The Neuroprotective Effects of Policosanol on Learning and Memory Impairment in a Male Rat Model of Alzheimer's Disease

Alzheimer's disease (AD) as a neurodegenerative disease is recognized with progressive cognitive function failure, which is determined by beta-amyloid (Aβ) accumulation in extracellular space and hyperphosphorylation of intracellular Tau protein. Aβ stimulates some kinds of active oxygen and causes oxidative stresses and apoptosis. Policosanol (PCO) is a reducing lipid complement, which has antioxidant and anti-inammatory activities. In the current research, the PCO effects on learning and memory impairment were investigated in a rat model of AD. Healthy adult male Wistar rats (230–250g) were divided randomly into 7 groups (n=6-7): Control, Sham (5 µL of phosphate-buffered saline, intracerebroventricular (ICV) microinjection), AD model (5 µL, ICV injection of Aβ), acacia gum (50 mg/kg, 8 weeks, gavage), PCO (50 mg/kg, 8 weeks, gavage), AD + acacia gum (50 mg/kg, 8 weeks, gavage), and AD + PCO (50 mg/kg, 8 weeks, gavage). Passive avoidance learning (PAL) and memory were assessed by shuttle box, cognitive memory by novel object recognition (NOR), and spatial memory by the Morris water maze (MWM) test. The oxidant and antioxidant parameters were examined at the end of the experiments. According to our results, ICV injection of Aβ caused reduced cognitive memory in NOR, spatial memory in MWM, and passive avoidance in PAL tests. PCO caused a recovery in cognitive memory, spatial memory, and PAL memory. Aβ plaques increased in the AD group, while PCO decreased it. Aβ increased total oxidant status and decreased total antioxidant capacity, whereas PCO reversed these parameters. Our results demonstrated that PCO has neuroprotective effects and can protect learning and memory impairments via its hypolipidemic and antioxidant effects.


Introduction
Alzheimer's disease (AD) as the sixth leading cause of death, is a multifactorial and progressive disorder (Reitz, 2012). It affects certain areas of the brain (Wenk, 2003). One of the neuropathological features of this disease may be the accumulation of neuronal plaques called beta-amyloid (Aβ) plaques and neuro bril coils resulting from the accumulation of microtubule-dependent protein hyperphosphorylation, such as intracellular Tau protein (Huang and Jiang, 2009). In AD, memory and learning can be impaired (Barone et al., 2014). AD affects the neurons, and consequently thinking, memory, and behavior and it has a signi cant effect on work and social life (Singhal et al., 2012;Klimova et al., 2015). However, the exact AD etiology and pathogenesis are still unknown (Tanzi et al., 1996;McNeilly et al., 2012). Oxidative stress has been shown associated with cognitive disorders, such as AD (Halagappa et al., 2007).
The important effect of oxidative stress as the main cause on AD pathogenesis has been reported (Andersen, 2004;Butter eld et al., 2006). In this regard, oxidative stress causes several neurological diseases, like Parkinson's disease, AD, and amyotrophic lateral sclerosis (Rojas and Gomes, 2013). The critical function of oxidative stress in the brain of AD cases has been also been indicated ( An unhealthy lifestyle leads to an increase in the incidence of obesity and hypertension, which are components of metabolic syndrome, which can be linked to AD (Halagappa et al., 2007). Metabolic defects lead to functional modi cations associated with cerebral aging and AD pathogenesis (Chen et al., 2016). Obesity and a diet high in fat are associated with cognitive impairment (Pistell et al., 2010; Kanoski and Davidson, 2011; Moy and McNay, 2013). It has been shown AD is highly observed in states, in which the consumption of diets high in and calorie is high ( Octacosanol, as a main constituent of the PCO, is very effective in lowering LDLs and increasing highdensity lipoproteins (HDLs), and also increases athletic performance (Taylor et al., 2003;Ma et al., 2018).
According to the various properties of PCO, including antioxidant, anti-in ammatory, cholesterol-lowering properties, and the fact that the effects of PCO on the treatment of AD have not yet been addressed, its effects on AD were assessed in the present experiment. We investigated the possible therapeutic effects of PCO as a therapeutic or protective compound in Aβ-related learning and memory impairment in an animal model of AD.

Material And Methods
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 con rmed 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): 1. Control group: This group had access to food and water inde nitely and did not undergo AD induction.
5. 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.

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 brils 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.

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 xed 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 lled 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 nd 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 oor covered by stainless-steel rods (

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 rst 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.

Retention test
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

Biochemical analysis
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.

