Male Sprague-Dawley rats (n=50, weighing 250-330 g) were used in this study. Rats were maintained under controlled hygienic conditions with a 12 hr light/dark reverse cycle at a constant temperature with free access to food and water. All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (Permit #: 10-115).

For each experiment, rats were given anesthesia [chloral hydrate 300 mg/kg, intraperitoneal (i.p)], followed by a single injection of STZ (1.5 mg/kg, dissolved in a vehicle consisting of 0.05 M citrate buffer) bilaterally into each lateral ventricle (5 µL/ventricle). Control animals were given an equal volume of intracerebroventricular (icv) vehicle via the same procedure. Agm was purchased from Sigma (Sigma, St. Louis, MO, USA), dissolved in normal saline (pH 7.4), and administered to rats via an intra-peritoneal route. Rats were injected with Agm (100 mg/kg) daily for up to two weeks after surgery. The concentration of Agm (100 mg/kg) was determined based upon the results of our previous study.25

The sham group (n=15) received bilateral icv injection of saline, 5 µL in each rat. The rats in the EC group (n=17) were given injection of STZ-icv (1.5 mg/kg, 1st and 3rd days after surgery) (Sigma, St. Louis, MO, USA) bilaterally.40 Rats in the STZ-Agm group (n=18) were given 5 uL injection of STZ-icv (1.5 mg/kg, 1st and 3rd days after surgery) (Sigma, St. Louis, MO, USA) bilaterally and treated with Agm [100 mg/kg, intraperitoneal (i.p.) daily for two weeks from the 1st day after surgery]. The heads of the rats were positioned in a stereotactic frame (coordinates of 1.5 mm posterior to the bregma, 1.5 mm lateral to the sagittal suture, 2.5 mm ventral to the surface of the brain).

Cognitive evaluation of rats was tested using a Morris water maze.41 Before conducting the Morris water maze test, we conducted pre-training. For all trials, a single rat was placed in a pool, facing the wall, at a different starting point (NW, NE, SW, or SE), and was then allowed to swim for a maximum of 60 seconds or until it reached the platform. If the rat failed to find the platform during the trial, it was manually guided to the platform by the investigator and placed on top of it for 15 seconds. Next, an apparatus consisting of a circular pool (200 cm diameter, 60 cm high) filled with water (depth 30 cm; 24±1℃) was placed in a room with consistently positioned spatial cues. An escape platform (15 cm diameter) was placed in the middle of one of the quadrants, 1.5 cm below the water surface, equidistant from the sidewall and the middle of the pool. The time required to reach the platform (escape latency) was measured in each trial. After the acquisition stage, a probe test was performed after removing the platform. We measured the latency up to the point when the rat traversed the location where the platform used to be as a measure of memory.

Rats were anesthetized and perfused as previously mentioned.42 Rat brains were cut into coronal slices of 2 mm thickness using a rat brain matrix (Ted Pella, Redding, CA, USA). Next, the brain slices were fixed with 4% paraformaldehyde (pH 7.4) for 3 days and subsequently embedded in a paraffin block. The paraffin blocks containing the hippocampus and cortex were deparaffinized and re-hydrated with different concentrations of alcohol and xylene. After hematoxylin and eosin (H&E) staining, stained hippocampus and cortex sections were examined under a microscope.

Five µm thick frozen brain sections were harvested onto clean glass slides (Thermo Scientific, Bremen, Germany), air-dried, and fixed in cold acetone for 10 minutes at -20℃. The slides were washed first in Tris-buffered saline (TBS) and then incubated with 0.3% H₂O₂ in methanol to quench endogenous peroxidase activity. Followed by a series of washes (three times with distilled water), the sections were blocked with 10% normal rabbit serum. Frozen brain sections (20 µm) were fixed in ice-cold acetone for 20 minutes. To block specific staining, sections were incubated in 5% bovine serum albumin (BSA) (Sigma-Aldrich, Sigma, St. Louis, MO, USA) diluted in PBS for 30 minutes before addition of primary and secondary antibodies. Primary antibodies for phosphorylated insulin receptor substrate-1 (p-IRS-1) (1:200, Santa Cruz, CA, USA), 8-oxo-2'-deoxy-guanosine (8-OHdG) (1:200, Chemicon, Billerica, MA, USA), and anti-Aβ (1:200, Millipore, Billerica, MA, USA) were applied to the samples for 24 hours at 4℃, followed by a 90-minute incubation with appropriate florescence secondary antibody (1:100, Invitrogen, Carlsbad, CA, USA) and three washings in PBS for 10 minutes each. After three washes in 0.1% phosphate buffered saline with Tween-20 (PBST), the sections were incubated with rhodamine-conjugated sheep anti-rabbit or sheep anti-mouse secondary antibody that was diluted to 1:200 with 5% BSA fraction V in 0.1% PBST for 2 h in the dark at room temperature. After three washing in PBS, all sections were incubated with 1 µg/mL of 4',6-diamidino-2-phenylindole (Sigma-Aldrich, Sigma, St. Louis, MO, USA) and 2 µg/mL of propidium iodide (Sigma-Aldrich, USA) for a counter staining. Tissues were then visualized under a confocal microscope (Zeiss LSM 700, Carl Zeiss, Thornwood, NY, USA).

