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TrkB signaling following pilocarpine-induced status epilepticus
Neurochemistry International 63 (2013) 405–412
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The PPARc agonist rosiglitazone prevents neuronal loss and attenuates development of spontaneous recurrent seizures through BDNF/TrkB signaling following pilocarpine-induced status epilepticus Hong Sun a,⇑, Xin Yu b, HaiQin Wu a, GuiLian Zhang a, Ru Zhang a, ShuQin Zhan a, HuQing Wang a, Li Yao a, Ning Bu a, YongNan Li c a
Department of Neurology, The Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China Department of Neurology, People’s Liberation Army 401 Hospital, Qingdao, Shandong 266071, China c Department of Neurology, The Fourth Affiliated Hospital, Harbin Medical University, Harbin 150001, Heilongjiang Province, China b
a r t i c l e
i n f o
Article history: Received 1 February 2013 Received in revised form 14 July 2013 Accepted 22 July 2013 Available online 6 August 2013 Keywords: Epilepsy BDNF TrkB PPARc Rosiglitazone Spontaneous recurrent seizures K252a
a b s t r a c t Hippocampal neuronal loss plays an important role in epileptogenesis, and it is considered a trigger of repeated spontaneous recurrent seizures (SRS). The BDNF/TrkB signaling pathway regulates neuronal plasticity in the CNS, and promotes epileptogenesis. Previous studies have shown that Peroxisome proliferator-activated receptor gamma (PPARc) agonists exert neuroprotective effects by inhibiting oxidative stress and inflammation in epilepsy. In the present study, the PPARc agonist rosiglitazone inhibited increases in BDNF and TrkB after status epilepticus (SE), and also prevented hippocampal neuronal loss. More importantly, our study showed that rosiglitazone suppressed SRS. However, the effects of rosiglitazone were significantly reversed by cotreatment with K252a, an antagonist of TrkB. Additionally, rosiglitazone did not affect the development and severity of SE. Thus, our data provide evidence that rosiglitazone exerts neuroprotective and antiepileptic effects involve BDNF/TrkB signaling. Our study also offers new perspectives for the treatment of epilepsy. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Hippocampal neuronal loss is a feature of the neuropathology of epilepsy. Specifically, it is characterized by profound neuronal loss in the CA1, CA3 and the hilus regions of the hippocampus, and glial cell activation and neurogenesis in different animal models of epilepsy (Siebzehnrubl and Blumcke, 2008; Holopainen, 2008). These pathological changes may contribute to alterations in neuronal circuits of the epileptic brain, and may play a role in epileptogenesis (Walker, 2007). Growing evidence indicates that neuronal degeneration is critical for plastic changes in hippocampal neuronal networks, and it is considered a powerful trigger for repeated spontaneous recurrent seizures (SRS) (Ben-Ari and Dudek, 2010; Zhang et al., 2002), but the molecular mechanisms are still poorly understood.
⇑ Corresponding author. Address: Department of Neurology, The Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, No.157 West 5 Road, Xi’an 710004, Shaanxi Province, China. Tel./fax: +86 29 87679249. E-mail addresses: [email protected], [email protected] (S. Hong). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.07.010
Brain-derived neurotrophic factor (BDNF) is mediated activation of the TrkB signal transduction pathway regulates neuronal plasticity in the CNS (Autry and Monteggia, 2012). In recent years, studies have suggested that BDNF can enhance neurogenesis and regulate neuronal circuits in the hippocampus in an epileptic model (Kuramoto et al., 2011). Previous studies have demonstrated that BDNF and TrkB mRNA and protein are up-regulated in the hippocampus after status epilepticus (SE), suggesting that BDNF may have a protective role against seizure-mediated excitotoxicity (Danzer et al., 2004; Paradiso et al., 2009; Kuramoto et al., 2011). Chronic intrahippocampal infusion of BDNF delayed amygdala kindling-induced seizure development, and reduced the increase in after discharge duration (Osehobo et al., 1999). However, other studies have suggested that increased BDNF could contribute to lasting structural and functional changes, and establish neuronal hyperexcitability in the hippocampus, thus promoting epileptogenesis (Kotloski and McNamara., 2010; Scharfman, 2005). Similarly, it has been demonstrated that transgenic mice overexpressing BDNF show increased SE severity in a KA-induced model of epilepsy (Barton and Shannon, 2005). In addition, BDNF up-regulation also accelerated neuronal apoptosis in the hippocampus and increased SRS (Heinrich et al., 2011). Thus, the role
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of BDNF/TrkB signaling in the development of epilepsy is controversial. Moreover, it is still uncertain whether BDNF upregulation and TrkB activation are a result of initial seizure induction and play a primary role in the epileptogenic process, or are a secondary consequence of excitotoxicity from repeated SRS. Peroxisome proliferator-activated receptor gamma (PPARc) is a nuclear membrane-associated transcription factor. In the past several years, a number of studies have shown that PPARc agonists exert neuroprotective effects in models of neurological damage and disease (Bordet et al., 2006; Kapadia et al., 2008), and some clinical data also confirming these experimental results (Gamboa et al., 2010). Importantly, our previous study showed that the PPARc agonist rosiglitazone attenuated seizure-induced neuronal loss and improved cognitive impairment by inhibiting inflammation and oxidative stress in lithium pilocarpine-induced SE in rats (Sun et al., 2008, 2012). Indeed, most studies have focused on the role of PPARc in anti-inflammatory and anti-oxidative actions, but no studies have attempted to address whether PPARc can influence BDNF/TrkB signaling in diseases of the CNS. In addition, it is not clear if PPARc agonists can influence SRS and SE in epilepsy. Thus, the aim of the present study was to determine whether rosiglitazone inhibits neuronal loss via-regulation BDNF/TrkB signaling in the epilepsy. And further to observe influence of rosiglitazone in the SRS. First, we examined BDNF and TrkB expression after SE, and the role of rosiglitazone in modulation of BDNF and TrkB expression. Next, we evaluated the effect of rosiglitazone on SRS and SE through observation of behavior and EEG in the lithium–pilocarpine model of epilepsy.
