Materials
MDMA was synthesized by the Department of Medical Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences according to the previous method [21]. It showed an acceptable purity value (> 95%), and its structure was fully confirmed with H-NMR, C-NMR, and Mass spectrometry methodologies. In our study, the following chemicals were used: KCl, MgCl2, Tris–HCl, dimethylsulfoxide (DMSO), sodium succinate, Na2HPO4, sucrose, Ethylene glycol-bis (2-aminoethyl ether)-N, N, N′, N′-tetraacetic acid (EGTA), 4–2-hydroxyethyl piperazine ethanesulfonic acid (HEPES), Rhodamine 123 (Rh 123), 2′, 7′- dichlorofluorescein diacetate (DCFH-DA), rotenone (Rot), Coomassie blue, ketamine, xylazine, and carboxymethylcellulose sodium (CMC). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) except for atorvastatin (Darou Pakhsh Pharmaceutical Co., Tehran, Iran) and rosuvastatin (Abidi Pharmaceutical Co., Tehran, Iran).
Atorvastatin and rosuvastatin were suspended in an aqueous solution of CMC 1%, and MDMA was dissolved in saline. Solutions and suspensions of chemicals were always prepared on the day of the experiment.
Animals
In this study, adult male Wistar rats weighing 180–200 g were purchased from the Faculty of Pharmacy, Tehran University of Medical Sciences. Animals were housed in separately standard polypropylene cages under a standard temperature-controlled environment (23 ± 1°C), 12-h light/12-h dark cycle schedule, humidity (55% ± 10%), and they had free accessibility to tap water and food. Experimental protocols were performed in accordance with the Tehran University of Medical Sciences Animal Ethics Committee and designated to reduce animals suffering stress and decrease the number of rats used.
Spatial learning and memory assessment
Spatial memory and learning were evaluated by the Morris water maze (MWM) training protocol, the same as described previously [13, 22]. Concisely, the MWM test consisted of four trials per day for four consecutive days and one test session (probe test). In each trial, animals were randomly placed in one of the four quadrants of the MWM pool (north, east, south, and west). Then they were allowed to find the hidden target platform, which was located 1 cm beneath the surface of the water within 90 seconds. MDMA (2.5, 5, and 10 mg/ kg) or saline was administered intraperitoneally to rats 30 minutes before the first trial in each four training days. Probe test was performed 24 h after the last training trial by removing the platform from the tank. A video camera was installed above the MWM pool to record the animal's behavior. Different parameters of the MWM test, including escape latency (time spent to reach the platform), swimming speed, traveled distance (path length to find the hidden platform), and the time spent in the target quadrant (in the probe test), were calculated by the Ethovision 7 tracking system (Noldus Information Technology, Wageningen, the Netherlands).
After the probe test of MWM, each group (n = 4) were anesthetized by intraperitoneal injection of ketamine/xylazine (100 and 10 mg/kg, respectively) until loss of consciousness and loss of any response, then sacrificed under general anesthesia, and their hippocampuses were dissected on the ice-cold surface and stored at − 80°C until analysis.
Experimental groups
The animals were allocated to the following groups. Each group contained eight rats.
CMC 1% + MDMA groups received CMC 1% w/v orally via a gastric metallic tube, once daily for 21 consecutive days. After this period, they were administered different doses of MDMA (2.5, 5, and 10 mg/kg) intraperitoneally 30 min before the first training trial during 4 training days in the MWM task.
Atorvastatin + MDMA groups, which were divided into three groups according to the different oral doses of atorvastatin (5,10 and 20 mg/kg) in CMC1% for 21 days, followed by MDMA (5 mg/kg, I.P) during acquisition trials (i.e., 4 days of training in MWM).
Rosuvastatin + MDMA groups, which were divided into three groups according to the different oral doses of rosuvastatin (5,10 and 20 mg/kg) in CMC1% for 21 days followed by MDMA (5 mg/kg, I.P) during acquisition trials (i.e., 4 days of training in MWM).
Atorvastatin/Rosuvastatin + saline groups, which received atorvastatin or rosuvastatin orally once daily for 21 consecutive days, then were injected saline rather than MDMA, 30 minutes before the MWM task.
Control group was treated orally with a single daily dose of vehicle (CMC 1%) for 21 successive days, followed by intrapersonal injection of saline 30 minutes before training sessions in MWM. Different experimental groups are explained in Table 1.
