrations in cortical and hippocampal tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHypothyroidism induced by PTU increased MDA but decreased total thiol levels in both cortex and hippocampus (P\u0026lt;0.001 for all) (Fig. 5 and 6). FA at all doses reduced the MDA concentration while increased thiol content in the hippocampus (P\u0026lt;0.05 to P\u0026lt;0.001). Also, FA 10 and 15 mg/kg decreased MDA while increased thiol content in the cortex of hypothyroid rats compared to the hypothyroid group (P\u0026lt;0.05 to P\u0026lt;0.001) but there was no significant difference between hypothyroid-FA 5 and hypothyroid groups in the cortical levels of MDA and thiol. There was no significant difference among the three FA-treated groups in the hippocampal levels of MDA and thiol but in the cortex of the hypothyroid-FA 15 group, MDA concentration was lower (P\u0026lt;0.01) while thiol content was higher (P\u0026lt;0.05) than the hypothyroid-FA 5 group (Fig. 5 and 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSOD activity in hippocampal and cortical tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 7 shows that hippocampal and cortical SOD activity significantly decreased following hypothyroidism induced by PTU administration (P\u0026lt;0.001 for both tissues). SOD activity in hypothyroid-FA 10 and hypothyroid-FA 15 groups significantly increased compared to the hypothyroid group (P\u0026lt;0.01 and P\u0026lt;0.001) and it was higher in the hypothyroid-FA 15 group than the hypothyroid-FA 5 group (P\u0026lt;0.001 in the hippocampus and P\u0026lt;0.01 in the cortex). There was no significant difference between the hypothyroid-FA 5 and the hypothyroid group in both hippocampus and cortex SOD activity (Figure 7). Both hippocampal and cortical SOD activity in all FA-treated hypothyroid groups were still lower than that of the control group (P\u0026lt;0.05 to P\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAChE activity in hippocampal and cortical tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFig. 8 shows that hippocampal and cortical AChE activity significantly increased following hypothyroidism induced by PTU (P\u0026lt;0.001 for both cortex and hippocampus tissues). Administration of 10 and 15 mg/kg of FA reduced AChE activity in the hippocampus and cortex compared with the hypothyroid group (P\u0026lt;0.05 to P\u0026lt;0.001) but there was no significant difference between the hypothyroid-FA 5 and the hypothyroid group. AChE activity in the cortex of both hypothyroid-FA 10 and hypothyroid-FA 15 groups was lower than that of the hypothyroid-FA 5 group (P\u0026lt;0.01 for both) but there was no significant difference in the hippocampal AChE activity among FA-treated hypothyroid groups. The results also showed that AChE activity in the hippocampus of all FA-treated hypothyroid groups and in the cortex of hypothyroid-FA 5 group was higher than that of the control group (P\u0026lt;0.05 to P\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eThyroxin level in the serum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that serum thyroxin level in the hypothyroid group was significantly lower than the control group (P\u0026lt;0.001). The results also showed that FA was not able to reverse serum thyroxin level as there was no significant difference between FA-treated hypothyroid groups and the hypothyroid group. Serum thyroxin level in all FA-treated hypothyroid groups was lower than the control group (P\u0026lt;0.001 for all cases).\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eIn the present study, PTU administration induced a hypothyroidism state as reflected by a low serum level of thyroxin in the hypothyroid group compared to the control group. PTU as a well-known drug used for hyperthyroidism treatment, has been frequently used to produce hypothyroidism in rodents (Asiaei et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Beheshti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hypothyroidism, especially during developmental and growth periods has been shown to negatively affect the central nervous system (Asiaei et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Beheshti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e, Baghcheghi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Studies in humans and animals have shown that hypothyroidism during developmental period impairs cognitive functions such as attention, learning and memory (Hosseini et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Beheshti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of the current study showed that PTU-induced hypothyroidism was accompanied with learning and memory impairment as confirmed by both MWM and PA tests. Also, we found that the rats of the hypothyroid group spent longer time to reach the platform during the five-day learning period in the MWM test. The rats of the hypothyroid group also traveled longer distance to reach the hidden platform than the control group. Interestingly, the rats of the hypothyroid group could not remember the location of the platform and spent less time and traveled shorter distance in the target area in the probe trial in the MWM. The results of PA test also showed that the rats of the hypothyroid group had a shorter delay but a higher frequency in entering the dark compartment, and spent longer time there, than the control group. These results are consistent with the previous studies which showed that hypothyroidism during lactation, infancy or developmental periods causes cognitive impairments such as learning and memory dysfunction (Hosseini et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Beydoun et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Farrokhi et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thyroid hormones are known to be important for non-hippocampal and hippocampal-related learning and memory, synaptic flexibility, and neurogenesis (Cooke et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Asiaei et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe exact mechanism(s) responsible for adverse effects of hypothyroidism on learning and memory have not been well elucidated. It is suggested that hypothyroidism causes cerebral atrophy and cerebral neuro-inflammation as it is also accompanied with amyloid beta (Aβ) production, tau hyperphosphorylation, and impairment of signaling pathways responsible for hippocampal-dependent spatial memory (Beydoun et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chaalal et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). An imbalance between the production of peroxidants and antioxidants and production of high levels of ROS and RNS (reactive nitrogen species) are also suggested to have a role in negative impacts of hyperthyroidism on learning and memory (Venditti and Di 2006).