Enduring amnesia induced by ICV scopolamine is reversed by sesame oil in male rats 1

PURPOSE: To evaluated the long-term effect of scopolamine and sesame oil on spatial memory. METHODS: Memory impairment induced by Intracerebroventricular (ICV) injection of scopolamine hydrochloride (10 μg/ rat). Animals were gavaged for 4 weeks with saline, sesame oil (0.5, 1, or 2 mL/kg/day), or 3 weeks with memantine (30 mg/kg/day) in advance to induction of amnesia. Morris water maze (MWM) test was conducted 6 days after microinjection of scopolamine. Then, blood and brain samples were collected and evaluated for the malondialdehyde (MDA) levels, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities, and total antioxidant status (TAS) and ferric reducing ability of plasma (FRAP). RESULTS: Scopolamine significantly decreased traveled distance and time spent in target quadrant in probe test. Pretreatment of rats with sesame oil (0.5 mg/kg) mitigated scopolamine-induced behavioral alterations. Measurement of MDA, SOD, and GPX in brain tissue, and FRAP and TAS in blood showed little changes in animals which had received scopolamine or sesame oil. CONCLUSIONS: Intracerebroventricular injection of scopolamine has a residual effect on memory after six days. Sesame oil has an improving effect on spatial memory; however this effect is possibly mediated by mechanisms other than antioxidant effect of sesame oil.


Introduction
Alzheimer's disease (AD) is the most prevalent, severe, and disabling cause of dementia and an important cause of morbidity and mortality in the world [1][2][3] .Dementia of the Alzheimer type is a progressive, fatal neurodegenerative condition characterized by deterioration in cognition and memory, progressive impairment in the ability to carry out activities of daily living 4 .A number of neuropsychiatric symptoms are reported with AD such as problems with memory, thinking, and behavior 5 .Similarities in the memory deficits between Alzheimer patients and scopolamine treated animals have been reported 6 .Effects of cholinergic antagonists such as scopolamine are very like the destruction of cholinergic neurons in the hippocampus or performance associated with cognitive impairment caused by Alzheimer's disease 7 .Some studies have shown that oxidative stress plays an important role in the occurrence of neurodegeneration in Alzheimer's disease 8 .
Oxidative stress is a critical detriment factor stimulating neuronal cell death 9 .Recently it is stated that scopolamine-induced disorders are associated with changes in the state of oxidative stress in the brains of rats 10 .
Sesame oil is a strong antioxidant dietary source for human 11 .Previous studies have shown that sesame oil has improving effects on memory and learning.Consumption of sesame oil during pregnancy and lactation increased passive avoidance learning of rats' offspring 12 .Also, sesame oil improved learning and memory in diabetic rats and in a streptozotocin induced Alzheimer's model in rats 13,14 .It is believed that neurophysiological effects of sesame oil on the learning process may be due to its antioxidant effects 15 .
Since, scopolamine-induced memory impairment is associated with brain oxidative stress and oxidative stress is one of the risk factors in Alzheimer's disease 10 , we aimed to investigate the effect of sesame oil on scopolamine model of dementia.chosen based on a previous study 16 .

Morris Water Maze Test
The water maze apparatus was a black circular pool with a diameter of 130 cm and a height of 80 cm, filled with water 20-1°C to a depth of 60 cm.The maze was divided into four equal quadrants, and release points were designated at each quadrant as N, E, S, and W. A hidden circular plexiglas platform (10-cm diameter) was located in the center of the northeast quadrant, submerged 1.5 cm under the surface of the water.Fixed, extra maze visual cues were mounted at different locations around the maze.Performances were recorded with a video tracking system (HVS Image, Hampton, United Kingdom).
One week after induction of dementia, rats were trained and tested in the water maze according to our two days protocol.
On the first day, the single training session consisted of eight trials within two consecutive blocks (four trials in each block and 5 min intervals between the blocks).A probe trial was performed on the second day, consisted of 60 s free swimming period without a platform.Time spent and traveled distance in the target quadrant was measured.In order to assess animal's sensory and motor coordination or the animal motivation the capability of rats to escape to a visible platform was tested 5 min after completion of probe tests 18 (Figure 1).

Tissue processing and homogenate preparation
At the end of experiments, rats were deeply anesthetized with ketamin (60 mg/kg) and xylasin (6 mg/kg), blood samples were collected from heart and then animals were decapitated.
Immediately, brains were excised, frozen in liquid nitrogen and stored at deep freeze (-70°C) for later measurements.For antioxidant activities measurement, the brain samples were homogenized in 1.15% KCl solution.The homogenates were centrifuged at 1000 rpm for 1 min at 4°1 9 .Then, brain tissue homogenate were used for determination of MDA levels, and activities of SOD and GPX.
MDA, FRAP, and TAS of blood samples were also measured.

