Aspirin attenuates spontaneous recurrent seizures and inhibits hippocampal neuronal loss, mossy fiber sprouting and aberrant neurogenesis following pilocarpine-induced status epilepticus in rats

brain research 1469 (2012) 103–113

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Aspirin attenuates spontaneous recurrent seizures and inhibits hippocampal neuronal loss, mossy fiber sprouting and aberrant neurogenesis following pilocarpine-induced status epilepticus in rats Lei Ma1, Xiao-Li Cui1, Ying Wang, Xiao-Wei Li, Feng Yang, Dong Wei, Wen Jiangn Department of Neurology, Xijing Hospital, Fourth Military Medical University, 17 Changle West Road, Xi’an 710032, China

art i cle i nfo

ab st rac t

Article history:

Accumulating data suggest that inflammation may contribute to epileptogenesis in

Accepted 31 May 2012

experimental models as well as in humans. However, whether anti-inflammatory treat-

Available online 2 July 2012

ments can prevent epileptogenesis still remains controversial. Here, we examined the anti-

Keywords:

epileptogenic effect and possible mechanisms of aspirin, a non-selective Cyclooxygenase

Epilepsy

(COX) inhibitor, in a rat model of lithium-pilocarpine-induced status epilepticus (SE).

Aspirin

Epileptic rats were treated with aspirin (20 mg/kg) at 0 h, 3 h, or 24 h after the termination

Inflammation

of SE, followed by once daily treatment for the subsequent 20 days. We found that aspirin

Epileptogenesis

treatment significantly reduced the frequency and duration of spontaneous recurrent

Mossy fiber sprouting

seizures during the chronic epileptic phase. Hippocampal neuronal loss five weeks after SE

Neurogenesis

was also attenuated in the CA1, CA3 and hilus following aspirin administration. Furthermore, the aberrant migration of newly generated granule cells and the formation of hilar basal dendrites were prevented by aspirin. Treatment with aspirin starting at 3 h or 24 h after SE also suppressed the development of mossy fiber sprouting. These findings suggest the possibility of a relative broad time-window for aspirin intervention in the epileptogenic process after injury. Aspirin may serve as a potential adjunctive therapy for individuals susceptible to chronic epilepsy. & 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Currently available antiepileptic drugs (AEDs) are mainly seizure suppressing, while they do not affect the underlying pathology or the progression of the disease (Pitkanen and Sutula, 2002; Rogawski and Loscher, 2004). Therefore, a medical need exists to develop alternative therapeutics that not only alleviate the symptoms, but also inhibit the process of epileptogenesis n

Corresponding author. Fax: þ86 29 84771319. E-mail address: [email protected] (W. Jiang). 1 Contributed equally to this work.

0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2012.05.058

(Perucca et al., 2007; Vezzani et al., 2011). However, the details of the mechanisms underlying epileptogenic process remain largely unclear (Jensen, 2009; Rakhade and Jensen, 2009). This hampers the development of better preventive treatments and cures for epilepsy cases that prove resistant to current therapies. Recent experimental and clinical evidence highlights that the activation of inflammatory pathways may contribute to the development of epilepsy (Aarli, 2000; Ravizza et al., 2008;

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Fig. 1 – COX enzymes expression in the epileptic rat hippocampus after chronic aspirin treatment. Representative immunoblots of COX-1 (A) and COX-2 (C) from hippocampus of epileptic and control rats are shown. Increased level of COX-1 and COX-2 abundance was observed at 14 day after status epilepticus. Densitometric analysis revealed that aspirin administration significantly decreased the expression of COX-1 (B) and COX-2 (D) in the hippocampus of epileptic rats compared with untreated epileptic rats (Po0.01, n ¼3 for each group). Values are expressed as a percentage of the control value (mean7SEM). The equivalence of loading protein in all samples was confirmed by immunoblots of b-actin.

