2.1. Animal study
Before starting the experiment, seventy-two adult male Wistar rats (weighing 175-200 gr) were purchased from the breeding institute of the animal house of Hamadan University of Medical Sciences (UMSHA). The animals were housed tree in a cage in a temperature-controlled room with 23-25 °C and 50–70% relative humidity under a 12:12 light-dark cycle from 19 till seven that treatment protocol and behavioral tests started in light cycles. Also, every animal accessed to food (rodent pellets consisted of 23% protein, 47% carbohydrate, 5% lipids, 5% cellulose, 20% water, and vitamins and minerals with a caloric density of approximately 3.0 kcal/g) (32) and water freely. All experimental processes and animal care procedures were confirmed by the Veterinary Ethics Committee of UMSHA and were confirmed with the Guidelines of the principles of laboratory animal care in the National Center of Health (code: IR.UMSHA.REC.1400.458).
2.2. Experimental design
At first, Rats for seven days were domesticated using handling and accustoming to circumstances. Then they were randomly assigned into nine groups(n=8 per group): (group1) control health (Control); they received saline 9% (as Ecdy dissolvent) for eight weeks through oral gavage (33). (group2) Sham; they received a five μL vehicle of beta-amyloid (phosphate-buffered saline (PBS)) via an intrahippocampal injection after stereotaxic surgery. (group3) Ecdy; they received Ecdy (10mg/kg/day) for eight weeks through oral gavage(33). (group4) HIIT; they performed HIIT on a special treadmill for eight weeks (5 sessions per week) (34). (group5) Ecdy + HIIT; they received Ecdy (10mg/kg/day) for eight weeks through oral gavage also performed HIIT on a treadmill for eight weeks. (group6) control Alzheimer’s (Alzheimer's); they received five μL beta-amyloid via an intrahippocampal injection after stereotaxic surgery, then received saline 9% for eight weeks through oral gavage (35). (group7) Alzheimer + Ecdy; they received Ecdy (10mg/kg/day) for eight weeks through oral gavage after receiving five μL beta-amyloid via intrahippocampal injection. (group8) Alzheimer + HIIT; they performed HIIT on a special treadmill for eight weeks after receiving five μL beta-amyloid via intrahippocampal injection. (9) Alzheimer + Ecdy + HIIT; they received Ecdy (10mg/kg/day) for eight weeks through oral gavage also performed HIIT on a treadmill for eight weeks after receiving five μL beta-amyloid via intrahippocampal injection (Fig.2). Although studies also have reported some other dosages such as 1 and 100 mg/kg/day, with due attention to studies, we decided to use 10mg/kg/day of Ecdy as an efficient dosage (33).
2.3. Aβ preparation, injection and stereotaxic surgery
The Amyloid-β1-42 peptide (100 μL) (cat number SCP0038, bought from Sigma Aldrich, USA) dissolved in 100 μL PBS (as vehicle solution) then incubated at 37 °C for seven days before in vivo utilizing. This action leads to fibril formation, which has neurotoxic properties (36). After preparing Aβ1-42 to induce AD model, the rats were anesthetized with intraperitoneal (i.p) injection of ketamine (100mg/kg) and xylzine (10mg/kg). Afterward, rats were stuck in the stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA), and their scalp was split. Then Bregma and Lambda adjusted to balance level in terms of the horizontal plane and a hole drilled above the ventricular area in the skull surface (coordinates: AP: -1.2 mm posterior to the Bregma, ML, ±2 mm lateral, and DV: 4.0 mm below the dura)(37). The injection was conducted with a five μL microsyringe (Hamilton Laboratory Products, Reno, NV, USA). Aβ solution (5 μL) was gradually injected into the region on the right side at a rate of 0.5 μL/min. Then the syringe was left in place for 5 min after the injection before being dislodged to allow diffusion of Aβ. The next scalp was stitched. Also, Sham group rats operated like this surgical protocol with the same injection volume, but they received PBS (vehicle solution) instead of Aβ. Animals are allowed to have one week of recovery after surgery before beginning treatment (37).
