Adult male Wistar rats weighing 250–280 g were purchased from Experimental Animal Center of Shandong University, China. The rats were housed under controlled temperature and light conditions (12 h light; 12 h dark cycle, with lights on at 08:00 a.m.), with free access to food and water. The experimental procedures were approved by the ethics committee of Shandong University and performed in accordance with international standards for experiments on animals.

The rats were randomly divided into three groups (15 rats in each group, n = 45): the control group, the pilocarpine-treated group, and the FK506-treated group. The rats in the control group were injected with a single administration of 0.9% saline, intraperitoneally. The rats in the pilocarpine-treated group received a single administration of 0.9% pilocarpine, intraperitoneally. The rats in the FK506-treated group received an intraperitoneal injection of 2 mg/kg FK506 (Fujisawa, Japan) 24 h prior to the administration of pilocarpine, and another injection of 2 mg/kg FK506 were given 1 h before the administration of pilocarpine. The dose of pilocarpine in the FK506-treated group was the same as that in the pilocarpine-treated group.

To prevent peripheral cholinergic effects on the central nervous system, lithium chloride (3 mEq/kg, intraperitoneal), which does not cross the blood–brain barrier, was administered 24 h prior to subcutaneous injection (s.c.) of 1 mg/kg scopolamine methyl nitrate, followed by the treatment of pilocarpine for 30 min. All efforts were made to reduce the number of animals used and minimize animal suffering. After 1 h, the rats developed convulsive seizures at stage 4 or 5 according to Racine as follows (Racine, 1972): stage 0, no spasm indicated; stage 1, facial myoclonus represented; stage 2, head nodding suggested; stage 3, forelimb clonus represented; stage 4, rearing along with severe forelimb clonus indicated; stage 5, rearing and falling together with severe forelimb clonus. SE was terminated by diazepam (10 mg/kg, intraperitoneal).

After the induction of SE, the experimental animals put on black belts and were trained in a Morris water maze every day for 7 days to assess their spatial learning and memory abilities. The water maze consisted of a tank filling with opaque powdered milk (0.5–1.5%, 22–25°C). The tank with the diameter of 1.2 m and the height of 0.5 m was separated into four parts (marked as A–D) in the form of clockwise. A hidden rigid platform (12 cm in diameter) was placed 1 cm below the surface of the water in quadrant D. An automated video tracking system (Ethovision 2.0; Noldus, Wageningen, Netherlands) was used for synchronous tracking of the animals' behavior. The Morris water maze tests included place navigation tests, which examine the ability of the rats to perform a specific location-based task, and spatial probe tests, which examine their ability to retain spatial memory (Pouzet et al., 2002). In the place navigation tests, the rats took part in four trials every day for 5 consecutive days. The escape latency (time from start point to reach the hidden platform) was used to assess the learning and memory performance of the rats. The platform is in quadrant D of the tank. The rats were placed in a determined start point and the longest escape latency was 120 s on each trial. The trial was terminated if the rats found a platform or the escape latency is over 120 s. If the rats failed to find the hidden platform within 120 s, they were allowed on the platform for a short time. In the experiment, we mainly selected the performance in the zone where the rat was released at the starting position farthest from the hidden platform was assessed. One day after place navigation tests, the hidden platform was removed. The rats were placed randomly in a determined start point to assess spatial memory. Each rat swam for 120 s. Swimming distance and trace in quadrant D and time spent in quadrant D were recorded to assess the cognitive ability of rats. The swimming speed was also recorded to find out whether the differences between escape latency and swimming distance resulted from different swimming abilities.

The rats were anesthetized with chloral hydrate (500 mg/kg, i.p.) at 12 d post-seizure and perfused via the left cardiac ventricle with 4% paraformaldehyde in phosphate buffered saline (PBS). The brain was removed and placed in the same fixative at 4°C for at least 24 h. The cerebellum and olfactory bulbs were removed. The first 6 mm of the frontal cerebral hemispheres was removed, and the next 8 mm, which contained the hippocampus, was collected and fixed in paraffin. The brains were placed in a 20% sucrose solution in phosphate buffer at room temperature for 48 h and in a 30% sucrose solution for at least 48 h. The brains were positioned on a freezing microtome stage, cut to a nominal thickness of 10 μm in the horizontal direction, followed by preserving at −20°C. HE staining was performed later. Surviving cells were defined as those with nuclei that were round shaped, with an intact cytoplasmic membrane and no nuclear condensation or distortion. Live pyramidal cells distributed in the area of hippocampal CA3 were observed using high magnification (×400).