Vezzani et al., 2011; Vezzani and Granata, 2005). The upregulation of pro-inflammatory signals are observed during epileptogenesis in brain areas of seizure generalization (Friedman, 2011; Marchi et al., 2009). Furthermore, experiments in animal models suggest that antiepileptogenic effects might be achieved by pharmacological interventions that targeting of specific pro-inflammatory pathways (Ravizza et al., 2011). Cyclooxygenases (COXs) are rate-limiting enzymes in the metabolic pathway that converts arachidonic acid to prostaglandins (PGs), which are potent mediators of inflammation (Simmons et al., 2004). A recent study by Jung et al. (2006) has demonstrated that chronic treatment with celecoxib, a selective COX-2 inhibitor, resulted in a remarkable decrease in the frequency of spontaneous recurrent seizures (SRS) after pilocarpine-induced prolonged seizures. This preclinical data support further attempts to use or development of various antiinflammatory drugs for preventing seizures after epileptogenic injuries. However, long-term inhibition of COX-2 increases the risk of heart attacks, stroke, and related conditions (Flier and Buhre, 2008). These undesirable side effects may limit the use of celecoxib in the treatment of chronic epilepsy. Aspirin, the most widely used medications in the world, represents one of the non-selective classical COX inhibitors (Vane and Botting, 2003). The drug is inexpensive and has been proved to be relatively safe in the long-term prevention of transient ischemic attacks, strokes, and heart attacks (Rothwell et al., 2011; Varughese, 1989). Previous studies have demonstrated that aspirin itself or in combination with AEDs dose-dependently decreased the incidence of seizures (Dhir et al., 2006; Srivastava and Gupta, 2001; Tandon et al., 2003; Wali and Patil, 1995). Although a large number of patients with epilepsy take aspirin while they are affected by a broad

spectrum of other conditions, the effects of aspirin on the development of epileptogenesis have not been specifically addressed yet. In the present study, we employed a Li-Pilocarpine rat model to investigate whether aspirin treatment after the induction of status epilepticus (SE) interferes with the development of SRS. The effects of aspirin on hippocampal neuronal loss, mossy fiber sprouting (MFS) and neurogenesis following SE have also been examined.

2.

Results

2.1. COX enzymes expression in the epileptic rat hippocampus after chronic aspirin treatment Representative immunoblots of COX-1 (Fig. 1A) and COX-2 (Fig. 1C) from hippocampus of epileptic and control rats are shown. Increased level of COX-1 and COX-2 abundance was observed at 14 day after status epilepticus compared with control rats. Densitometric analysis revealed that aspirin administration significantly decreased the expression of COX-1 (Fig. 1B) and COX-2 (Fig. 1D) in the hippocampus of epileptic rats compared with untreated epileptic rats (Po0.01, n¼ 3 for each group).

2.2. Aspirin attenuated the development of spontaneous seizures following SE Eighty rats injected with lithium and pilocarpine developed SE and seventeen rats out of these died from SE. Seizures could be detected behaviorally within 15 min after lithium and pilocarpine injection and the duration of SE was

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controlled within 70 min. After the induction of SE we measured the frequency and duration of observed SRS for 10 days during the chronic epileptic phase (from day 20 to day 30) using video recordings. All rats developed SRS during the monitoring period. We monitored 8.470.3 seizures per day in SE rats (n¼ 14) on average. Compared with SE rats, the frequency of observed seizures was significantly decreased in aspirin-treated groups (Fig. 2A; SE-0 h ASP, 1.570.2; SE-3 h ASP, 2.170.2; SE-24 h ASP, 1.270.1; Po0.05; n¼ 16 for SE-0 h ASP group, n¼18 for SE-3 h ASP group, n¼ 15 for SE-24 h ASP group). The average duration per seizure was 29.771.9 s in SE group. Aspirin treatment dramatically reduced the duration of observed seizures during the chronic epileptic phase (Fig. 2B; SE-0 h ASP, 12.370.8 s; SE-3 h ASP, 13.970.7 s; SE24 h ASP, 8.570.7 s; Po0.01). The frequency and duration of observed SRS were not significantly different among aspirintreated groups (P40.05).

