2.1 Subjects and housing
A total of 96 adult male Wistar rats ranging in age from 4 to 6 weeks old were used. Rats were housed with free access to food and water under a 12 h light/dark cycle. The Institutional Animal Care and Use Committee of the Kerman Medical University approved all procedures, and the National Institutes of Health ‘Guide for the Care and Use of Laboratory Animals’ were followed. The animals were randomly divided into 12 groups (n = 8, in each group): Control, Saline + DMSO, 3AP, WIN-55,212-2 (WIN) (0.1, 0.5, 1 mg/kg), AM251 (AM) (1mg/kg), 3AP+WIN (0.1, 0.5, 1 mg/kg) and 3AP+ AM (1mg/kg). WIN (cannabinoid agonist) and AM (cannabinoid antagonist) were dissolved in DMSO. WIN was administered in three different doses (0.1, 0.5, 1 ml/kg) 30 min prior to 3AP (55 mg/kg, i.p.) once daily for 3 days. In a group of animals, AM (1mg/kg) was also administered 30 min prior to 3AP for three consecutive days. In the Saline +DMSO group, animals received the treatment once daily for 3 days. In the 3AP groups, rats received (55 mg/kg) only on the first day. In the 3AP+WIN+AM group, the rats received co-administration of WIN (0.1, 0.5, 1 mg/kg) and AM 30 minutes before 3AP injection once daily for 3 days. Following completion of experiments, and 24 hours after the last treatment, the animals were deeply anaesthetized by CO2 gas, their brains were removed, and the vermis of the cerebellum was rapidly separated and brain samples (n=4) of each group were protected in %10 formalin for histological tests.
2.2 Behavioral assays
2.3 Open field test
Locomotor activity was measured by movement within an open field (90 90 30 cm) for 5 min. Animals were transported to the experimental room about 1h before any session started in order to allow acclimatization to the room. At the beginning of the test session, the rat was placed in the center of the testing field. The number of times the animal reared and groomed, the time spent in the center of the open field (s), the total distance moved (TDM, cm), the total duration of mobility (s), and velocity (cm/s) were recorded. The floor was cleaned between sessions. A video camera was mounted 1.5 m above the floor of the open-field arena in a position which allowed the entire arena to be within view. The entire 5-min session was recorded on videotape and analyzed off line using Ethovision software (version 7.1, Noldus Information Technology, Ethovision, Wageningen, the Netherlands) (Shabani et al., 2012a).
2.4 Foot print
This test was used to evaluate differences in gait. To obtain footprints, the hind feet of the rats were coated with nontoxic paints. The animals were then allowed to move freely across the walkway of a white sheet. To characterize the walking pattern, stance width and the distance between left and right hind footprints were analyzed.
2.5 Grip-strength test
The grip-strength test was used to quantify skeletal muscular strength (Shabani et al., 2011). Each rat was suspended with both forepaws on a horizontal steel wire [80 cm long, diameter 7 mm]. The animal was held in a vertical position when its front paws were placed in contact with the wire. When the rat grasped the wire, it was released, and the latency to fall was recorded with a stopwatch. Rats were randomly tested, and each animal was given three trials with a 30 min rest interval.
2.6 Evaluation of motor coordination.
To evaluate balance deficits of rats, we used the rotarod test (Hugo Sachs Electronik, Germany), which measures balance, coordination, and motor control. The rotarod accelerated from the minimum speed of 10 rpms to the maximum speed of 60 rpm. Rats were given three trials using a maximum time of 300 s with a 30 min rest interval. The trial was terminated when rats fell from the apparatus or after a maximum of 5 min. Rats were scored with disturbances in coordination if they fell from the test apparatus within 5 min (Shabani et al., 2012b).
2.7 Histopathology analysis
Following fixation with 10% formalin, the vermis of the cerebellum was dissected and embedded with paraffin, serially sectioned (4 μm). The slides were dewaxed and boiled (in a 600 W microwave oven) at 120 °C for 10 minutes. The sliced samples were stained with hematoxylin-eosin (H&E) and observed with a bright field microscope (Olympus CX31) (Abbasloo et al., 2015). Neurons that had distinctive nuclei and cell bodies were counted.
2.8 Statistical analyses
For the statistical analysis, two-way analysis of variance (ANOVA) was used, followed by the post-hoc Tukey test for comparisons among groups. Student’s t-test was used to compare Control and DMSO groups and since there was no different between these groups, we compared other groups to DMSO instead of Control group. Data are expressed as mean ±SEM (All row data are presented in an Excel file as a supplementary file). The minimum level of significance was P < 0.05.