For Western blot analysis, rats from all groups were sacrificed and their brains were perfused with saline through the heart aorta to rinse away traces of blood. Next, the portion of the brain comprising the hippocampus and cortex was dissected for extraction of proteins and treated with lysis buffer (1% Triton X-100, 0.5% NP-40, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM Ethylene-di-amine tetra acetic acid, 1 mM ethylene glycol tetraacetic acid (pH 8.0), 0.2 mM sodium orthovanadate, 0.2 mM phenyl methyl sulfonyl fluoride, and protease inhibitor cocktail). Isolated proteins were centrifuged at 12000 rpm at 4℃. Equal amounts of protein (20 µg) from the supernatants were separated on a 10% acrylamide gel and proteins were electrophoretically transferred onto nitrocellulose membranes. After blocking with 5% skim milk for 2 hrs, the membranes were incubated with primary antibodies against Bcl2 (1:1000, Santa Cruz, San Jose, CA, USA), Bax (1:1000, Santa Cruz, San Jose, CA, USA), cleaved caspase-3 (1:1000, Cell signaling, Billerica, MA, USA), PI3K (1:2000, Millipore, Billerica, MA, USA), Nrf2 (1:200, Santa Cruz, San Jose, CA, USA), γ-GCS (1:500, Thermo Scientific, Bremen, Germany), and β-actin (1:1000, Millipore, Billerica, MA, USA) at 4℃ overnight. Later the membranes were washed three times for 5 min each with TBST. The detection of secondary anti-rabbit and anti-mouse antibodies (1:3000, New England Bio labs, USA) was conducted for 1 hour at room temperature. After washing with PBST (0.05% with Tween 20) three times, immunoreactive signals were detected by chemiluminescence with an ECL detection system (Amersham Life Science, London, UK) using the LAS 4000 program.

Statistical analyses were carried out using SPSS 18.0 software (IBM Portsmouth, IBM North Harbour, Portsmouth, UK). All data are expressed as means±S.E.M. Statistical significance in intergroup differences was determined by one-way analysis of variance, followed by Scheffe's post hoc multiple comparison test. Each experiment included at least three replicates per condition. Differences with a p value less than 0.05 were considered statistically significant.

To determine the accumulation of Aβ, on the 21st day following STZ injection, Aβ staining was performed in the sham, EC, and STZ-Agm groups. The expression of Aβ was considerably decreased in the STZ-Agm group compared to the EC group (Fig. 1A). To confirm phosphorylation of IRS-1 by Agm treatment in STZ-icv rats, we conducted immunohistochemistry using phospho-IRS-1 antibody, because IRS-1 plays a key role in transmitting signals from the insulin receptors to intracellular pathways. In the STZ-Agm group, both hippocampus (Fig. 1B) and cortex regions (Fig. 1C) showed an increase in IRS-1 phosphorylation compared to the EC group. These data indicated that Agm treatment could promote IRS-1 phosphorylation in STZ-icv rat models.

In previous studies, Morris water maze tests have been commonly applied for the assessment of cognition and memory functions. To confirm the enhancement of memory function in our STZ-induced Alzheimer rat model upon treatment with Agm, we conducted Morris water maze tests. The swimming times of four trials per day for 5 days in each group are shown in Fig. 1. Escape latency time (days 1-5) (to find a hidden platform) was significantly prolonged in the EC group compared to the sham group (Fig. 1D). Fig. 1D shows that the STZ-Agm group animals presented a significantly lower latency to find the platform than the EC group. The animals of the STZ-Agm group demonstrated improved Morris water maze acquisition performance. Our findings suggest that Agm treatment could improve learning and memory dysfunction in STZ-induced Alzheimer rats.

To investigate morphological differences in cells, we conducted H&E staining. The H&E staining of Cornu Ammonis (CA)1, CA2, and CA3 regions of the hippocampus revealed a large number of degenerated cells in the EC group compared with STZ-Agm group (Fig. 2A), which were evidenced by a decrease in the number of H&E stained cells. The EC group exhibited greater shrinkage of the nuclei of cells than those in the STZ-Agm group. Fig. 2B shows that the number of abnormal cells in the cortical region were higher in the EC group than the STZ-Agm group. Fig. 2 indicates that Agm treatment reduces STZ-induced histological abnormalities in hippocampus and cortex regions, compared with the EC group.

To confirm apoptotic cell death in the EC group and the STZ-Agm group, western blot analysis was conducted to check expression of apoptotic proteins. Fig. 3A indicates that the presence of cleaved caspase-3, an active form of caspase-3, was attenuated in the STZ-Agm group compared with the EC group. Additionally, in the present study, the anti-apoptotic effect of Agm was investigated by checking the expression of proteins, such as Bax, Bcl2, and PI3K, using western blotting. Among the quantitative western blot results, the expression of Bax, known as a pro-apoptotic protein, was higher in the EC group than the STZ-Agm group (Fig. 3C). In contrast, Bcl2, known as an anti-apoptotic protein, was expressed more in the STZ-Agm group than the EC group (Fig. 3B). Also, the STZ-Agm group showed increased expression of PI3K, known to be related to survival pathways and insulin signal transduction, compared with the EC group (Fig. 3D). Fig. 3 suggests that Agm treatment inhibits cell death signaling in STZ-induced Alzheimer rats.

To detect the generation of Reactive Oxygen Species (ROS), which causes DNA damage to the cells, we conducted 8-OHdG staining. The immunohistochemical localization of 8-OHdG revealed a decreased number of 8-OHdG positive cells in the STZ-Agm group compared to the EC group (Fig. 4A). Fig. 4A indicates that ROS generation is decreased in the STZ-Agm group compared with the EC group. The western blot data for Nrf2 (known as an antioxidant transcription factor) (Fig. 4B) and γ-GCS (an important enzyme in glutathione synthesis) (Fig. 4C) show that Agm treatment promotes Nrf2-mediated antioxidant pathway signaling. Fig. 4 indicates that Agm treatment prevents cell death and promotes Nrf2-mediated antioxidant signaling in STZ-induced Alzheimer rats.