2.3. Aspirin inhibited hippocampal neuronal loss following SE Hippocampal neuronal loss was estimated in control (n¼ 5), SE (n¼ 5) and aspirin-treated rats five weeks after SE. NeuN

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staining demonstrated a substantial neuronal loss in the CA1 (32% of control), CA3 (44% of control) and hilus (46% of control) of the hippocampus following SE, but there was no apparent neuronal loss in the CA2 area (Fig. 3). Aspirintreated rats showed less neuronal loss in the CA1, CA3 and hilus than rats from the SE group. In CA1 area, neuronal cell count in SE-0 h ASP (n ¼5), SE-3 h ASP (n ¼6) and SE-24 h ASP (n¼ 5) groups were 56%, 86% and 89% of control, respectively (Fig. 3G; SE-0 h ASP group, Po0.05, compared with SE group; SE-3 h ASP and SE-24 h ASP groups, Po0.01, compared with SE group). Cell loss in SE-0 h ASP group were more evident than that of the other two aspirin-treated groups (Po0.01, by Dunnett’s post hoc tests). NeuN positive cells of CA3 area in SE-0 h ASP, SE-3 h ASP and SE-24 h ASP groups were 79%, 91% and 87% of control, respectively (Fig. 3H; SE0 h ASP group, Po0.05, compared with SE group; SE-3 h ASP and SE-24 h ASP groups, Po0.01, compared with SE group). Similarly, NeuN positive cells of hilus in SE-0 h ASP, SE-3 h ASP and SE-24 h ASP groups were 84%, 86% and 83% of control, respectively (Fig. 3I; Po0.01, compared with SE group). We did not detect a significant difference of cell loss in the CA3 and hilus region among aspirin-treated groups (P40.05).

Fig. 2 – Aspirin treatment decreased the frequency and duration of recurrent spontaneous seizures following status epilepticus. Seizures were monitored for 10 days during the chronic epileptic phase (from day 20 to day 30 after the induction of seizures) using video recordings. Quantification of the average frequency (A) and duration (B) of observed seizures in aspirin-treated groups showing significantly decrease compared with status epilepticus (SE) rats (Po0.01). No significant differences were observed among aspirin-treated groups (P40.05).

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Fig. 3 – Aspirin treatment abated seizure-induced cell death in the hippocampus. Hippocampal neuronal loss was assessed in control (A), SE (B), SE-0 h ASP (C), SE-3 h ASP (D), and SE-24 h ASP (E) rats five weeks after SE by NeuN staining. SE rats showed a substantial neuronal loss in the CA1, CA3 and hilus of the hippocampus. (F) is magnified view of boxed area in (B). Quantitative analysis demonstrates that aspirin treatment significantly attenuated the neuronal loss in the CA1 (G), CA3 (H) and hilus (I) after SE. Po0.01 compared with control group; #Po0.05 compared with SE group; ##Po0.01 compared with SE group. Scale bars: A, B, C, D and E, 400 lm; F, 50 lm.

2.4. Aspirin suppressed seizure-induced mossy fiber sprouting The effects of aspirin treatment on MFS five weeks after SE were visualized by Timm staining. Prominent staining both in the dentate hilus and inner molecular layer of the hippocampus were observed in SE rats (n¼ 4, Fig. 4B). There was no such sprouting in the control group (n¼ 5, Fig. 4A).There were also remarkable Timm’s stained fibers in the dentate hilus in rats receiving aspirin injection 0 h after SE (n¼ 5, Fig. 4C). However, rats in the SE-3 h ASP (n¼ 6) and SE-24 h ASP (n¼ 5) groups (Fig. 4D and E) presented a low density of Timm’s staining in the inner molecular layer when compared with SE group. Quantification of Timm index confirmed the above findings (Fig. 4F). The scores of MFS in the SE-3 h ASP (1.57 0.3) and SE-24 h ASP groups (1.770.3) were significantly lower than that in SE (3.970.3) and SE-0 h ASP groups (3.170.2) (Po0.01).

