Adult male Sprague-Dawley rats (180–220 g) were used in the study. All the experimental procedures were approved by the Animal Research Committee of the Ningxia Medical University (Ningxia, China) and performed in accordance with the Chinese Animal Welfare Act for the use and care of laboratory animals. Rats were divided into four groups at random: Normal control (control), fear conditioning (fear), epilepsy (epilepsy) and epilepsy treated with fear groups (epilepsy + fear). The rats with epilepsy were triggered with pilocarpine and raised for 30 days after the epilepsy seizure. Each group comprised of six rats.

The epileptic model was established with LiCl/pilocarpine as previously described (10). The rats were injected intraperitoneally (IP) with LiCl (127 mg/kg) on day 1 and administered pilocarpine injection (30 mg/kg) 18–24 h later. Epileptic seizures of racine Ⅳ-V grade were chosen for the experiments. Epileptic rats were administered chloral hydrate (300 mg/kg) injection IP after 1 h of seizures to suppress the seizures. The normal control rats were administered the saline injection, respectively.

The rats were placed in the conditioning chamber (23×23×35 cm) for 5 min before the loudspeaker stimulus. A sonic wave (1 min, 1,000 Hz, 75 Db) was delivered through the loudspeaker as the conditioned stimulus. Electrical foot shock (3 sec, 1 mA) stimulation occurred from the metal grid floor as the unconditioned stimulus. The context was a 1 min tone followed by the 3 sec foot shock, which was repeated 30 times (Fig. 1A). Following training in the chamber, the rats were returned to their home cages. The training was performed in 4 consecutive days. The rats of the fear and the epilepsy treated with fear groups underwent this training procedure. The control and the epilepsy groups had the same conditioned stimulus of the tone, but without the unconditioned electric stimulation at the foot shock.

Freezing time was used as an index of fear conditioning. Freezing was defined as immobility, excluding respiratory movements with a freezing posture. Rats remained still, sluggish, curled or crouched whilst breathing, and had a slight rocking motion. The total time of the freezing posture was called the freezing time and was measured starting from day 5 of training. The freezing time was measured immediately after the tone stimulation (without foot shock) and within 30 min. The behavioral activities were recorded by video.

The rats were anaesthetized and subsequentely decapitated to collect the whole blood with centrifuge tubes. Blood was incubated at 37̊C for 30 min. Serum was collected after 10 min centrifugation at 1,688 x g. Serum COR and CRP concentrations were measured by the radioimmunoassay kits, following the manufacturer's instructions. COR and hs-CRP radioimmunoassay kits were purchased from the Beijing Huaying Biotechnology Research Institute (Beijing, China). The radioimmunoassay was analyzed on the BN II Nephelometer (Dade Behring Marburg GmbH, Marburg, Germany) analyzer.

Brains were dissected and fixed routinely. A stainless steel rat brain matrix (175–300 g, coronal; RWD Life Science Co., Ltd., Shenzhen, China) was used to define the brain tissue blocks, including the hippocampus and insular cortex, respectively. A total of 15-µm coronal serial sections were prepared routinely. The boundary of the hippocampus and insular cortex was defined in accordance with the atlas of Paxinos and Watson (11). Ten sections, including the hippocampus or insular cortex, from each animal were chosen.

Immunohistochemistry was performed routinely. The stathmin protein 1 antibody (1:80; Proteintech, Inc., Chicago, IL, USA) and microtubule-associated protein (MAP2) antibodies (1:100; Bioworld, Visalia CA, USA) were used. The avidin-biotin kit (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) was used for the second antibody incubation and 3,3′-diaminobenzidine staining. All the sections were counterstained with hematoxylin. The immunohistochemistry-positivity of the images was assessed semi quantitatively by automatically measuring the optical densities.

Western blotting was performed routinely. Antibodies were used at the following concentrations: Stathmin 1 (1:300; cat no. ab47328); phosphorylated stathmin 1 (1:500; cat no. ab47398; Abcam, Cambridge, MA, USA); microtubule-associated protein (1:800; cat no. 17490-1-AP; Bioworld); α-tubulin (1:5,000; cat no. 66031-1-Ig Proteintech); GAPDH (1:5,000; cat no. TA-08; Bioss, Beijing, China); and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (cat no. ZB-2301; ZSGB-BIO, Beijing, China). Quantization of relative band densities was performed by scanning densitometry.

