Animals and experimental design
Healthy adult male Wistar rats (230–250 g) were prepared from Hamadan University of Medical Sciences. Environmental conditions in the animal included the temperature of 22 ± 2°C and the optical cycle was 12 hours of light and 12 hours of darkness (from 7 am to 7 pm). The rats were given adequate water and food during the experiment and all tests were performed throughout the day. The research protocol was confirmed by the Animal Ethics Committee Guidelines for the Use of Experimental Animals (IR.BASU.REC.1398.029), following the “NIH Guide for the Care and Use of Laboratory Animals”.
Adaptation to the environment was done one week the experiments and then, animals were randomly divided into seven experimental groups: (n=6-7):
- Control group: This group had access to food and water indefinitely and did not undergo AD induction.
- Sham group: The rats received phosphate-buffered saline (intracerebroventricular (ICV); 5 μL).
- AD group: The rats received Aβ1-40 (5 μL; ICV).
- PCO group: 50 mg/kg of PCO was given once daily by oral gavage for 8 weeks.
- Acacia gum group (vehicle): 50 mg/kg of acacia gum was given once daily by oral gavage for 8 weeks.
- AD + acacia gum group: The rats received Aβ1-40 (5 μL; ICV) and 50 mg/kg of acacia gum was given once daily by oral gavage for 8 weeks.
- AD + PCO group: The rats received Aβ1-40 (5 μL, ICV) and 50 mg/kg of PCO was given once daily by oral gavage for 8 weeks.
Clinical dose of Policosanol: or Administration and dosage
Administration of PCO was done at 50 mg/kg body weight (Guerra et al., 2015). PCO is a water-insoluble substance (Luz et al.), and it was prepared by oral suspension with the use of acacia gum, which is a solvent of PCO (Guerra et al., 2015; Elseweidy et al., 2016). The rats were then gavaged by this suspension for 8 weeks (Elseweidy et al., 2016).
Aβ injections and surgery
Aβ1-40 (100 µg; Tocris Bioscience, Bristol, UK) was dissolved in 100 µL of PBS (vehicle solution), followed by incubation (37◦C / 7 days) before usage. As a result of this process, neurotoxic amyloid fibrils were obtained (Lorenzo and Yankner, 1994; Yaghmaei et al., 2013). The rats to generate an AD model were anesthetized using ketamine and xylazine (100 and 10 mg/kg, respectively) and transferred to the stereotaxic apparatus (Stoelting Co., Wood Dale, IL, USA). Using an electrically shielded heating pad, the rats’ body temperature was kept at 37.0 ± 0.2◦C during Aβ injection. Their skulls were uncovered and over the ventricular regions, the holes were drilled based on the coordinates of the appendix: 2 mm lateral to the midline, 1.2 mm posterior to bregma, and 4 mm ventral to the surface of the cortex (Paxinos and Watson, 2005). The rats were injected (1 μL/min) slowly using a 5 µL microsyringe (Hamilton Laboratory Products, USA). The injections lasted for 5 min and then, syringes were left untouched for 5 min after the injection and removed painlessly. The sham group received a vehicle solution. The recovery for rats lasted for 7 days (Asadbegi et al., 2017; Ahmadi et al., 2021a; Ahmadi et al., 2021b) (Fig.1).
Locomotor activity in the open field (OF)test
Locomotor activity was assessed using the OF test. The apparatus is made of white acrylic with a surface area of 50 × 50 cm, and the walls are 38 cm in height. The field is lit by low ambient room lights (Bisagno et al., 2004). An overhead video camera recorded the time spent by rats in the central and peripheral zones and the data were analyzed through a video track software. The square-shaped central zone locates 25 cm from each wall. Animals were located in the middle of the central zone and could explore for 10 min (Drews et al., 2005). The total distance moved (locomotor activity) and average velocity in the apparatus were measured (Lalonde et al., 2003).