2.5. Aspirin inhibited seizure-induced cell proliferation in the dentate gyrus and prevented aberrant migration of newborn cells The effects of aspirin on the proliferation of neuronal progenitors in the dentate gyrus (DG) are shown in Fig. 4. After lithium and pilocarpine treatment, SE (n¼5) induced a significant increase in the number of BrdU-labeled cells in the DG in comparison with control rats (n ¼5, Fig. 5A and B; Po0.01). This increasing in the number of new-born cells was remarkably attenuated by aspirin treatment following the induction of seizures (Fig. 5C–E). Consistent with previous reports (Jiang et al., 2003; Liu et al., 1998; Yang et al., 2008), many BrdU labeled cells were observed in the hilus in SE group (Fig. 5B). Notably, we found that the number of newborn cells in the hilus was significantly decreased in aspirintreated groups compared with SE rats (Fig. 5C–E), raising the possibility that aspirin might inhibit the aberrant migration

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Fig. 4 – Aspirin treatment prevented seizure-induced mossy fiber sprouting. Mossy fiber sprouting (MFS) was visualized by Timm staining. Compared with control group (A), prominent stained fibers both in the dentate hilus and inner molecular layer of the hippocampus were observed in SE (B) and SE-0 h ASP rats (C). Quantification of Timm’s index demonstrated that aspirin treatment 3 h or 24 h after SE reduced the degree of MFS (D, E, and F). Po0.05 compared with control group; Po0.01 compared with control group; ##Po0.01 compared with SE group. Scale bar¼ 100 lm. m: Molecular layer, g: granular layer, h: hilar.

of those newborn cells in the SGZ into the hilus. Quantitative analysis revealed that SE induced a strong increase in BrdU positive cells in the hilus of animals compared with normal control group (Fig. 5F; Po0.01). Animals receiving aspirin at different time points (n ¼6, 6, 5 for SE-0 h ASP, SE-3 h ASP and SE-24 h ASP groups, respectively) after SE displayed reduced levels of BrdU positive cells in the hilus compared with epileptic animals without aspirin treatment (Fig. 5F; Po0.01).

2.6. Aspirin prevented seizure-induced formation of hilar basal dendrites One of the structural changes involving newly generated dentate granule cells is the formation of hilar basal dendrites (Shapiro et al., 2008). We then analyzed the extent of basal

dendrites of newborn cells reaching into the hilus using DCX (a marker of migrating neurons) labeling. Most DCX-positive neurons had their soma located in either the SGZ or the inner one-third of the granule cell layer, with a significant dendritic growth into the outer two-third of the dentate molecular layer in control rats (n ¼5, Fig. 6A). Five weeks after SE, many DCX-positive neurons were found to migrate into the hilus and processes projecting in varying directions (n¼ 5, Fig. 6B). Confirming previous results (Jessberger et al., 2007; Yang et al., 2010), quantitative analysis revealed a significant increase in the length and number of dendritic processes reaching into the hilus compared with controls (Fig. 6B, G and H; Po0.01). Strikingly, aspirin treatment starting at different time points (n ¼5, 6, 5 for SE-0 h ASP, SE-3 h ASP and SE-24 h ASP groups, respectively) reduced the ectopic hilar processes of newborn neurons and prevented them from extending

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Fig. 5 – Aspirin treatment suppressed seizure-induced cell proliferation in the dentate gyrus and inhibited aberrant migration of newborn cells. Cell proliferation is assessed by BrdU labeling of dividing cells. Representative microphotographs show BrdUpositive cells in the dentate gyrus (DG) in control (A), SE (B), SE-0 h ASP (C), SE-3 h ASP (D), and SE-24 h ASP (E) rats. Note that more labeled cells in the hilus were observed in SE rats as compared with control and aspirin-treated rats. Quantitative analysis demonstrated the significant inhibitory effect of aspirin on cell proliferation in the in the hilus (F). Po0.01 compared with control group; ##Po0.01 compared with SE group. Scale bar: 100 lm.

deeply into the hilus (Fig. 6C–E, G and H; Po0.01, compared with SE group).

3.

