Epilepsy is a common and often deleterious neurological condition. Emerging evidence has demonstrated the roles of innate immunity and the associated inflammatory processes in epilepsy. In a previous study, we found that Toll-like receptors (TLRs) are upregulated and promote mossy fiber sprouting (MFS) in an epileptic model. As downstream effectors of TLRs, the activating transcription factor 3 (ATF3) and p53 proteins were shown to be involved in neurite outgrowth. In the present study, we hypothesized that ATF3 and p53 participate in the process of epilepsy and can affect MFS. To investigate this hypothesis, we examined the expression of ATF3 and p53 in hippocampal tissues of rats kindled by pentylenetetrazole (PTZ) using immunofluorescence, immunohistochemistry and western blotting. MFS was evaluated by Timm staining in the hippocampus. Results from these experiments revealed that expression of ATF3 and p53 is significantly higher (p<0.05) in the CA3 area of the hippocampus in the PTZ-treated group compared to the control group. ATF3 expression gradually increased from 3 days to 4 weeks, peaked at 4 weeks and decreased slightly at 6 weeks in the PTZ group, while the expression of p53 was maintained at similar levels at different time-points following PTZ treatment. No obvious difference in the expression of these proteins was observed between the PTZ and the control group in the dentate gyrus (DG) area (p>0.05). The degree of MFS in the PTZ group peaked at 4 weeks and was maintained at a high level until 6 weeks post-PTZ treatment. In conclusion, ATF3 and p53 may be involved in the occurrence of seizure and play critical roles in MFS in the PTZ kindling model.
Epilepsy, a disorder of recurrent seizures, is a common and often deleterious neurological condition. It has considerable impact on the patients’ quality of life and greatly increases the risk of injury, socioeconomic disadvantage, and even mortality (
The roles of the inflammatory system in the occurrence of seizure are currently heavily investigated. Louboutin
A previous study in an epileptic rat model suggested that aberrant mossy fiber sprouting (MFS) may contribute to spontaneous seizures (
Rats were treated following the Guidelines for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH; Bethesda, MD, USA). All protocols were approved by the Animal Ethics Committee of the Central South University in China. A total of 180 adult male Sprague-Dawley rats (6–8 weeks of age, 180–220 g) were purchased from the Animal Experimental Center of the Central South University (Changsha, China). They were housed in quiet rooms with a 12–12 h light-dark cycle (light from 07:00 a.m. to 19:00 p.m.) and a 22–24°C temperature, and were given standard laboratory food and tap water
At different time-points, the rats were deeply anesthetized with 10% chloral hydrate and perfused intracardially with 300 ml of normal saline and 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C. The brains were removed and placed in 4% paraformaldehyde overnight, then transferred into 0.1 M phosphate buffer containing 20–30% sucrose. Subsequently, serial, 20-μm-thick sections were performed for immunohistochemistry and immunofluorescence analysis. The sections were subjected to conventional rewarming and heat-induced antigen retrieval in 10 mM sodium citrate buffer (0.01 mol/l; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at boiling temperature for 24 min; cool sodium citrate buffer was added every 6 min. Peroxidase and lipids were eliminated by the admixture of 1% hydrogen and methanol at 4°C for 30 min. After rinsing in 0.01 M phosphate-buffered saline (PBS), the sections were blocked using 5% goat serum at room temperature for 2 h and incubated overnight at 4°C with the rabbit anti-rat monoclonal antibodies anti-ATF3 and −p53 (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). After rinsing in 0.01 M PBS, the sections were incubated with a biotinylated goat anti-rabbit secondary antibody (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at room temperature for 60 min. To visualize peroxidase labeling, the sections were stained with diaminobenzidine (Boster Biological Technology Ltd., Wuhan, China), dehydrated and mounted. The sections were observed under a fluorescence microscope (Leica DM5000B microscope; Leica Camera Co., Solms, Germany). Images were processed with a Leica DM5000 B image analysis system (Leica Microsystems, Glattbrugg, Switzerland).
Rewarming and antigen recovery of brain tissue sections were performed as described above. Each section was permeabilized with 1% Triton X-100 in Tris-buffered saline with Tween-20. After blocking with 10% goat serum (Zhongshan Golden Bridge Biotechnology Co., Ltd.) at room temperature for 2 h, the samples were incubated at 4°C overnight with anti-ATF3 or −p53 at 1:12.5 dilution. After rinsing in 0.01 M PBS, the sections were incubated in the dark for 1 h at room temperature with Alexa Fluor 555-conjugated goat anti-rabbit IgG (1:1,000; Invitrogen, Carlsbad, CA, USA). The fluorescence intensity was measured on a Leica DM5000 B system.