Novel object recognition test (NOR)
This test measures the visuospatial memory of rodents (Hansen et al., 2010; Ganji et al., 2017). Adaptation to the apparatus (60 × 60 × 45 cm) was done 24 h prior to test by placing animals in the device for 20 min. After 24 h, we placed two identical objects in the apparatus and the animals were placed independently in the middle part and close to the walls, and their heads were fixed to be opposite to the objects. In this phase, animals were given 10 min to explore the objects, and then they were transferred to their cages. After 24 h, one of the familiar objects was replaced with a new one, and the animals were placed in the device with a new and familiar objects for 10 min (Cohen and Stackman Jr, 2015). A video camera recorded this phase. The discrimination index was considered as the time taken to explore the new object to the total time spent with both objects. The experiment timeline represents the time taken exploring both objects. Objects were presented randomly between the groups and rats. Cleaning of the objects and the box was done during intervals using 70 % ethanol to get rid of olfactory cues (Hansen et al., 2010).
Morris water maze test
Spatial memory was tested using Morris water maze (MWM), which is a black circular pool with a diameter of 180 cm and a depth of 60 cm filled with water (22±1°C) (42 cm of depth). The pool has four quadrants and starting sites with an equal distance from each other called north, east, south, and west. There is an invisible platform (diameter: 10 cm) that is 1 cm below the water in the northern quadrant center. Training sessions were performed from 10:00 AM to 13:00 PM for four days, in which two blocks with four trials were considered. In the training phase, each rat could swim to find the invisible platform for 90s. Training was done from all starting sites. The rats could stay on the platform for 30 s between the two trials. A 5-min resting time was considered between two blocks. The parameters, such as time spent to reach the platform (escape latency) and traveled distance were recorded using a video camera (Nikon, Melville, NY, USA) installed above the pool and attached to a tracking software. The probe trial was conducted on day 5, on which the platform was removed and animals could swim for 60s. Then, we recorded the time spent in the target quadrant (Zarrinkalam et al., 2016; Zarrinkalam et al., 2018).
Passive avoidance learning (PAL) test
Passive avoidance apparatus
A step-through device measured passive avoidance learning (PAL) and memory (Zarrinkalam et al., 2016), which has two light (transparent plastic) and dark (dark opaque plastic) compartments (both 20 cm × 20 cm × 30 cm) . Both chambers have a floor covered by stainless-steel rods (3 mm in diameter) spaced 1 cm apart. A shock generator (Borj Sanat, Tehran, Iran) electrifies the floor of the dark compartment. A rectangular opening guillotine door (6cm×8cm) separates two compartments (Komaki et al., 2015; Shiri et al., 2017).
Passive avoidance training
The habituation phase was done by giving the groups two primary trials. After placing the animals in the light compartment facing away from the door, the guillotine door was raised after 30 s. Rats prefer dark environments. Following the entrance of the rats to the dark chamber, the door was closed and 30 s later, they were transferred to their cages. This trial was repeated after 30 min, and 30 min later, the first acquisition trial was done. The latency to enter the dark chamber (step-through latency, STLa) was recorded after placing all four paws in the dark chamber. After entrance to the dark chamber, the guillotine door was closed and the animal received an electrical shock (50 Hz, 1.5 s, 0.4 mA intensity). After 30 s, the animal was transferred to its home cage and the process was repeated after 2 min. The training was finished when the animal remained in the light chamber for 120 s. The number of light-dark transitions was also noted (Zarrinkalam et al., 2016; Shiri et al., 2017).
The retention test was conducted 24 h following the acquisition trial. Animals were located in the light compartment and after 30 s, the door was raised. The STLr and time spent in the dark compartment (TDC) were noted for 300 s. When the rats did not enter the dark chamber during 300 s, the retention test was finished and the animal received a ceiling score of 300 s (Barzegar et al., 2015; Zarrinkalam et al., 2016).
After all behavioral tests, 5 ml of portal vein blood specimens were collected into heparinized tubes. The specimens were then centrifuged (3500 rpm / 10 min / 4°C) and serums were frozen at −80°C and transferred for biochemical assessments. Finally, total antioxidant capacity (TAC) and total oxidant status (TOS) were determined.
After all experiments, rats were deeply anesthetized using urethane and perfused via the heart using formol–saline (Komaki and Esteky, 2005; Komaki et al., 2007). Regarding Congo red staining, hippocampal coronal sections (5 μm) were prepared. Then, the slides were assessed using an optic microscope and Image J software. Congo red staining was applied to indicate Aβ plaque generation in the brain tissue (Mirzaei et al., 2018).
Data analysis was done by one-way and two-way analysis of variance (ANOVA), followed by Tukey's post-hoc test to compare groups. Data are presented as mean ± SEM. Statistical signiﬁcance was set at P < 0.05.