Aβ1-42 (Sigma-Aldrich, USA) stock solution was diluted by PBS to a concentration of 1 µg/µl and was incubated at 37 °C for 7 days to allow the formation of fibril aggregation. SEW2871 was purchased from Cayman Chemical and dissolved in Dimethyl sulfoxide (DMSO) to the final concentration of 1 mg/ml.

The rats used in this study were males of the Wistar albino strain weighing 250-300 g that were housed in cages (four per cage) with ad libitum food and water. The animals were maintained under a controlled environment (12 h light-dark cycle with lights on from 07:00 to 19:00 h and 25 ± 2 °C room temperature). All animal manipulations were carried out according to the Ethical Committee for the use and care of laboratory animals of Shahid Beheshti University of medical sciences in compliance with the standards of the European Communities Council directive (86/609/EEC).

Animals were randomly divided into 4 groups as following (n=10 per each):

Control: the rats receiving intrahippocampal physiological buffer solution (PBS) (Aβ vehicle) injection and daily i.p. injection of DMSO (SEW2871 vehicle)

The reagents used for AD induction or to stimulate S1PR1 in animals, were administered according to experimental schedule summarized in Figure 1(Fig. 1).

Rats were anesthetized with chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic instrument (Stoelting, USA).

The following coordinates were used for bilateral intrahippocampal injection: AP: -3.84 mm, L: ± 2.2 mm and V: 2.5 mm according to the atlas of rat brain (Paxinos and Watson, 2007[44]). A solution containing 2 µg aggregated Aβ1-42 or PBS (for control and SEW groups) of the same volume was injected. Solutions were administrated via Hamilton microsyringe. All injections were made at the rate of 2 μl/4min. To minimize leakage of the solutions, the needle was kept in the position for 2 min after injection and the scalp was then closed with a suture.

Started on the first day after the stereotaxic surgery, rats were injected with i.p. DMSO (the SEW2871 solvent) or SEW2871 (0.5 mg/kg) for 14 consecutive days (Park et al., 2010[43]). At the end of drug treatment period (14^th day after surgery) animals were sacrificed.

The behavioral studies were carried out on days 9-14 after the surgery. A circular water tank (140 cm diameter, 55 cm high) was divided into four equally spaced quadrants. A transparent platform was set at the southeast quadrant of the tank, 1 cm below the surface of the water. Extra-maze cues such as some racks, bookshelves, pictures on the walls, surrounded the room where the water maze was performed. On day 9^th, rats were habituated to the pool by allowing them to perform a 120 s swimming without the platform.

The task was conducted for four consecutive days. In each trial (that constitutes four trains), a rat was placed in the water at one of the four starting positions, with the sequence of the positions being selected randomly. The rat was allowed to swim until it found the platform or till the 90 s had elapsed. In this latter case, the trial was terminated and the animal was put on the platform for 20 s. The distance moved (cm) and escape latency (s) to reach the platform was recorded by a computer.

On day 14^th, the platform was removed from the pool and animals underwent a 60 s spatial probe trial. The time spent (s) in the target quadrant that the platform was formerly located was recorded and compared between groups (Moradpour et al., 2006[38]).

After behavioral tests, rats were sacrificed and hippocampus (n=6) was used to determine the S1PR1-3 and cleaved caspase-3 expression by Western blotting method. Hippocampal tissues were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 % Nonidet P-40, 50 mM NaF, 20 mM EDTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 0.1 % SDS, and 2 mM phenylmethylsulfonyl fluoride). The homogeneate tissues were then centrifuged at 5,000 rpm for 10 min at 4 °C. The supernatants were collected as total protein extracts. Protein concentration was determined by using Bradford's method (Bradford, 1976[5]). Individual samples were subjected to SDS polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membranes. Blotting membranes were incubated with nonfat milk and then incubated overnight at 4 °C with the primary antibodies against cleaved caspase-3 (Cell Signalling, Beverly, MA, USA; 1:1000), S1PR1 (Abcam, USA; 1:1500), S1PR2 (Cayman Chemical, USA; 1:1500), S1PR3 (Abcam, USA; 1:1500). Then the membranes were incubated with secondary antibodies and processed with enhanced chemiluminescence reagent kit (Amersham Biosciences Europe GmbH, Germany). The images were scanned and analyzed semiquantitatively in a blind fashion using the Image J software. The expression of β-actin was used as an internal control for every experiment.