Measurement of oxidative stress
To determine the levels of lipid peroxidation, MDA as a marker of oxidative stress was used.MDA levels were measured using the thiobarbituric acid-reactive substances (TBARS) method 20 .Glutathione peroxidase activity in brain tissue and blood was measured according to Paglia and Valentine using Randox (Randox, United Kingdom) 21.Tissue superoxide dismutase was measured by a spectrophotometric method based on the inhibition of a superoxide-induced reduced nicotinamide adenine dinucleotide (NADH) oxidation according to Paoletti et al. 22 .

Statistical analysis
Results were analyzed using the SPSS version 16.0.Data for traveled distance and escape latency of block 1 and block 2 were compared by paired samples t-test.One-way analysis of variance (ANOVA) followed by the least significant difference (LSD) test was used to compare differences between means in more than two groups.Results are expressed as Means± SEM.The significant level was set at p<0.05.

Effect of scopolamine and sesame oil on Morris Water Maze task
During acquisition (first day of experiment including block 1 and block 2), escape latency and traveled distance indicated learning in locating the hidden platform, while more time spent and long distance traveled in the target quadrant in probe test (second day without platform) indicated better spatial memory.
Results of our study indicated that during acquisition, the performance of all groups improved with subsequent block of training.The Student t-test showed a significant difference in traveled distance and escape latency between the block 1 and 2 in all groups (Figure 2).The paired t-test for the traveled distance revealed a significant difference between two blocks in all groups; sham (p<0.05),scopolamine (p<0.05),sesame oil 0.5 ml/kg (p<0.001),sesame oil 1 ml/kg (p<0.05),sesame oil 2 ml/kg (p<0.01), and memantine (p<0.01)groups.The paired t-test for the escape latency revealed a significant difference between two blocks in all groups, in sham (p< 0.05), scopolamine (p< 0.01), sesame oil 0.5 ml/kg (p<0.001),sesame oil 1 ml/kg (p<0.05),sesame oil 2 ml/kg (p<0.01), and memantine (p<0.05)groups.enhancing effect on these parameters.Whereas, animals receiving 1ml/kg and 2 ml/kg doses of sesame oil and memantine did not show any significant differences with scopolamine group, indicating that memory has not improved in these group (Figure 3).

A B FIGURE 4 -Effects of scopolamine and different doses of sesame oil on traveled distances (A) and escape latency (B) in the visible platform test.
There were no significant differences between the various groups.

Effect of scopolamine and sesame oil on oxidative stress parameters
Measurement of serum and brain MDA levels 6 days after injection of scopolamine did not show a significant change.
The effect of sesame oil treatment on serum and brain MDA levels also was not significant (Table 1) except for the sesame oil with dose of 0.5 mg/kg that significantly (p<0.05)reduced brain SOD levels in comparison with sham and scopolamine groups and sesame oil with dose of 2 mg/kg that significantly reduced brain GPX activity in comparison with sham group.Three weeks of memantine pretreatment significantly (p<0.05).Asle Iranifam and colleagues also indicated that diet containing 10% sesame oil during pregnancy and lactation increased passive avoidance memory in rats' offspring 12 .Furthermore, findings of Hovayda's study showed that sesame oil increased learning in both castrated and intact animals 30 .
In the present study MDA levels (as a lipid peroxidation marker) of serum and brain tissue did not significantly change after six days in scopolamine treated animals.Also, GPX and SOD activities of the brain tissue were not significantly influenced by scopolamine.Three possibilities are likely in this regard.First, whole brain, nor cortex and hippocampus, was used for oxidative stress assays because their amount to measure various biochemical parameters was insufficient.Therefore, changes in the MDA and Also antioxidant assays on serum FRAP and TAS, and brain SOD and GPX did not show significant alterations except for sesame oil 0.5 mg/kg and 2 mg/kg that reduced SOD and GPX levels, respectively.Sesame oil is one of the best resources for polyunsaturated fatty acids (PUFA) 32 .Sesame oil protects against lipid peroxidation by increasing enzymatic and non-enzymatic antioxidant mechanisms 13 although in our study enzymatic mechanisms were negligible.Therefore, vitamin E (tocopherol) in sesame oil may have provided a non-enzymatic defense against the free radicals damage 33 .
Furthermore, in this study it is possible that oral administration of sesame oil has directly corrected scopolamineinduced cholinergic dysfunction 34 .Alternatively, sesame oil as a