Histology
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
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.

Effect of PCO and Aβ on locomotor activity in the open eld test
Comparing the mean velocity and the distance moved showed no signi cant difference between the different groups (distance moved: F (6, 55) = 2.018, P=0.0786 and mean velocity F (6, 55) = 2.015, P=0.0791). Also, the motor activity did not change signi cantly after Aβ injection and PCO did not affect the motor activity (Fig. 2).

Effect of PCO and Aβ on NOR test
DI as an index of the NOR test is considered as the time spent to explore the new object divided by the total time to explore both familiar and new objects on the second day of the test (F (6, 40) = 8.204, P<0.0001). The time spent around the new object in the AD group decreased signi cantly in comparison with the control and sham groups (P<0.001). A signi cant decrease was detected in DI of the AD group in the comparison with PCO and acacia gum groups (P < 0.001). A signi cant increase was found in DI in the AD+PCO group in comparison with the AD group (P < 0.01) (Fig. 3).

Effect of PCO and Aβ on MWM test
The escape latency and the distance moved to reach the invisible platform on the rst to fourth days of training were the criterion for learning in animals. In this period, the AD group showed a signi cant increase in the time spent to nd the invisible platform compared with the control and sham groups. During this four-day learning period, signi cant differences were observed between the escape latency in the AD rats and the control and sham groups (( rst day: P < 0.05), (second day: P < 0.0001), (third day: P < 0.001, P < 0.01), and (the fourth day: P < 0.001 and P < 0.0001, respectively)). Also, a signi cant difference was detected between the AD and PCO group on the second to fourth days (P < 0.01, P < 0.01, and P < 0.0001, respectively), in the acacia gum group on the second, third, and fourth days (P < 0.05, P < 0.01, and P < 0.0001, respectively), and in the AD+PCO group on the second and fourth days (P < 0.05) (Fig. 4A). The distance traveled to nd the invisible platform in the AD group during the training days on the third and fourth days was signi cantly different from the other groups tested (Fig. 4B). The average time spent in the target quadrant on the test day (the fth day) was measured in the probe trial. The AD group had the least time spent in the target quadrant than all groups, but this parameter was signi cantly different between the AD group and other groups (Control, P < 0.01; PCO, P < 0.001; and AD+PCOP < 0.01) (Fig. 4C).

Effect of PCO and Aβ on PAL test
Comparing the latency to enter the dark compartment in the compromise stage (STLa) indicated is no signi cant difference between the groups, which indicates that the rats did not differ from each other in terms of entering the dark chamber before the shock (F (6, 38) = 1.825, P=0.1202) (Fig. 5A). The groups showed no signi cant difference regarding the number of shocks received until the learning criteria were met (NTa) (F (6, 59) = 2.682, P=0.0228) (Fig. 5B).

Effect of PCO and Aβ on TAC and TOS
TOS is considered an oxidative indicator. As illustrated in Fig. 7A, PCO and AD+PCO groups were found with a markedly lower TOS level in comparison with the AD group (F (6, 35) = 9.379, P<0/0001). In general, the AD group had a signi cantly higher concentration of TOS compared with other groups. TAC is considered an antioxidant indicator. The AD group was found with a significantly lower TAC mean level in the plasma than the PCO group (F (6, 31) = 6.064, P=0.0003) (Fig. 7B).

Effects of PCO on brain Aβ plaques
To approve generation of Aβ plaques animals' brains, Congo Red staining was done. Fig. 8 indicates the Aβ plaques (red spots) in the hippocampal coronal sections. These plaques were found in the brain sections related to the Aβ group. After staining, the PCO-treated Aβ rats were found with a signi cant decrease in Aβ plaque deposits in than the Aβ rats. No signi cant plaque was detected in the control and sham groups.