After the behavioral assessment, 6 rats of experimental groups were anesthetized and sacrificed by decapitation. The brains of animals were removed and one hemisphere was fixed in 4 % paraformaldehyde and embedded in paraffin. Serial coronal sections of 5 µm thickness were cut (beginning from 2.7 mm posterior to Bregma through the hippocampus), a total of 9 sections were collected from each brain and Nissl's staining was performed. Briefly, the sections were dipped into aqueous solution of cresyl violet (1 %) for 7 min and then washed with distilled water followed by placing in 95 % ethanol. The sections were then dehydrated in absolute alcohol, cleared and mounted with permount mounting medium and analyzed under the light microscope. The number of survived pyramidal cells was counted in a 1 mm length of CA1 region of hippocampus under ×40 magnification of light microscopy as performed previously (Xu et al., 2009[51]) and the number of neurons in this region was counted in different groups and percentage of neuronal loss was compared.

All data were expressed as mean ± SEM. Data were analyzed by using one-way ANOVA, followed by Tukey HSD post hoc analysis and P<0.05 was assumed to be statistically significant. All statistical studies were carried out with SPSS program (version 16.0, SPSS Inc., USA).

In order to determine the effect of intrahippocampal injection of Aβ1-42 on S1PRs protein levels, the expressions of S1PR1-3 proteins (that are highly expressed in the brain) were evaluated by Western blotting method. Compared with control, the expression of S1PR1 was reduced prominently in rats hippocampus two weeks after Aβ1-42 (P<0.001) (Figure 2A(Fig. 2)), while the expression of S1PR2 was significantly elevated then (P<0.05) (Figure 2B(Fig. 2)) and no change was observed in S1PR3 expression (Figure 2C(Fig. 2)).

The expression of S1PR1-3 was also determined in AD model rats chronically treated with SEW2871. According to the S1PR1-3 immunoblotting results, the expression of S1PR1 increased significantly in Aβ+SEW group compared with Aβ group (P<0.01) (Figure 2A(Fig. 2)). However, treatment with SEW2871 could not affect the S1PR2 and S1PR3 levels (Figure 2B, C(Fig. 2)).

Regarding the positive influence of the SEW2871 on S1PR1 expression in Aβ1-42 injected rats, we decided to examine the effects of chronic administration of SEW2871 on cognitive deficit in AD model rats.

In order to survey the spatial learning and memory performance in rats, the Morris water maze task was accomplished. Results showed that, comparing with control group, learning and memory ability of Aβ1-42 injected rats were significantly impaired. The average escape latency in searching for the hidden underwater platform, decreased with the increase of training days. Injection of Aβ1-42 caused a significant decline in spatial learning as indicated by significant increases in distance moved and escape latency (P<0.001) in searching for the underwater platform (Figure 3A, B(Fig. 3)). In addition, the time spent in the target quadrant (in the probe test) decreased significantly (P<0.001) (Figure 3C(Fig. 3)). These results showed that the application of Aβ1-42 impaired rat's spatial learning and memory. The effects of SEW2871 administration on spatial learning and memory are shown in Figure 3(Fig. 3). In the hidden platform test, administration of SEW2871 slightly improved the learning task in Aβ1-42 injected rats. The distance moved and escape latency to reach the hidden platform were slightly shorter in SEW2871-administrated AD model animals on 4^th day of training, compared with rats were injected with Aβ1-42 only (P<0.05) (Figure 3A, B(Fig. 3)). In addition, after removing the platform, the time spent in the target quadrant increased significantly in Aβ injected animals which received SEW2871 compared with AD rats that did not receive the drug (P<0.05) (Figure 3C)(Fig. 3).

Chronic administration of SEW2871 to PBS injected group resulted in no significant change in learning task, but caused deficit in the memory performance. In probe test, the time spent in the target quadrant in PBS injected rats which chronically received SEW2871 was reduced significantly, compared with control group (P<0.05) (Figure 3C(Fig. 3)).

The results of microscopic examinations of the hippocampus sections showed that Aβ injection reduces neuronal layer thickness in CA1 area of hippocampus and administration of SEW2871 prevents the Aβ-induced damage (Figure 4(Fig. 4)). Furthermore, intrahippocampal Aβ injection resulted in significant neural loss in CA1 area compared with control animals (P<0.001) which was markedly attenuated by SEW2871 treatment (P<0.05) (Table 1(Tab. 1)).

To suggest about the apoptotic pathways, the intracellular levels of cleaved caspase-3 was measured to estimate caspase-3 activation. The hippocampi Western blot analysis showed a significant increase in Aβ injected rats (P<0.001), while administration of SEW2871 in AD rats reduced the Aβ1-42 induced caspase-3 cleavage (p<0.05) (Figure 5(Fig. 5)). All together, these results suggest that both Aβ apoptotic effects and SEW2871 anti-apoptotic benefits may be mediated at least in part through caspase-3 modifications.