Animals and general procedures
In this experimental study, 63 female Sprague-Dawley rats with an age range of 8-10 weeks and a weight range of 200-220 gr were provided from the Center for Breeding and Reproduction of Laboratory Animals and after transferring to the sports physiology animal laboratory of the university, they were kept in the laboratory for one week for adaptation. Inclusion criteria were being able to perform treadmill running (trainability) and not being affected with any kind of disease and clear diagnose of EAE in EAE groups. Exclusion criteria were, not being able to continue exercise programs and affecting with any kind of disease. All ethical principles of working with animals in this study were regarded according to the ethical principles of working with animals of Shiraz University as well as the Helsinki Agreement. The graduate and ethic committee at Shiraz University approved the study proposal and procedures (number: 34579). During the study, the animals were kept in standard conditions of light (12-hour dark-light cycle), temperature (22-24°C), and humidity (55-60%) in clear polycarbonate cages with autoclave capability. Sterile shaved woods were used to change the bedding of the animals, and they had ad libitum access to water and food throughout the study.
Induction of EAE disease
After a seven-day adaptation period, to induce EAE, 20 guinea pigs were provided from the Pasteur Institute of Iran and transferred to the animal laboratory. The guinea pigs were anesthesized by ketamine and xylazine, and they were then dissected and their spinal cord tissues were extracted. The spinal cord tissues were immediately immersed in a nitrogen tank and were then pounded in a nitrogen-filled mortar. Considering the previous studies, guinea pig's spinal cord was used as an antigen to further enhance the complete freund's adjuvant in the nervous system. To homogenize the spinal cord tissue, it was mixed with an equal amount of normal saline, and placed in a shaker at 5°C until it was completely homogenized. The homogenized solution was then made into an emulsion solution in a ratio of 1 to 1 with complete freund's adjuvant (CFA). To prepare this suspension, two glass syringes were used, being connected by a steel interface. One of the syringes contained the homogenized guinea pig's brain and spinal cord and the other syringe contained the same volume of complete CFA; the solution was mixed in equal proportions and its color was uniformed and whitened using a shaker. After rats' complete anesthesia with ketamine and xylazine, 400 μl of the antigen and adjuvant mixture was injected subcutaneously in the back and 100 μl into the cushion area of each animal with needle number 25. To diagnose induction of the disease, the daily disease process was evaluated and the disease scale was set as follows: zero: no disease, 1: tail movement disorder, 2: tail paralysis, 3: gait disorder, 4: one-leg paralysis, 5: two-leg paralysis, 6: hands and legs paralysis, and 7: death[34, 35]. Due to the research requirement for animals' minimal daily activities, rats on scales 6 and 7 were typically excluded from the study.
Grouping and research design
After ensuring the induction of EAE in rats, based on the standards and their homogenization and on mobility and disability scale as well as inclusion and exclusion criteria, 49 rats with EAE were divided into seven groups of equal number (n=7), including: (1) experimental autoimmune encephalomyelitis (EAE), (2) sham (Sh), (3) 50 mg / kg royal jelly consumption (RJ50), (4) 100 mg / kg royal jelly consumption (RJ100), (5) aerobic training (AT), (6) AT + RJ50, and (7) AT + RJ100. Allocation to each group was through simple randomization method. The number of animals in each group was according to statistical approach for animal studies . In addition, eight healthy rats were included in the healthy control (HC) group to evaluate the effects of EAE induction on the research variables. Rats in the royal jelly consumption groups received peritoneally doses of the prescribed royal jelly (dissolved in normal saline) every day for 5 weeks . Rats in the endurance training groups performed endurance training on the treadmill at a speed of 11 meters per minute, five sessions per week and each session of 30 minutes for 5 weeks [38, 39].
Familiarization of animals to treadmill training
Endurance training began approximately 10 days after induction of the EAE experimental model. To familiarize animals to treadmill, rat performed endurance training on the treadmill every day at a speed of 6 meters per minute and a slope of 11 degrees for 5 to 25 minutes for a week [38, 39].
Main aerobic training protocol
Rats performed endurance training every day at a speed of 11 meters per minute for 25-35 minutes for five weeks. In other words, the training period was 25 minutes in the first week, and 2 minutes were added to the duration of exercise each week so that duration of training reached 35 minutes in the fifth week. One of the reasons for choosing this training protocol was the neuroprotective effects of this type of training in rats with cognitive impairments and rats and mice with the experimental model of Parkinson's encephalomyelitis [38, 39].
Royal jelly consumption
In this study, royal jelly was used with doses of 100 and 50 mg / kg during five weeks. RJ was prepared from Marvdasht Agricultural Jihad Center and dissolved daily in normal saline as required; then it was injected peritoneally into rats similar to a recent study.
Evaluation of anxiety-like behaviors
Elevated plus-maze behavioral model was used to measure anxiety. This evaluation was based on a model first proposed by Pellow et al, using an elevated + -maze consisting of two open arms and two enclosed arms. The dimensions of the open and closed arms are 10 × 50, with two sides and the end of the enclosed arms height is 40 cm. The four arms lead to a central area are 10 × 10 cm. The maze was placed at a height of 50 cm above the ground. The rats were placed in the central area of the maze, facing an open arm. During the 5 minutes, animals moved freely in different parts of the maze, and the frequency of entries to the open arm and closed arm, and the duration of placement in the open and closed arm were measured. The test and related evaluations were performed by a research assistant who were blind about the group allocations.
Forced swimming test
Forced swimming as a valid test was used to measure depression. 24 hours before the test, the animals were placed in water for 15 minutes. The behavior of rat was recorded during five minutes testing. Inertness of the mouse's limbs and its buoyancy were considered as immobility and its duration is considered as immobility time. All test procedures were performed according to the available guidelines. The test and related evaluations were performed by a research assistant who were blind about the group allocations.
Rats weight was were measured once a week throughout the study. 48 hours after the last training session, the rats were anesthetized with ketamine (15 mg/kg) and xylazine (70 mg/kg) and killed by the drug, and their hippocampus tissue was isolated and homogenized and stored in nitrogen fluid at -80 ° C for further analysis. Real time PCR was used to measure the gene expression levels of the research variables.
To measure IL-17, IL-23, IL-10 and TGF-β gene expression levels by qPCR, 20 mg of tissue was isolated from the hippocampus; RNA extraction from tissues in all study groups was performed according to the protocol of the manufacturer (Kiagen, Germany). To ensure the quality of RNA, electrophoresis was performed using agarose gel and light absorption property at 260 nm with Sigma's Picop Drop device (made in USA). Also, the formula (C (μg / μl) = A260 × ε × d / 1000) was used to evaluate RNA quality. Following cDNA synthesis, a reverse transcription reaction was performed using the fermentase kit manufacturer protocol (K1621) and implementing the designed primers (Table 1). To determine the efficacy and specificity of the primers, the pre-primers were evaluated using the software available on the NCBI site. To measure the gene expression levels, the research variables were used using the TBP internal control gene. After confirming the completion of the qPCR and after reaching the expression threshold (Cycle Treshold), the formula 2-ΔΔCT was used to quantify the ratio of the desired gene to the reference gene.
Insert table 1
The Shapiro-Wilk test was used to investigate the normality of the findings of the study. Regarding the normal distribution of findings, in order to investigate difference between the groups, one-way analysis of variance (ANOVA) was used and in the case of significant difference, for paired group comparisons, Tukey's post hoc test were used in Graphpad Prism 8.3.6 software (P≥ 0.05).