The One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, China) was used to detect the neuronal apoptosis. Continuous brain sections were dewaxed, hydrated, and incubated with proteinase K solution (20 μg/mL, without DNase) for 25 min at 37°C. Then, the sections were washed in PBS three times, 5 min each time. Next, DNA fragments were labeled by incubation with 50 μl TUNEL detection solution for 1 h at 37°C in a humidified chamber. The reaction was terminated by 2 × SSC termination solvent provided in the kit. After washed in PBS three times, the sections were mounted with anti-fluorescence quenching agent and examined under the fluorescence microscope.

The 10-μm thick coronal sections were incubated with 3% H₂O₂ for 5 min to inhibit endogenous peroxidase activity and then rinsed in PBS for 5 min, followed by incubation with primary anti-glial fibrillary acidic protein (GFAP) antibody (Abcam, USA) diluted by 1:200 (glial fibrillary acidic protein) at 4°C overnight. Following incubation with species-specific biotinylated secondary antibody, the sections were incubated with peroxidase-labeled streptavidin, and the color was developed with diaminobenzene (Brusco et al., 1997). Finally, the sections were counterstained with hematoxylin. Negative controls were processed simultaneously by omitting the primary antibodies.

The dissected hippocampus tissues were washed with cold PBS, lysed in SDS lysis buffer and separated by SDS-PAGE. Then, the proteins were transferred to polyvinylidene difluoride membrane and immunoblotted with anti-GFAP primary antibody and horseradish peroxidase-conjugated secondary antibodies (all from Cell Signaling Technology, USA). Finally, detection was performed using the enhanced chemiluminescence reagents (Plus-ECL, PerkinElmer, MA, USA). β-actin was used as a control. The ImageJ software was used for the quantification of the bands.

Statistical analyses were performed using SPSS version 19.0 (SPSS Inc., Chicago). The data were presented as the mean ± standard deviation (SD). Differences between two groups were determined using the t-Test. Differences among more than two groups were evaluated by one-way analysis of variance (ANOVA) followed by a Newman–Keuls post-hoc test. Chi-square test was applied to analyze the amount of subjects in stage 4 and 5 seizures. Differences were considered statistically significant at a P < 0.05.

Behavioral episodes induced by pilocarpine injections showed typical increases in their intensity and duration, gradually progressing toward SE. Compared with the pilocarpine-treated group, the FK506-treated group exhibited a longer latency period and a smaller percentage at stage V (Table 1). Control animals did not exhibit any behavioral seizure activity.

HE staining was used to detect neuronal changes in the hippocampus. The results showed that dead neurons with pyknotic nuclei were mainly located at hippocampal CA3 zone. Different from dead neurons, the live cells have round and palely stained nuclei. Compared to the control group, the pilocarpine-treated group had fewer surviving cells. However, when FK506 was given before pilocarpine injection, more neurons survived after SE (Figure 1A). The results from TUNEL staining also showed that the number of apoptotic neurons in pilocarpine-treated group was higher than that in control group. FK506 treatment could decrease the number of apoptotic neurons after seizures (Figure 1B).

As shown by the results obtained in the Morris water maze test, the FK506 treatment improved the learning and memory ability of the rats. In the place navigation tests, the escape latency was significantly prolonged in the pilocarpine-treated group compared to the control group. In contrast, the prolonged latency was significantly reduced in the FK506-treated group (Figure 2A). As shown in Figure 2B, in the spatial probe tests, there were no significant differences in the swimming speeds of the rats between the indicated groups. Compared with the control group, the swimming distance in target zone was significantly decreased in the pilocarpine-treated group, while the swimming distance of the FK506-treated rats in the target zone was longer than that of the pilocarpine-treated rats (Figure 2C). Consistently, the rats in the FK506-treated group spent more time in the target zone and made more crossings to the position of the former platform than pilocarpine-treated group (Figures 2D,E).

GFAP, a cytoskeletal protein present in astroglial somata and projections, is used as a specific immunomarker of astrocytes. The immunohistochemistry analysis revealed that GFAP was expressed in cells 72 h after SE and that the number of GFAP-labeled cells increased 7 d after SE. As shown in Figure 3A, the GFAP expression was significantly increased in the pilocarpine-treated group compared with the control group, while the GFAP expression significantly decreased in the FK506-treated group when compared with the pilocarpine-treated group. Consistent with immunostaining data, the results from western blot showed that FK506 remarkably inhibited the increase of GFAP protein expression level after SE (Figure 3B). These results suggested that FK506 could significantly attenuate SE-induced reactive astrogliosis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.