Discussion

In this study, we demonstrated that aspirin treatment over a latent period suppressed recurrent spontaneous seizures during the chronic epileptic phase in a rat model of temporal lobe epilepsy (TLE). Furthermore, aspirin dramatically inhibited seizure-induced neuronal loss, MFS, and aberrant neurogenesis in the hippocampus. Thus, our findings suggest that aspirin might substantially prevent the epileptogenic process and the underlying pathological alterations after SE in rats. Clear evidence supports that PGs are important inflammatory mediators that are involved in seizure generation and

exacerbation (Cole-Edwards and Bazan, 2005; Vezzani and Baram, 2007; Vezzani and Granata, 2005). COXs are ratelimiting enzymes in PGs synthesis and are major targets of nonsteroidal anti-inflammatory drugs (NSAIDs) (Takemiya et al., 2007). An up-regulation of hippocampal COX-2 expression was observed in epileptic rats and in patients with TLE (Desjardins et al., 2003; Okada et al., 2001). More recent data indicates that the expression of COX-1 in microglia was also enhanced in the hippocampus and areas around the third ventricle during the progression of seizures (Tanaka et al., 2009). Furthermore, most animal studies confirmed the anticonvulsant effect of COXs inhibition (Akarsu et al., 2006; Akula et al., 2008; Oliveira et al., 2008; Zandieh et al., 2010). Thus, these data suggests that both COX-1 and COX-2 isoenzymes may participate in the genesis and maintenance of seizures.

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Fig. 6 – Aspirin treatment blocked seizure-induced aberrant formation of hilar basal dendrites. Representative microphotographs show the extent of DCX-positive basal dendrites reaching into the hilus in control (A), SE (B), SE-0 h ASP (C), SE-3 h ASP (D), and SE-24 h ASP (E) rats. Note that many newborn neurons alter the direction of migration and projected dendrites to the hilus five weeks after SE. Aspirin treatment inhibited the formation of basal dendrites reaching into the hilus. (F) is magnified view of boxed area in (B). Quantification of DCX-positive cells demonstrates that aspirin significantly decreased the length and number of dendritic processes reaching into the hilus following SE (G and H). Po0.01 compared with control group; ##Po0.01 compared with SE group. Scale bars: A–D and E, 100 lm; F, 50 lm. h: Hilar.

Brain inflammation induced by SE can become chronic and contribute to epileptogenesis in experimental models as well as in humans (Vezzani and Granata, 2005). Anti-inflammatory treatments may thus constitute a novel approach for the prevention of epileptogenesis. While the anticonvulsant action of a variety of COX enzyme inhibitors have been tested in rats, only a few recent experiments have examined the antiepileptogenic effects of three different selective COX-2 inhibitors. Celecoxib treatment, starting 24 h after SE for 42 days, attenuated the likelihood of developing SRS following SE (Jung et al., 2006). Parecoxib given for 14 days after SE, reduced the severity of seizures in epileptic rats (Polascheck et al., 2010). In contrast, no effect on spontaneous seizures was observed after 7 days of treatment with SC58236 (Holtman et al., 2009). Nevertheless, the comparison and interpretation of these conflicting results are complicated

by differences in the type of seizures, in the treatment protocols as well as differences in the observing periods. Several clinical observations have described the occurrence of epileptic convulsion following aspirin withdrawal, raising the possibility that aspirin might have anticonvulsant properties (Antonaci et al., 1996; Scheepers et al., 2007). Consistent with these clinical data, animal studies have demonstrate the anticonvulsant effect of aspirin itself or in combination with AEDs (Dhir et al., 2006; Srivastava and Gupta, 2001; Tandon et al., 2003; Wali and Patil, 1995). Although a large number of patients with epilepsy take aspirin while they are affected by a broad spectrum of other conditions, effects of aspirin on epileptogenesis have not been reported so far. We found that aspirin treatment, starting at different time points (0 h, 3 h and 24 h) following seizure induction until day 20, led to a significant decrease in the frequency and duration of observed

