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).
Animalsand 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).
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 × 105)
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 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.