PC12 cells were cultured in MEM/F-12 (1:1) medium (Invitrogen, USA) supplemented with 10% fetal calf serum (FCS, Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin. The PC12 cells were cultured in a humidifed atmosphere containing 5% CO₂ at 37˚C. When the cells reached the proper confluence, they were placed into a serum-free medium. The cells were incubated with PAF (0.1, 0.3, 0.6 μmol/L) for 24 hours or incubated with PAF (0.3 μmol/L) for three hours. Without changing the culture medium, the cells were then treated with BN52021 (Long He Co.; low dose group: 1 mg/L; high dose group: 5 mg/L) for an additional 48 hours.

PC12 cells were plated at 5 × 10⁴ cells/well (200 μl) into 96 well plates, and allowed to adhere and grow until they reached the required confluence. After drug treatment, the survival of cells was determined by MTT assay. Briefy, after incubation with 20 μl MTT (5 mg/ml, Sigma) at 37˚C for 3 hours, living cells containing MTT formazon crystals were made soluble in 200 μl dimethylsulfoxide (DMSO, Sigma). The absorbance of each well was measured using a microculture plate reader with a test wave length of 570 nm and a reference wave length of 655 nm. The light absorption value was measured to determine formazan absorption.

To determine whether PAF-triggered cell death was apoptotic, we measured cell apoptosis using an AnnexinVFITC Apoptosis Detection Kit (BD Pharmingen, USA) according to the manufacturer’s instructions. Briefy, the cells were plated at a density of 5 × 10⁵ cells/ml into six well plates, and cultured until the cells reached 70% confluence. After drug treatment, cells were trypsinized, collected by centrifugation. The cells were then resuspended in 200 μl of binding buffer (provided by the manufacturer). This buffer contained 5 μl of annexin V-FITC and propidium iodide (PI). The cells were incubated for 15 minutes at room temperature. All samples were kept on ice and analyzed immediately with a FACSCalibur flow cytometer (BD, USA).

Forty Sprague-Dawley rats (approximately 4 months old and weighing 200 g) were divided into four groups, including a control group, a model group, a low dose group and a high dose group. The rats were anesthetized with 20% urethane (1300 mg/kg, i.p). The rats first were placed in a stereotaxic instrument with the incisor bar set 3 mm below the earbars (i.e., plat skull), and the scalp was incised and retracted. A hole was drilled in appropriate location in the skull with a dental drill. PAF (0.3 μg/kg, dissolved in artificial CSF, 5 μg/μl) was injected into the fourth ventricle. Eight control rats were injected with artificial CSF. The next day after operation, the low dose and high dose group rats were treated with BN52021. BN52021 was first dissolved in DMSO, and then PBS was added at the following concentrations (i.p.): 0.01 mol/L PBS, 1 mg/kg PBS (low dose), and 5 mg/kg PBS (high dose).

Behavioral testing began 6 days after injection (i.p.). The Morris water maze test was used to measure the rats’ ability to learn and remember spatial cues. The navigational test was conducted in a dark airtight room. The pool was divided into four quadrants, and a platform was placed in one of the quadrants. The rats were randomly placed in the water from four different directions. The rats were then allowed 90 seconds to find the platform. If the rats failed to find the platform within 90 seconds, they were placed on the platform where they stayed for 30 seconds. This test was conducted once per day for 4 days. The platform was then removed, and the rats were placed in the center of the pool to find the platform in order to test their special memory. A computer recorded (1) the number of times that the rat passed the platform and (2) the percentage of swimming distance in the platform quadrant to the total distance.

MTT assay of cell viability showed that the absorbance value decreased gradually with the increase of PAF concentration. The absorbance value of 4 PAF concentration groups was shown in Figure 1. Measurement of Apoptosis by Flow Cytometry was shown in Figure 2: As the concentration of PAF increased, the percentage of apopotosis of cells increased.

MTT Assay of Cell Viability to test PAF cytotoxicity was shown in Figure 3. Both low dose and high dose of BN52021 could effectively increase light absorption of A value. Measurement of Apoptosis by Flow Cytometry was shown is Figure 4. Compared with PAF group, both low dose and high dose of BN52021 could decrease the percentage of apopotosis of cells.

The rats appeared normal and did not lose weight following the PAF injection. The training results of the four tests in place navigation test (i.e., evasive latency) are shown in Figure 5. The percentage of swimming distance in the platform quadrant relative to the total dis-

tance and the number of times the rats passed the platform are shown in Figure 6. The control group and the two treatment groups showed significant increases in both the number of times passing the platform and the percentage of the swimming distance, as compared to the model group.