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SRS after drug discontinuation. This result suggests that chronic aspirin administration may interfere with the process of epileptogenesis after brain insult. In neurodegenerative diseases with a marked inflammatory component, COX-1 is predominantly localized in microglia whereas COX-2 is mainly expressed in neurons (Aid and Bosetti, 2007). Microglia activation is a hallmark of neuroinflammation in patients and in animal models of epilepsy. It has been suggested that COX-1 might be one of the major players in neuroinflammation (Choi et al., 2009). Since aspirin affects more the COX-1 variant than the COX-2 variant of the enzyme (Warner and Mitchell, 2002), the possible anti-epileptogenic effect of selective COX-1 inhibitors remains to be identified. Brain inflammation promotes increased neuronal excitability, decreases seizure threshold and is likely to be involved in the molecular, structural and synaptic changes characterizing epileptogenesis (Ravizza et al., 2011). The drugs with COX enzymes inhibitory activity could reduce hippocampal cell death and attenuate aberrant neurogenesis in several animal models of epilepsy (Baran et al., 1994; Jung et al., 2006; Kunz and Oliw, 2001). Consistent with these previous results, we found that aspirin inhibited the neuronal loss, aberrant migration of newly generated granule cells and the formation of hilar basal dendrites in the hippocampus following LiPilocarpine induced SE. Our results also demonstrated that aspirin treatment initiated at 3 h or 24 h after SE suppressed the development of MFS in rats. Interestingly, aspirin administration immediately (0 h) after the induction of seizures did not inhibit MFS in the chronic period. Similarly, the inhibitory effect of aspirin treatment on hippocampal CA1 cell loss in the SE-0 hAsp group was lower than that in the SE-24 hAsp group. It has been suggested that transient and adequate inflammatory response is linked to tissue repair processes, whereas uncontrolled inflammation may result in production of neurotoxic factors that amplify underlying disease states (Glass et al., 2010). Thus, our results may indicate that the optimal timing of pharmacological intervention is crucial to avoid suppression of beneficial aspects of inflammation in regenerative responses. The possible mechanisms underlying the counteractive effects of aspirin on seizure-induced neuronal death, MFS, and aberrant neurogenesis in the hippocampus were not investigated in the current study. NF-kB, a transcription factor complex, plays a central role in many biological processes, including inflammation. Recent data suggests that salicylic acid and its derivatives modulate signaling through NF-kB (McCarty and Block, 2006). In addition, COXs, secretases, NFkB, peroxisome proliferator activated receptor (PPAR), and RhoGTPases in neurons, glia, and endothelial cells are potential molecular targets of NSAIDs that may mediate the therapeutic function of these drugs in neurodegeneration (Ajmone-Cat et al., 2008; Lleo et al., 2007). Such common upstream pathways of brain inflammation may therefore account for the molecular mechanisms underlying the anti-epileptogenic effect of aspirin. Collectively, our data demonstrated that aspirin treatment, which is initiated as late as 24 h following the induction of SE, attenuated the development of SRS and inhibited hippocampal neuronal loss, MFS and aberrant neurogenesis in rats.

These initial observations may have important clinical implications that aspirin may serve as a potential adjunctive therapy to prevent the epileptogenetic process. Future epidemiological studies in epileptic patients who take aspirin while affected by other conditions and clinical trials could provide further insight into the relationship between aspirin and epileptogenesis.

4.

Experimental procedures

4.1.

Animals and induction of status epilepticus

Adult male Sprague-Dawley rats weighing between 200 and 250 g at the time of first treatment were used in this study. The rats were housed under controlled temperature and light conditions (12 h light/dark cycle with lights on at 8:00 AM), with ad libitum access to food and water. All procedures used were in strict accordance with the guidelines established by the U.S. NIH and were approved by the Fourth Military Medical University Animal Care Committee. Every effort was made to minimize the number of animals used and their suffering. Status epilepticus was induced by lithium-pilocarpine intraperitoneal (i.p.) injection as described previously (Yang et al., 2010). Lithium chloride (3 mEq/kg, i.p.; Sigma-Aldrich, St. Louis, MO) was injected 18–20 h prior to the administration of pilocarpine (30 mg/kg, i.p., dissolved in saline; Sigma-Aldrich). Rats were pretreated with scopolamine methyl bromide (1 mg/kg, i.p.; Sigma-Aldrich) 20 min prior to pilocarpine injection to reduce its peripheral effects. Behavioral changes were recorded and graded according to Racine’s (1972) scale: stage 1, immobilization, staring, mouth and facial movements; stage 2, wet dog shakes and head nodding; stage 3, forelimb clonus; stage 4, rearing; stage 5, rearing and falling. Seizures were terminated with diazepam (10 mg/kg, i.p.) when rats experienced stage 4 seizures for 70 min. Control rats were given an equal volume of physiological saline. Approximately 2 h after pilocarpine injection, rats were injected subcutaneously with 0.9% NaCl solution (10 ml) to restore volume loss.