The entire hippocampi, including both CA3 and dentate gyrus (DG) areas, were used for western blot analysis. Rats in the control and the PTZ groups were deeply anesthetized with chloral hydrate (350 mg/kg), and cervical dislocation was performed at different time-points. Tissues were snap-frozen in liquid nitrogen, and protein samples were extracted directly from the hippocampi by homogenization in admixture of 1 mM phenylmethyl sulfonyfluoride (PMSF) and RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Following heating at 100°C for 10 min in 5X SDS-PAGE loading buffer, (Beijing Cowin Biotech Co., Ltd., Beijing, China), equal amounts of denatured protein were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the protein bands were electrotransferred onto polyvinylidene fluoride (PVDF) membranes (Pall Corp., Port Washington, NY, USA) and stained with the appropriate antibody (anti-ATF3, 1:1,000; anti-p53, 1:1,500). Immunostaining with the 3-phosphate dehydrogenase (GAPDH) antibody (1:2,000; Sigma-Aldrich) was used to normalize the expression data. The immunoreactive bands were visualized by enhanced chemiluminescence using Image Lab™ software with the gel imaging analysis system (Bio-Rad, Hercules, CA, USA).
At different time-points, the rats were deeply anesthetized with 10% chloral hydrate (Laboratory of The Second Xiangya Hospital, Central South University, Changsha, China) and perfused intracardially with 300 ml of normal saline, followed by addition of 200 ml of 0.1 M phosphate buffer (pH, 7.2–7.6; Sinopharm Chemical Reagent Co., Ltd.) containing 0.4% sodium sulfide (Shanghai Aibi Chemistry Preparation Co., Ltd., Shanghai, China) and 400 ml of 4% paraformaldehyde (Tianjin Chemical Reagent Co., Ltd., Tianjin, China), at 4°C. The brains were removed, fixed in 4% paraformaldehyde for 24 h, transferred to 0.1 M phosphate buffer containing 30% sucrose (Sinopharm Chemical Reagent Co., Ltd.), and cut into 30-μm coronal sections. The sections were stained in the dark for 90 min in a solution containing 60 ml of 50% arabic gum (Sinopharm Chemical Reagent Co., Ltd.), 10 ml of 2 M citrate buffer (Sinopharm Chemical Reagent Co., Ltd.), 30 ml of 0.5 M hydroquinone (Shanghai Aibi Chemistry Preparation Co., Ltd.) and 0.5 ml of 17% silver nitrate (Sinopharm Chemical Reagent Co., Ltd.). The glass slides were washed in de-ionized water and counterstained by Nissl solution (Beyotime Institute of Biotechnology, China) for 5 min. Subsequently, the glass slides were dehydrated with gradient ethanol between 50 and 100%. They were made transparent by xylene and mounted with permount mounting medium (Sinopharm Chemical Reagent Co., Ltd.). Mossy fiber sprouting was evaluated by rating the distribution of supragranular Timm granules (TG) at a standard location in the dorsal and the ventral hippocampus. Timm scoring scale ranged between 0 and 5 according to the following criteria: 0, no TG in the supragranular region; 1, sparse TG in the supragranular regions in a patchy distribution; 2, several TG in a continuous distribution; 3, prominent TG on a continuous distribution with occasional patches of confluent TG; 4, prominent TG forming a confluent dense laminar band and 5, a confluent dense laminar band of TG that further extends into the inner molecular layer.
Statistical analysis was performed with the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA), and the data were expressed as the mean ± standard deviation (SD). Differences among multiple groups were assessed by a one-way analysis of variance (ANOVA), and differences between 2 groups were evaluated using the independent samples t-test. Differences with p<0.05 were considered significant.
With the exception of 5 rats in the PTZ group that died as a result of status epilepticus or generalized clonic-tonic seizures at 1 or 2 weeks, the remaining rats in this group developed seizure activity of stage 3, 4 or 5 after continuous PTZ injection for 18–22 days. The PTZ-induced seizure activity generally occurred 5–10 min after the PTZ injection, and had a duration of 5–30 min. Spontaneous recurrent seizures of grade 2–3 were detected in kindled rats as early as 23 days after the first injection. No epileptiform activity was observed in the control groups.