\u003c/p\u003e \u003cp\u003eThe results of the current study also showed that hypothyroidism-associated learning and memory was accompanied with a decrease in thiol content and SOD activity but an increase in MDA level in both hippocampus and cortex tissues. These findings confirmed the occurrence of an oxidative stress state in the brain of hypothyroid rats which may have a role in learning and memory impairing effects of hypothyroidism seen in the present study. The results of previous researches have shown that MDA levels are reduced in patients with controlled hypothyroidism due to antioxidant mechanisms mediated by thyroid hormones (Villanueva et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It has also been previously reported that hypothyroidism is accompanied with a decrease in thiol content and SOD and CAT activities in the brain.\u003c/p\u003e \u003cp\u003eInterestingly, AChE activity in the hippocampus and cortex of hypothyroid rats was significantly decreased compared to the control ones. Acetylcholine is one of the major neurotransmitters involved in cognitive function. It has been previously reported that hypothyroidism is associated with cholinergic system dysfunction (Smith et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). It was also shown that thyroxin increased choline acetyltransferase activity and acetylcholine level in the brain and consequently, improved learning and memory (Fu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Considering the results of the present study and the mentioned evidence, negative effects of hypothyroidism may at least in part be due to its effects on the cholinergic system.\u003c/p\u003e \u003cp\u003eSupplementation with THs is widely done to treat hypothyroidism. Levothyroxine has been reported to reduce hippocampal cognitive impairment in hypothyroid mice (Smith et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Recently, vitamins C and E and anti-oxidant natural products were suggested to reduce the adverse effects of hypothyroidism on brain functions including learning and memory (Beheshti et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018b\u003c/span\u003e; Baghcheghi et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the current research, treatment by three doses of FA (i.e. 5, 10 and 15 mg/kg) improved learning and memory of the hypothyroid rats. The results showed that the hypothyroid rats treated with different doses of FA spent less time and traveled shorter distances to reach the platform during the 5-day learning period in the MWM. They also better remembered the location of the platform and spent more time and traveled longer distances in the target area of the MWM in the probe trial. The results of PA test also showed that treatment by different doses of FA prolonged the latency to enter the dark compartment and increased the total time spent in the light compartment while decreased the time in the dark chamber and reduced the number of entries to the dark compartment. To the best of our knowledge, the effects of FA on hypothyroidism-associated learning and memory impairment were not previously reported. Previous studies have shown that administration of vitamin B improves cognitive function in people with low FA levels by decreasing homocysteine (Shooshtari et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). FA deficiency has been reported to be followed by increases in homocysteine and cognition and learning and memory impairments (Dam et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results also showed that FA attenuated MDA while increased thiol and SOD in both hippocampus and cortex tissues. It has been previously reported that FA improves the level of glutathione (GSH), but causes a significant reduction in brain MDA levels, indicating suppression of lipid peroxidation (Singh et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). FA administration in patients with Alzheimer's disease could improve cognition and reduce inflammatory factors (Chen et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Calder\u0026oacute;n et al. 2020). FA deficiency increases the risk of other neurological disorders including stroke, through induction of oxidative DNA damage associated with morphological damage and increased cell autophagy function (Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Short-term administration of FA for 7 weeks in patients with metformin-treated type 2 diabetes was able to significantly decrease serum MDA levels (Aghamohammadi et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Moreover, FA with or without vitamin B12 given for 30 days could prevent mitochondrial dysfunction and DNA damage caused by short-term treatment with arsenic trioxide in rats (Majumdar et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). FA treatment in stressed rats was able to reduce depressive-like behaviors and brain oxidative damage, and ameliorate hippocampal antioxidant imbalance (Budni et al. 2013; R\u0026eacute;us et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Menegas et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo better understand the responsible mechanism(s), AChE activity was also evaluated in the brain. The results showed that FA decreases AChE activity in the hippocampus and cortex. Considering these results, it seems that learning and memory improving effects of FA seen in the present study are at least in part due to its attenuating effect on AChE activity. It has also been reported that FA deficiency is associated with cholinergic system dysfunction and increased AChE activity (Crivello et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNevertheless, FA was not able to restore the thyroxin concentration to normal levels. Thus, it seems that FA was not able to protect the thyroid grain from damaging effects of PTU. To better understand the effects of FA on serum thyroxin level, further investigations using other animal models including thyroidectomy model need to be done.