4.2.

Drug administration

Rats were randomly allocated into the following five groups: (1) SE-0 h ASP group, these rats were administrated with aspirin (20 mg/kg i.p.; Sigma-Aldrich) immediately after the termination of SE, followed by once-a-day treatment for the subsequent 20 days; (2) SE-3 h ASP group, these rats received their first aspirin injection 3 h after the termination of seizures; (3) SE-24 h ASP group, these rats received their first aspirin injection 24 h after the termination of seizures; (4) SE group, these rats received injections of identical volumes of saline instead of aspirin; (5) Control group were age-matched rats without SE.

4.3.

Monitoring of spontaneous seizures

Epileptic rats were video-monitored with video cameras for 12 h per day from day 10 to day 30 after SE. The recordings were analyzed independently by two observers who did not know the results of group allocation. For rating of seizure

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severity, Racine’s scale was used. The frequency and duration of stage 4 and 5 seizures were measured.

4.4.

Immunoblot analysis

Additional epileptic rats were sacrificed at 14 day after SE. The brain was gently removed, frozen in liquid nitrogen and stored at 80 1C. Tissues were placed in cold buffer containing 250 mM sucrose, 18 mM Tris-Hepes, pH 7.4, 1 mM EDTA, Complete protease inhibitor (Roche Applied Science, Indianapolis, IN) and were disrupted using a PRO 200 homogenizer (PRO Scientific Inc., Monroe, CT). Total protein concentration of samples was measured by using the Bio-Rad RC/DC reagent kit (Bio-Rad Laboratories, Hercules, CA, USA). Samples of the protein (30 mg) were loaded per lane on 12% SDSpolyacrylamide gels and transferred to nitrocellulose membranes. The samples were blocked with 5% non-fat milk in TBST for 1 h and incubated for 2 h at room temperature with goat-derived polyclonal anti-COX-1 or anti-COX-2 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). As a protein loading control, the samples were also incubated with anti-b-actin antibody (1:5000, Sigma). After washed with PBS, the samples were incubated for 1 h with donkey anti-rabbit IgG conjugated to horseradish peroxidase (1:2000, Sigma). Protein bands were visualized by using Super Signal West Pico chemiluminescent substrate kit (Pierce).

4.5.

Immunohistochemistry

Under anesthesia with sodium pentobarbital (50 mg/kg, i.p.), rats were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The brains were immediately removed, postfixed for 2 h in the same fixative, and placed in 20% sucrose at 4 1C until they sank. Coronal sections (30 mm thickness) through the entire hippocampus were cut on a freezing microtome, stored in PBS. For immunohistochemical labeling, we used mouse anti-NeuN antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and goat anti-DCX antibody (1:500; Santa Cruz), respectively. Sections were incubated with their primary antibodies diluted in PBS with 1% BSA at 4 1C for 36 h. After incubation, they were washed with PBS and incubated in biotinylated goat anti-mouse secondary, or rabbit anti-goat antibodies (1:500; Vector laboratories, Burlingame, CA) for 2 h at room temperature, followed by rinsing in PBS and incubation in avidin-biotin-peroxidase complex (1:500; Sigma-Aldrich) for 2 h. After a final wash, sections were reacted for peroxidase enzyme activity using 3, 3-diaminobenzidine. The specificity of immunolabeling was verified by controls in which the primary antibody was omitted.

4.6.