The Timm scores in the CA3 area of the PTZ group were significantly different from those of the control group in all time-points (p<0.05;
The expression of the ATF3 and p53 proteins was mainly observed in the pyramidal cells in the CA3 region and in hilar neurons in the DG within the hippocampus. Compared to the control group, expression of ATF3 and p53 in the CA3 area of the PTZ group was significantly increased (P<0.05). Immunohistochemistry analysis showed that the expression of ATF3 in the PTZ group gradually increased from 3 days to 4 weeks, peaked at 4 weeks, and slightly decreased at 6 weeks (
Increasing evidence has highlighted the roles of immunity and inflammatory processes in epilepsy (
The expression of both ATF3 and p53 proteins was increased in the PTZ compared to the control group, indicating that these proteins may participate in the occurrence of epilepsy. ATF3 is encoded by an early-response gene, the expression of which is induced in cells exposed to a variety of stress stimuli (
In addition, it has been reported that p53 is involved in ATF3-mediated injury response, potentially via ATF3-mediated regulation of the p53 stability, by interference upon p53 ubiquitination (
In summary, our results demonstrated an increase in the levels of ATF3 and p53 in the PTZ groups compared to the control groups in the rat hippocampus. This study indicated that ATF3 may play a role in aberrant MFS in a p53-dependent manner during the early stage of epileptogenesis. We did not knockout or overexpress the rat ATF3 and p53 genes or proteins in this study; these experiments may provide additional evidence to explain our results and to elucidate the functions of the two proteins. Further studies are needed to elucidate their roles in the neurons, both in rat models and epileptic patients. The results reported herein provide important perspectives for future studies on ATF3 and p53, aiming to identify more effective treatments for epilepsy.
This study was supported by the 2011FJ4271 grant from the Natural Science foundation of Hunan Province. We thank Zhao-hui Luo for technical assistance.
Timm staining in the (A) CA3 and (B) dental gyrus (DG) inner molecular layer areas (IML) of the control and the pentylenetetrazole (PTZ) groups. Upper panels show Timm granules from the control and the PTZ groups (different time-points following treatment), as observed under a fluorescence microscope (magnification, ×100). Scale bars (green lines), 200 μm. Lower panels show quantified intensities of Timm granules (Timm scores), calculated in 28 PTZ and 30 control rats. **P<0.05. Timm scores gradually increase following PTZ injection in the CA3 area. No significant increase in Timm scores, indicative of fiber sprouting (MFS), is observed in the CA3 area of the control group, and in the IML of either the control or the PTZ group.
The expression of activating transcription factor 3 (ATF3) in the CA3 area of the hippocampus, as assessed by immunohistochemical staining in the control and the PTZ groups (different time-points post-treatment). The upper panel shows AT3 staining as observed under a fluorescence microscope (magnification, ×100; scale bars, 200 μm). The lower panel shows quantified intensities from 29 PTZ rats and 30 control rats. Compared to the control group, ATF3 expression is significantly increased as early as 1 week post-PTZ injection, reaches a peak at 4 weeks, and shows a slight decline at 6 weeks. **P<0.05.
The expression of p53 in the CA3 area of the hippocampus, as assessed by immunohistochemical staining in the control and the PTZ groups (different time-points post-treatment). The upper panel shows p53 staining as observed under a fluorescence microscope (magnification, ×100; scale bars, 200 μm). The lower panel shows quantified intensities from 29 PTZ and 30 control rats. p53 shows higher expression in the PTZ group compared to the control, but was expressed at similar levels (P>0.05) from 3 days to 6 weeks.
The expression of activating transcription factor 3 (ATF3) and p53 in the hippocampus. (A) Western blot of samples from 28 PTZ and 30 control (con) rats. The 3-phosphate dehydrogenase (GAPDH) was used as the loading control. D, days; w, weeks. Quantification of the western blot results showed that (B) ATF3 expression is increased at all time-points in the pentylenetetrazole (PTZ) group compared to the control; it gradually increases from 3 days to 4 weeks, and decreases from 4 to 6 weeks; and that (C) the expression of p53 is increased in the PTZ compared to the control group.
Immunofluorescence staining of activating transcription factor 3 (ATF3) and p53 in the CA3 area of the hippocampus, as observed under a fluorescence microscope (magnification, ×200). ATF3 mainly accumulates in the cytoplasm of the neurons, while p53 mostly locates in the cell nuclei. 29 PTZ and 30 control rats were assessed by immunofluorescence. Scale bars, 100 μm.