Animals
30 female Lewis rats with an age of 8–10 weeks and a weight of (100–125 kg) were purchased from DarouPakhsh Pharmaceutical Company, Iran. Individually housed in special cages, the animals are kept in a controlled environment (12/12 h light and dark) and fed standard rodent chow and water. The animals were acclimated to their new surroundings for one week before being divided into one of three groups (10 rats each): control (Co), EAE only (EAE) and an EAE exercise group (EAE+Ex).
Estrous cycle evaluation.
As reported previously [1], Female rats estrous cycles were observed every day. The rats with estrous cycles that began on the same day were chosen. Between 7 and 8 in the morning, vaginal lavage was carried out with a mild manual restraint. A plastic pipette with the tip inserted inside the animal vagina and 10μl of normal saline (0.9%) was added to collect vaginal excrement. The glass slides were covered with vaginal fluid. Each rat had one drop of vaginal fluid removed with a clean tip.
Morris Water Maze
The Morris Water Maze (MWM) is considered to be the gold standard to assess spatial learning as well as memory capacity [24]. This approach is based on the observation that animals have developed an efficient manner of navigating their surroundings and escaping danger that allows them to attain the desired outcome with the least amount of effort [25, 26]. The MWM used in this study was a black circular tank with a diameter of about 136 cm and a height of 60cm filled with water (21oC) at a depth of 25 cm. Maze was divided into four quadrants: northeast, northwest, southeast, and southwest as well as starting positions: north, south, west, and east. A concealed circular platform with a diameter of 10 cm was situated in the SW quadrant's center, 1.5 cm below the water's surface. Each test was recorded and assessed using a video camera connected to a tracking device (EthoVision XT) situated above the pool to calculate the time in each quadrant and swimming velocity of each rat. Animals were allowed to adapt to the WMM test for two days. The MWM test was implemented across five consecutive days: four days for learning and one day as prop test. All sessions began at 8:00 a.m. For the training days, each rat was placed at one of the four predetermined beginning locations in the water, facing the tank's wall, and then given free reign to swim until they reached the platform that was concealed inside the tank. Every trial required every rat to locate the hidden platform within 60 seconds. If the rat discovered the platform, it was permitted to stay there for 30 seconds. If the rat did not find the platform within 60 seconds, it was placed on platform 30 seconds. The prop test was performed on day 5 to assess memory. On the prop test day, the platform was removed from the tank and the time it took the rat to reach the area in which the platform was previously placed was measured as well as time spent in that specific area. The test was discontinued at 60 seconds. Assessments were completed at baseline prior to EAE induction and again 30 days post inoculation.
Induction and Clinical Disability Assessment
As previously described [27], 50 ug of guinea pig spinal cord homogenate and 2 mg of heat-mycobacterium (Difco H37Ra) were added to incomplete Freund's adjuvant (Sigma). A subcutaneous injection of 50 ul of the inoculation was given into the base of the tail. The presence of any indicators of impairment was assessed using EAE scores previously described [28].
Exercise Training and Treatment
For seven days, the animals practiced running for 15 minutes each day at a belt pace of 5 to 10 meters per minute without slop. Rats were made to run by administering an electric shock (0.3 mA). As described by Kim and colleagues [29] exercise training was started 2 weeks prior EAE immunization (preconditioning protocol) and continued until 2 weeks post EAE immunization. Exercise was prescribed starting at 17 m/min and increased each week (week 1: 17 m/min, week 2: 18 m/min, week 3: 19 m/min, and week 4: 20 m/min) for a total duration of 30 minutes per day, five days a week. Rats in the non-exercise groups were placed on the treadmill lane with the machine turned off and were subjected to the same schedule as the rats in the exercise groups.
Anaesthesia and euthanasia
Rats were euthanized by ketamine and xylazine (3/1) and the animal brains were extracted. Hippocampal tissue was quickly dissected and frozen in liquid nitrogen immediately. Frozen tissues were homogenized in the Trizol reagent at a 1 ml Trizol/100 mg tissue ratio. The left hippocampal was dissected and frozen at 80°C for gene expression analysis. The right hippocampal was also dissected for morphology investigation, post-fixed in 4% formaldehyde and immersed in double-distilled water.
Gene expression analysis.
Real-time polymerase chain reaction (PCR) was utilized to analyse TrKB and BDNF mRNA in the hippocampus. Total RNA was extracted from samples using the TRIzol kit (Invitrogen, USA) in accordance with the manufacturer's instructions. After RNA purification, TrKB and BDNF gene primers were made using the NCBI website (Table 1.). Real-time PCR was carried out using the Applied Biosystems TM Real-Time PCR apparatus with Master Mix and SYBR Green I (Fluka, Switzerland) as the starting materials. Using a standard curve, the efficacy of each gene was determined. GAPDH was used as a checkpoint.
Table 1. The nucleotide sequences of sense and antisense primers.
Cresyl violet staining method
Cresyl violet staining was used to count the number of neurons in the right hippocampus. Brain slices are dried in three sequential steps: 70% alcohol (for 10 min), 95% alcohol (with a few drops of 10% acetic acid; for 2-3 min) and finally 100% alcohol after creating 5 m-long microtome incisions and undergoing tissue processing (for 10 min). Slices were washed in xylene for five minutes, colored with cresyl violet, covered with DPX, and then sealed with a cover slip.
Statistical analyses
The mean and standard error are used to represent the data. The data were analyzed using a two-way variance analysis (ANOVA). Repeated measures were used in the MWM and significance was assessed using P values <0.05. The normality and homogeneity of the data was determined using Tukey post-hoc test.