Timm’s staining

To evaluate the degree of MFS into the inner molecular layer (IML), we employed a modified Timm procedure (Cavazos et al., 1991) to label the zinc-containing axons of the granule cells five weeks after SE. In brief, rats were deeply anesthetized and perfused transcardially with an aqueous solution of 500 ml of 0.4% sodium sulfide, followed by 500 ml of 1% paraformaldehyde/l.25% glutaraldehyde solution. The brains

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were removed and left overnight in a 30% solution of sucrose in fixative. Coronal sections (30 mm) were mounted on slides, dried, and developed for 60–80 min in the dark until the molecular layer in the DG was clearly stained in 120 ml of 50% gum arabic, 20 ml of 2 M citrate buffer, 60 ml of 0.5 M hydroquinone, and 1 ml of 19% silver nitrate. The extent of MFS was evaluated by two observers who were blinded to the experimental groups according to a standardized 0–5 scale (Cavazos et al., 1991). 0: No zinc staining between the tips and crest of the DG; 1: sparse zinc staining in the supragranular region in a patchy distribution between the tips and crest of the DG; 2: more abundant staining in the supragranular region with patchy distribution between tips and crest of the DG and a continuous pattern near the tips; 3: prominent staining in the supragranular region in a continuous pattern between the tips and crest of the DG; 4: prominent staining in the supragranular region that forms a confluent dense laminar band in the IML but does not completely fill it; 5: confluent dense laminar band of staining in the supragranular region that completely occupies the IML.

4.7.

BrdU labeling

Rats received four BrdU injections (50 mg/kg per injection), with 12 h intervals starting from day 6 after SE. The animals were allowed to survive for 28 days after the last BrdU injection. Free floating sections were pretreated in 50% formamide/2  SSC buffer (0.3 M NaCl, 0.03 M sodium citrate) at 65 1C for 2 h and were incubated in 2 M HCl at 37 1C for 30 min. After a 10 min wash in 0.1 M borate buffer (pH 8.5) to neutralize the HCl, sections were incubated in mouse antiBrdU antibody (1:500; Santa Cruz) at 4 1C for 36 h. Sections were washed again and incubated in biotinylated goat antimouse secondary antibody (1:250; Vector Laboratories, Burlingame, CA) for 2 h at room temperature, followed by wash in PBS and incubation in avidin-biotin-peroxidase complex for 2 h. After a final wash, sections were reacted for peroxidase enzyme activity using 3, 3-diaminobenzidine.

4.8.

Cell quantification

In a microscope (Olympus BX51, 400  , microscopic field diameter 440 mm), the number of hippocampal neurons was counted in every sixth section in CA1 (four fields), CA2 (two fields), CA3 (two fields), and the whole hilus of the DG in a mediolateral direction. NeuN-positive cells were counted only if structures of appropriate size and shape were demonstrated clearly. This was done to determine the relative change in number of neurons in different groups. The BrdU-labeled dividing cells were counted in the whole DG. Although we did not use a stereological method for cell counting, our cell counting analysis provided data complementary to our qualitative observations on the hippocampal sections. The hilar basal dendrites of newborn neurons were examined for all DCX-labeled cells. The quantitative analysis of the number and the length of the hilar processes was reported previously (Yang et al., 2010). In brief, the cells chosen for measurement had a well-defined cell body located in the subgranular zone (SGZ). The length of the dendrites was determined by measuring the process from the site of

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origin at the soma to the point where it was no longer visible or had terminated as a growth cone. The pictures were analyzed in Image Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD). The investigator was blinded to the treatment of the respective animal.

4.9.

Statistical analysis

All data are expressed as mean7SEM. The significance of differences between groups was calculated by analysis of variance (ANOVA), followed by post hoc testing for individual differences by the Bonferroni or Dunnett’s tests, depending on whether data were normally distributed or not. Data management and statistical analyses were performed in SPSS v16.0. Statistical significance was set at Po0.05.

Acknowledgments This work was supported by Grants from the Natural Science Foundation of China (Nos. 30870840 and 81071051) and the Program for New Century Excellent Talents in University (NCET-07-0146 to W. Jiang). The authors thank Ming Shi and Dong-Yun Feng for their technical assistance.

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