Discussion
The present study showed that ICV scopolamine microinjection has long-lasting effects on the rats' Morris Water Maze performance.Scopolamine did not significantly change acquisition phase of learning, however the retrieval phase was significantly impaired.Also, four weeks of pretreatment with dose of 0.5ml/kg of sesame oil significantly reversed memory deficit induced by scopolamine and this indicates that sesame oil restored the spatial memory in demented rats.
Scopolamine-induced amnesia is a very well recognized animal model of memory dysfunction, widely used to test potential drugs of anti-Alzheimer's properties 23 .Enduring effect of scopolamine on memory impairment has been shown by Seifhosseini and colleague recently 24 .Scopolamine as a muscarinic cholinergic receptor antagonist impairs memory function 6 .It is well established that the cholinergic neurotransmission system in the basal forebrain plays an important role in learning and memory 25 .Also, destruction of cholinergic neurons or their function in the hippocampus is associated with cognitive impairment caused by Alzheimer's disease 26 .Moreover, scopolamine-induced amnesia is connected with increased oxidative stress in structures associated with learning and memory 27 .Oxidative stress, in turn, is a critical detriment factor leading to neuroinflammation and loss of cognitive function in Alzheimer's disease, Parkinson's disease, and multiple sclerosis 28 .Therefore, utilization of antioxidants or substances that boost cholinergic neurotransmission can improve learning and memory and may be useful in prevention and treatment of Alzheimer's disease.
In support of our findings, memory improving effects of sesame oil have been shown in several studies.Six weeks of feeding with a diet containing sesaminol glucosides protected mice against beta-amyloid peptide-induced cognitive deficits 29 .
Fathi and colleagues showed that 28-day pretreatment of rats with sesame oil prevented spatial memory deficit in streptoztocin-neuroprotective agent which contains sesamin and sesamolin may have acted on the membrane fluidity of hippocampal neurons and number of dendrite branches, neural synapses, and synapse efficiency 12,13,35.However, further investigation is required to clarify this issue.

Conclusion
Sesame oil protects against cognitive deficits induced by intracerebroventricular scopolamine in rats, and this effect may be mediated by mechanisms other than its antioxidant properties.
used in this study were provided by the colony of Tabriz university of Medical Sciences.Adult male Wistar rats (200-250g) were housed in standard cages in a temperature controlled room (22-24ºC), humidity (40-60%), and light period (12h dark-12h light).Food and water were available ad libitum.All experiments were performed in agreement with guidelines of the Tabriz University of Medical Sciences for care and use of laboratory animals.Sixty male Wistar rats were randomly divided into six (n=10) experimental groups: • Sham-operated controls • Scopolamine group: these animals received ICV injection of scopolamine (10 μg) • Scopolamine-sesame oil groups: these animals received sesame oil (0.5, 1 or 2 ml/kg) for 4 weeks before scopolamine injection • Scopolamine -memantine group: these animals were treated with memantine (20mg/kg/day) for 3 weeks before injection of scopolamine Dose and duration of treatment with memantine were

FIGURE 1 -
FIGURE 1 -Time line of sesame oil treatment, behavioral test, and samplings.MWM (Morris Water Maze)

FIGURE 2 -
FIGURE 2 -Effects of scopolamine and different doses of sesame oil on traveled distance (A) and escape latency (B) to find the hidden platform.Each block represents the average of four consecutive trials.*p<0.05,**p<0.01,and***p<0.001indicates the difference between Blocks 1 and 2 (b1 and b2) in each group.

FIGURE 3 -
FIGURE 3 -Effects of scopolamine and different doses of sesame oil on the percentage of traveled distance (A) and time spent (B) in target quadrant.Values are the mean ±SEM (n= 8-10).*p<0.05 and **p<0.01indicates the difference relative to the control group and # p<0.05 indicates the difference relative to scopolamine group.
enzyme levels are not exclusive to learning and memory related structures.Second, non-enzymatic adaptive changes by the body; such as vitamin E mobilization 31 , may have counteracted the oxidative stress.Third, MDA and enzyme levels have returned to normal values by physiological body neutralizing scopolamineinduced oxidative stress after six days.Sesame oil pretreatment in the present study did not significantly change MDA levels of serum and brain tissue.

TABLE 1 -
Effect of sesame oil on biochemical parameters of serum and brain tissue six days after ICV injection of scopolamine (One-way ANOVA analysis).