\u003c/p\u003e "},{"header":"Conclusion","content":" \u003cp\u003eFA could improve learning and memory ability in hypothyroid rats. The observed protective effects may have been achieved by suppression of oxidative stress and regulation of AChE activity.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in this studies including animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by Ethical Committee of the Animal Research of Mashhad University of Medical Sciences (Ethical code: IR.MUMS.MEDICAL.REC.1399.639 ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Mahmoud Hosseini conducted conception and design of the project, Sabiheh Amirahmadi, Somaieh Ahmadabady, Mahsa Akbarain, Kataneh Abrari, Arezoo Rajbian, and Farzaneh Vafaee performed the experiments. Mahmoud Hosseini, Arezoo Rajbian, and Farzaneh Vafaee prepared a draft of the manuscript. Mahmoud Hosseini, Arezoo Rajbian, Farzaneh Vafaee performed statistical analysis. Mahmoud Hosseini, Farzaneh Vafaee, and Arezoo Rajbian provided final revision of the manuscript. All authors contributed to manuscript preparation and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Funding\u003c/strong\u003e This study was funded by Vice Chancellor for Research and Technology, Mashhad University of Medical Sciences with the following grant number: 991073\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors appreciate the Vice Chancellor for Research and Technology, Mashhad University of Medical Sciences for financial support (NO: 991073).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAghamohammadi V, Gargari BP, Aliasgharzadeh A (2011) Effect of folic acid supplementation on homocysteine, serum total antioxidant capacity, and malondialdehyde in patients with type 2 diabetes mellitus. 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J Nutr Biochem 38:41\u0026ndash;49. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jnutbio.2016.08.009\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"metabolic-brain-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebr","sideBox":"Learn more about [Metabolic Brain Disease](https://www.springer.com/journal/11011)","snPcode":"11011","submissionUrl":"https://submission.nature.com/new-submission/11011/3","title":"Metabolic Brain Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Cholinergic dysfunction, Folic acid, Hypothyroidism, Learning and memory deficit, Oxidative injury","lastPublishedDoi":"10.21203/rs.3.rs-383794/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-383794/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypothyroidism has been associated with cognitive decline. Considering the role that has been suggested for folic acid (FA) in cognitive performance, the present study was designed to investigate the effects of FA against hypothyroidism-induced cognitive impairment, oxidative damage and acetylcholinesterase (AChE) activity alterations in propylthiouracil (PTU)-induced hypothyroid rats.\u003c/p\u003e \u003cp\u003eIn this study, PTU (0.05% in drinking water) and FA (5, 10, and 15 mg/kg, oral gavage) were administered to the rats for a period of 7 weeks. Then, behavioral performance was tested using Morris water maze (MWM) and passive avoidance (PA) tasks. Finally, oxidative stress indicators and AChE activity were assayed in the brain tissues.\u003c/p\u003e \u003cp\u003eThe impairing effect of hypothyroidism on cognitive performance was markedly alleviated by FA especially at the higher doses. In the MWM test, FA reduced escape latency and travelled distance, compared to the non-treated hypothyroid group. In the PA test, the latency to enter the dark chamber was significantly enhanced by FA as compared to the non-treated hypothyroid group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05-p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Besides, FA attenuated AChE activity and malondialdehyde level but increased superoxidase dismutase enzyme activity and total thiol content (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05-p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eIn conclusion, FA could improve learning and memory ability in hypothyroid rats. The observed protective effects may be mediated through regulation of oxidative stress and AChE activity.\u003c/p\u003e","manuscriptTitle":"Folic Acid Attenuated Learning and Memory Impairment via Inhibition of Oxidative Damage and Acetylcholinesterase Activity in Hypothyroid Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-04-28 18:45:35","doi":"10.21203/rs.3.rs-383794/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2021-04-01T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Metabolic Brain Disease","date":"2021-03-31T08:00:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"metabolic-brain-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mebr","sideBox":"Learn more about [Metabolic Brain Disease](https://www.springer.com/journal/11011)","snPcode":"11011","submissionUrl":"https://submission.nature.com/new-submission/11011/3","title":"Metabolic Brain Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7676318a-fdc4-4784-ae06-a9f117119029","owner":[],"postedDate":"April 28th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":3948027,"name":"Molecular Biology"},{"id":3948028,"name":"Neurology"},{"id":3948029,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2021-07-30T07:40:11+00:00","versionOfRecord":[],"versionCreatedAt":"2021-04-28 18:45:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-383794","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-383794","identity":"rs-383794","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}