Table 1
Summarized the employed experimental protocol in our study.
Experimental groups | Treatments on days 1–21 | MWM on days 22–26 |
MDMA groups | CMC 1% orally | Different doses of MDMA (2.5, 5, and 10 mg/kg, I.P) 30 min before the first training trial during 4 training days in the MWM task | Sacrificed after the probe test and hippocampuses were dissected to evaluate ROS, LPO, and caspase3 and − 9 activities |
Atorvastatin + MDMA groups | Different oral doses of atorvastatin (5,10 and 20 mg/kg) in CMC1% | MDMA 5 mg/kg, I.P, 30 min before the first training trial during 4 training days in the MWM task |
Rosuvastatin + MDMA groups | Different oral doses of rosuvastatin (5,10 and 20 mg/kg) in CMC1% | MDMA 5 mg/kg, I.P, 30 min before the first training trial during 4 training days in the MWM task |
Atorvastatin group | atorvastatin 20 mg/kg in CMC 1%, orally | Saline |
Rosuvastatin group | rosuvastatin 20 mg/kg in CMC 1%, orally | Saline |
Control | CMC 1% orally | Saline |
ROS assay
production of ROS in hippocampal cells was measured by a fluorometric assay based on the conversion of 2′,7′–dichlorofluorescein diacetate (DCFH-DA), a non-fluorescent chemical to a fluorescent compound named 2′,7′–dichlorofluorescein (DCF). Samples were homogenized, 50 µl supernatant was added to 10 µl DCFH-DA and 162 µl assay buffer, then, solutions were incubated at 37°C for 15 min. The fluorescence of DCF was examined spectrometrically by an ELISA fluorometer (Biotec, Tecan U.S.) with maximum excitation of 488 nm and maximum emission of 525 nm in the spectra within 60 min. Finally, the results were reported as the percentage of control, which is considered 100% [23].
Lipid peroxidation (LPO) assay
The end product of LPO is malondialdehyde (MDA) which reacts with Thiobarbituric acid (TBA) and produces a new chemical complex named TBA reactive substances (TBARS). Briefly, the samples were homogenized and mixed with 800 µL trichloroacetic acid 20% (TCA) followed by centrifugation at 3500 g for 30 min. Then 600 µL of supernatant was mixed with 150 µL TBA (1% w/v). Finally, the cocktail was heated in a steaming water bath for 30 min followed by the addition of 400 µL of n-butanol to extract TBRAS adducts. After cooling down, the absorbance was evaluated at 532 nm by an ELISA reader [24].
Measurement of caspase-3 and − 9 activity
Caspase-3 and − 9 activities were evaluated using colorimetric assays based on the distinctive identification of specific amino acid sequences in these enzymes. A tetrapeptide substrate containing a particular caspase recognition sequence was labeled with the chromophore r-nitroaniline (ρNA). Then, ρNA was released from the substrate through cleavage by caspase and produced a yellow color, which its intensity was recorded using an ELISA reader at 405 nm. The amount of caspase activity is directly proportional to the production of yellow color upon cleavage. Concisely, the treated cells were lysed in the supplied lysis buffer and were incubated on ice for 10 minutes. Therefore, samples were incubated in caspase buffer (pH 7.4, 20% glycerol, 100 mM HEPES, 5 mM dithiothreitol, 0.5 mM ethylenediaminetetraacetic acid [EDTA]) containing 100 mM of caspase-3 and − 9-specific substrates (Ac-DEVD-ρNA and Ac-LEHD-ρNA, respectively) for 4 hours at 37°C. Then, absorbance was measured at 405 nm. The caspase-3 and − 9 activities in different groups were reported as the percentage of control, which was considered as 100% [25].
Protein Content Measurement
For normalizing data obtained from the mentioned tests, 10 µL Bradford reagent was added to 100 µL of homogenized and diluted samples; then, 5 min later, the absorbance was recorded at 595 nm by spectrophotometer. BSA was used as the standard [26].
Statistical analysis
Spatial memory performance parameters (escape latency, swimming speed, traveled distance, and the spent time in the target quadrant in probe test) and oxidative stress biomarkers were recorded for each group. Data were expressed as a mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) followed by the post-hoc Tukey test to illustrate the significant differences between groups. A level of P < 0.05 was considered statistically significant.