2.4. Preparation and gavage of Ecdy
The dry powder of Ecdysterone supplementation (purchased from Amazon company, USA) dissolved in distilled water at a concentration of 10 mg/cc and then gavaged orally with an insulin syringe accompanied by a gavage needle in the amount of 10 mg/kg. This protocol was carried out every day for eight weeks (33).
2.5. HIIT exercise protocol
One week after injection of Aβ, the exercise group rats were trained on a motorized rodent treadmill apparatus (Tajhiz Gostare Omide Iranian, Iran) (38). All rats were initially familiarized with the treadmill environment through walking at a speed between 5 to 10 m/min for 10 minutes 5 days. After animals adapted to the environment, Rats ran on a treadmill with a 15° inclination until exhaustion to measure their maximal oxygen uptake (V̇o2 max). They started the protocol at speed of 6 m/min and increased by 3 m/min every 3 min until rats were unable to run (39). After the determination of V̇o2 max,the main period of exercise began. The animals every day ran 1min intervals at 90% of maximal exercise capacity, followed by 1min intervals at 50% of maximal exercise capacity alternately at no incline (0%) for 30 minutes in the first week and every week, 5 minutes added to the time of exercise until the time reached to 60 minutes in the seventh week (39). This protocol lasted for eight weeks. Besides, the main period of exercise included a 5 min warm-up and a 5 min cool-down at 40% VO2max before and after the exercise period (40). At the same time, animals trained for five days over a week (they rested on Fridays and Mondays) between 9 AM and 2 PM.
2.6. Morris Water Maze (MWM)
2.6.1. Morris water maze apparatus
Spital memory and cognitive performance were assayed using a five days Morris water maze (MWM) test. The water maze device (180 cm in diameter and 60 cm in height) was full of 25 ± 1 °C water to a depth of 45 cm located in a room containing a variety of visual cues that was allocated into four equal quadrants. In addition, an invisible circular platform (10 cm diameter) was submerged in a stable position at 1.5 cm beneath the water's surface in the center of the Northwest quadrant of the pool. Low lights were used for illumination, and the room was sound insulated. On the other hand, the pool was separated into four specific start points as the East (E), West (W), North (N), and South (S). These adjustments remained consistent for all rats across the training trials (41).
Before starting the first training session, rats were put in the Morris water maze pool to swim without any platform for habituation to the environment for 60 seconds.
2.6.3. Hidden platform training
The training sessions were performed between 9 AM and 12 PM for four days, including. Eight trials were divided equally into two blocks (every block was four trials), that there was a five-minute break between every block for every rat. For each trial, the animals in all groups were placed in the pool (facing the pool wall) to start the trial at one of the four starting points in a different order in every trial and allowed to swim for 60 seconds from a start point (E, W, N, and S) in the pool to find the invisible platform. If animals could detect the platform in 60 seconds, they were let to remain on the platform to rest and discover the environment for 30 seconds, but if they failed to find it, they were guided to the invisible platform by a technician and then were let to rest and discovery for 30 seconds. A video camera (Nikon, Melville, NY) linked to a tracking system in the computer was directly installed above the pool to record specific parameters, such as Distance moved by swimming and time spent to reach the platform (Scape latency) from the training sessions (41).
2.6.4. Probe test
On the fifth day, a probe test was conducted to evaluate spatial memory retention twenty-four hours after the training phase. On this day, the platform was eliminated, and each rat was placed in the pool like the training method and was allowed freely to swim for 60 seconds. Then, as an assay of spatial memory retention, the number of entrances to the target quadrant, time spent in the target quadrant, distance traveled in the target quadrant, and Average Speed was analyzed (42).
2.6.5. visual test
Forty-five minutes after the probe test, the platform elevated above the water surface and also was made apparent by a piece of bright sponge and located in the SE quadrant, and rats were let to swim and explore the visible platform for 60 seconds in order to evince their visual ability (43).