All the data were processed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Mean ± standard deviation that the measurement data using one-way ANOVA; same object point in time measurement data using a repeated measures analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

Rats (n=6) in the normal control exhibited an extremely short freezing time, from 2 to 14 sec, with an average of 5 sec. Whereas the epileptic rats (n=6, and epilepsy after 30 days) had a longer freezing time, from 10 to 152 sec, with an average of 36 sec, which was ~5–6-fold longer compared to the normal control (Fig. 1B).

Rats with fear training by tone and foot shock (fear) only (n=6) exhibited an increased freezing time immediately following the shock, from 15 to 152 sec, with an average of 74 sec. This was ~10–12-fold longer compared to the control and ~2-fold longer compared to the epilepsy group. Therefore, the contextual fear has occurred in these rats (F=4.521, P<0.05).

The rats of the epilepsy + fear (n=6) group had the longest freezing time among the four groups. The average freezing time was 203 sec, which was approximately three times higher compared to the fear group. As these rats have experienced epileptic seizures during the past 30 days, the increased freezing time revealed a significantly strengthened effect of epileptic seizures on the learned fear of the tone-shock contextual. These results indicate that these rats not only have the memory regarding the context in which they received the electrical stimulation, but have also developed a strong aversive response to the tone and the environment associated with a painful experience.

Psychological processes, such as fear, are always accompanied with systemic stress responses. A systemic stress response includes objective signs of stress, such as high serum levels of adrenocorticotropic and cortisol, elevation of immunoreactions and an increase in arterial blood pressure (12). In the present study, the serum COR and inflammatory biomarker, CRP, were chosen as the biological indices of psychological stress caused by the fear.

The COR and CRP levels of the fear and the epilepsy + fear rats elevated when compared to the control. However, there was no statistical significance when compared to the control (Fig. 1C and D).

According to the results of the western blotting, as shown in Fig. 2, the expression of stathmin in the three areas of the brain increased in a similar sequence. The highest expression of stathmin was observed in the epilepsy + fear rats. Fear conditioning (the fear group) increased the expression of stathmin slightly, whereas the epilepsy + fear treatment increased it dramatically. The stathmin expression level of the epilepsy group was also significantly increased, more than that of the fear group, particularly in the insular cortex and hypothalamus, but less in the hippocampus (insula F=75.863, P<0.05; hippocampus F=36.698, P<0.05; and hypothalamus F=150.761, P<0.05).

Stathmin is a phospho-protein that binds to tubulin and regulates microtubule dynamics. The binding of stathmin to tubulin and its ability to disrupt the microtubules is modulated through its phosphorylation on multiple serine residues. These microtubule destabilizing activities are suppressed by phosphorylation of stathmin (9). Therefore, the expression levels of phosphorylated stathmin were examined by western blotting. The phosphorylated stathmin increased slightly in the epilepsy + fear conditioning rats, in all three areas of the brain. The increasing effects on phosphorylated stathmin were stronger in the epilepsy group compared to the fear group, suggesting that phosphorylated stathmin was more sensitive in the epileptic brain to the contextual stimulus of tone.

Western blotting analysis revealed that the expression of MAP2 decreased inversely with stathmin in the epilepsy and the fear groups in all three areas of the brain. The most significant decrease was observed in the epilepsy + fear group (Fig. 2). The expression of α-tubulin was not significantly changed in all the groups and in all three areas of the brain.

The microtubule stability was indicated by the ratio of MAP2 and α-tubulin/stathmin. In all three areas of the brain tested, epilepsy + fear had the lowest ratio, indicating that the microtubule stability was lowest. The most evident change of this ratio occurred in the insular cortex, instead of the hippocampus (Fig. 2).

To further analyze the variation of stathmin expression during the development of epilepsy, the immunohisotochemical patterns in the temporal cortex and the hippocampus were examined at 3, 30 and 60 days after the epileptic seizures. The stathmin expression started to increase at 3 days and reached its peak after 30 days of epilepsy in the local areas of the insular cortex. The reinforced stathmin expression in the dendrites was even more noticeable in the local areas of the insular, which was located at the IV layer of the cortex. Evident variation patterns were also observed in the hippocampus CA2 region, but not in the amygdale and temporal lobe cortex (Fig. 3). Stathmin expression declined to the normal control level in 60 days, in all the areas tested (insula F=52.018, P<0.05; hippocampus F=42.074, P<0.05; amygdala F=12.699, P<0.05; and temporal lobe F=21.469, P<0.05).