Discussion
In the current research, we studied the effects of an ICV injection of Aβ (1-40) on learning and memory, under the in uence of PCO as a cholesterol-lowering, anti-in ammatory, and antioxidant supplement in adult male rats. Our ndings showed that the use of PCO is not effective in motor activity in the open eld test in all groups. Consumption of PCO in AD male rats improved cognitive memory evidenced by the NOR test, spatial memory con rmed by the MWM test, and PAL and memory evidenced by the shuttle box test. Aβ plaques increased in the AD group, while PCO decreased the plaques. The ICV injection of Aβ increases TOS, which indicates an increase in and induction of oxidative stress, whereas the use of PCO increases TAC, which indicates an increase in antioxidant properties.
Prior to the behavioral tests, the rat's motor activity was assessed in an open eld test, and our ndings showed that PCO had no effect on motor activity. According to our previous ndings, cinnamaldehyde with antioxidant properties had no effect on the animal's motor activity (Etaee et al., 2019). Also, the chronic use of Cyanidin-3-glucoside in diabetic rats showed that it does not signi cantly alter the motor activity of animals (Nasri et al. Our results showed that memory impairment induced by Aβ was associated with a reduction in antioxidant capacity and an increase in oxidative stress. Evidence suggests that Aβ may directly impair mitochondrial function, and also energy de ciency and neuronal death can be seen in AD patients (Du and Yan, 2010). PCO by increasing TAC levels and its antioxidant properties to some extent prevents the effects of Aβ. The brain is susceptible to oxidative stress because of its low level of antioxidants and cell membrane lipids; thus, injecting Aβ (which leads to the induction of AD The PCO hypoglycemia effect may be due to the AMP-kinase activation that is similar to the mechanism, by which metformin (Met) acts. It activates the glucose absorption into the skeletal muscle, inhibits hepatic gluconeogenesis, and ultimately reduces circulating fat (Zhou et al., 2001; Shaw et al., 2005). In this regard, it has been shown that the pretreatment by Met via its neuroprotective effect can prevent the impaired synaptic plasticity caused by Aβ (Asadbegi et al., 2016). The biguanide Met, as a rst-line antidiabetic treatment for type 2 diabetes, can act as an insulin sensitizer and reduce blood glucose through an increase in glucose uptake into muscles and a reduction in liver gluconeogenesis by activating AMP-activated protein kinase (AMPK) (Campbell et al., 2017). Met is also a possible treatment for dementia by reducing pTau (Farr et al., 2019). It also signi cantly reduces in ammatory markers, like TNF-α and CRP. The hypoglycemic effect of PCO is mainly related to AMP-kinase activation, which is similar to the mechanism used by Met (Elseweidy et al., 2016).

Conclusion
In summary, our ndings obtained from the NOR, MWM, and PAL tests showed that Aβ impairs learning and memory, while PCO eliminates learning and memory impairment following the Aβ injection and even improves memory. The results of biochemical studies also showed that Aβ increases the TOS levels and decreases the TAC levels and vice versa, the consumption of PCO reduces the TOS levels and increases the TAC levels. These results indicate the antioxidant properties of PCO. Aβ plaques increased in the AD group, while PCO decreased these plaques. The effects of PCO on learning and memory can be due to its antioxidant, hypolipidemic, and anti-in ammatory properties.

Declarations
Compliance with Ethical Standards

Ethical Approval
The experiments were carried out according to Guidelines of the National Institutes of Health on the principles of laboratory animal care (NIH Publication 80-23, 1996). The Local Ethical Committee approved all planned experimental procedures.

Con ict of interest
The authors declare that they have no con ict of interest. The experimental timeline. Following 2 weeks of acclimatization, to generate an AD model, the rats in the experimental groups were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) and placed in a stereotaxic device. The Aβ solution (5 μL; 1 μL/1 min) was injected intracerebroventricularly (ICV).
Following recovery, policosanol (PCO) was received by animals by gavage daily for 8 weeks. Then, the open eld and novel object recognition (NOR) tests were performed. To measure spatial (acquisition and retention) and aversive (acquisition and retention) learning and memory following the training programs, the MWM and shuttle box tests were used, respectively. After the experiments, the biochemical parameters and the concentrations of the biomarkers of oxidative stress were calculated by serum assessment.

Figure 2
Comparing the mean velocity and the distance moved between groups. Data are represented as mean ± SEM. No signi cant difference was detected between groups (n=6-7).

Figure 4
The time spent to reach the hidden platform (latency) (A). The entire distance moved to reach the hidden platform (total distance) (B). The mean time spent in the target quadrant on the test day (C). Data are presented as mean ± SEM. ** p<0.01 and **** p<0.0001 versus the control group; &&& p<0.001 and &&&& p<0.001 versus the sham group; # p < 0.05, ## p < 0.01, and #### p < 0.0001 versus the AD group (n=6-7).

Figure 6
The effect of policosanol (PCO) administration after the ICV injection of amyloid-beta (Aβ). Comparing latency to enter the dark compartment on the test day (STLr) (A) and the time spent in the dark compartment on the test day (TDC) (B). Data are presented as mean ± SEM. * p < 0.05 versus the control group; && p<0.01 versus the sham group; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the AD group (n=6-7).