2.7. Passive Avoidance Task (PAT)
2.7.1. Passive avoidance apparatus
In the survey, a PAL device and a procedure (step-through method) were identical to the previous studies used to evaluate passive avoidance memory and learning (44-46). The device contained a bright cubical space with a dimension of (22_22_32 cm3) made of limpid plastic and a dark cubical space with dark opaque plastic walls (22_22_32 cm3). Also, the space floors were made of stainless steel shafts (3mm in diameter), placed at a 1-cm distance from each other. Besides, a shock generator was used for electrifying the dark chamber floor, and a rectangular opening (6_8 cm2) was placed between the two bright and dark spaces, which could be closed by an opaque guillotine door (41).
2.7.2. Passive avoidance training
In order to adapt the rats to the device, they were given two trials opportunity to adapt. At first, the rats entered into the bright section of the apparatus; then, after 30 seconds, the guillotine door picked up. With due attention to the natural trend of rats to the dark environment, they tried to enter the dark compartment (entrance defined as all the body entering the dark compartment). The guillotine door closed as the rats entered the dark compartment. After spending 30 seconds, they were deleted from the dark compartment and placed in their cages; the second trial repeated after 30min precisely in the same way, and the experiment was pursued after the same pause by the first acquisition trial. In the acquisition trial, the guillotine door was picked up immediately after locating rats in the bright compartment, the entrance delay to the dark compartment or step-through latency to acquisition (STLa) was evaluated, and after entering rat to the dark compartment, the door closed, an electrical shock used (0.4 mA) for 1.5 seconds. Then the rats were returned to a separate cage after 30 seconds; this protocol repeated for two more minutes after 120 seconds. Whenever the rats reentered in the dark compartment, they have received a foot shock, and the protocol was repeated, but by remaining them in the light compartment for all two minutes continuously, training interrupted, and the number of trials was recorded as the number of acquisitions. Also, the step-through latency in the acquisition trial (STLa) was recorded (41).
2.7.3. Retention test
Long-term memory as a retention trial 24 hours after the PAL acquisition trial was carried out. Like the PAL training session, the animals took in the light compartment and immediately opened the door. Then, the step-through latency in the retention trial (STLr) and the time spent in the dark compartment (TDC) were recorded for 10 minutes. Through the retention test, there were no electrical shocks. If animals did not enter the dark compartment within 300 seconds, the retention test terminated, and a ceiling score of 300 seconds was recorded (43).
2.8. Nissl staining protocol
A day after experiments finished, three rats in every group chose and were deeply anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg) and perfused at first with normal saline and then 10% formalin for fixation through the heart and post-fixed in the same solution. After some months, formalin-fixed brain samples were placed in a tissue processor in a 21 hours protocol and washed with tap water. Ethanol dilution (70%,80%,90%, and 99% respectively) was used for dehydration, cleared in xylene, embedded in paraffin wax (60 °C) till being cold, and next sectioned with a microtome (Leitz GmBH, Wetzlar, Germany) to obtain five μm thickness section and finally collected on glass slides. In staining protocol, brain sample slides deparaffinized in xylene (20 minutes), rehydrated in descending alcohol (96%, 90%, 80%, and 70%, 5 minutes each solution), washed in tap water, stained with cresyl violet solution for 5 minutes, rewashed in tap water and destained in ascending alcohol solutions (70%, 80%, 90%, and 96%, (just washing)), and finally cleared in xylene. The number of intact hippocampal CA1 pyramidal cells in 1mm length counted.
2.9. Assay of hippocampus biochemical parameters
At the end of the study, after behavioral tests, five rats in every group chose and were deeply anesthetized by ketamine (100 mg/kg) and xylazine (10 mg/kg) and brain them extracted from the skull, then hippocampus separated and froze at −80 °C after washing in PBS. Next, samples were sent for biochemistry measurements. Finally, hippocampus measurements were performed for Super Oxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx) and Glutathione Reductase (GRx) as antioxidant enzymes concerning the current protocols (47-49).
2.10. Statistical analysis
Data expressed as mean ± standard error of the mean (SEM) and The Graph Pad Prism version 8.0 (Graph Pad Software, San Diego, CA, USA) applied for statistical analysis. All data were analyzed by two-way and one-way analyses of variance (ANOVA) followed by Tukey’s post hoc test. Results considered significantly different if P < 0.05.