2.3. EEG recording and behavioral observation All undergoing Li–Pilo-induced SE was used for cortical electroencephalographic (EEG) recording. The bilateral EEG cortical activity was recorded during the acute (SE and 12 h after), and the chronic (14 days after SE) phases. In the chronic phase, a 3 h EEG recording was obtained every day until convulsive SRS at stages 3–5 was observed. SRS occurrence was evaluated through a 6 week video recording started 2 weeks after SE. All recordings for SRS were done during the light period. Epileptic rats were video recording for at least 12 h daily for 6 week from starting 2 week after SE. The frequency and duration of stage 4/5 seizures were recorded, and the severity of SRS was scored according to the Racine’s scale (Racine, 1972). The recordings were analyzed by observers who were blind to the treatment of each rat. 2.4. Drug administration Animals were randomly assigned into 4 groups: Control group, SE group, SE + rosiglitazone group and SE + rosiglitazone + K252a group. All drug solutions were prepared fresh daily. Rosiglitazone (Cayman Chemical, USA) was diluted in a 30% DMSO and 70% isotonic saline solution, and intracerebroventricularly injected (i.c.v) at doses of 100 lg/kg at 1 h before the injections of lithium chloride. K252a (10 lg in PBS containing 1% DMSO, Calbiochem, San Diego, CA) was administered i.c.v at 1 h before rosiglitazone, this dose was found that it blocked the effect of exogenously administered BDNF on serotonin clearance (Benmansour et al., 2008). For long-term survival studies (12–42 days), rosiglitazone and/or K252a were administered everyday as described above until every animal was sacrificed.
2. Materials and methods 2.5. Immunohistochemistry and histology 2.1. Lithium pilocarpine-induced model of epilepsy Male Sprague–Dawley rats (weight 200–220 g; 12 weeks old) were used for all experiments. The detailed procedure was performed as previously described (Sun et al., 2008). Lithium chloride (127 mg/kg, i.p., Sigma) was injected 24 h prior to the administration of pilocarpine. One single dose of pilocarpine hydrochloride (30 mg/kg, i.p., Sigma) was administered. Then, the rats received repeated injections of pilocarpine (10 mg/kg, i.p.) every 30 min until they developed seizures which were scored by Racine (1972))’s scale. Control rats (n = 4) received all treatments with saline instead of pilocarpine. In order to reduce the mortality of rats, we require that the duration of the SE is the seizure lasted for 1 h (Glien et al., 2001). All animal protocol conformed to the Guide for the Care and Use of Laboratory Animals (Declaration of Helsinki of the World Medical Association).
2.2. Electrodes and cannula implantation Intracerebroventriclar administration was performed as described previously (Rigoulot et al., 2004; Yananli et al., 2008). Briefly, the rats were fixed in a stereotaxic frame, anesthetized, and a midline incision was made along the scalp. The skull was exposed and a burr hole was made in the location over the left sensorimotor cortex, 2 mm posterior and 2 mm lateral to bregma. The guide cannula was implanted 1.5 mm ventral to the superior surface of the skull. Then six single-contact electrodes were placed epidural 2 mm lateral to sagittal suture on both sides: 2 mm anterior, 2 mm posterior, 6 mm posterior to the bregma. And the electrodes and guide cannula fixed with jeweler acrylic cement. Animals were allowed to recover for one week prior to EEG recordings.
At each time point, rats were deeply anesthetized with pentobarbital (1.8 g/kg, Sigma) and perfused transcardially with ice-cold phosphate buffer (PB, 0.1 M, pH 7.4) followed by ice-cold fixative (4% paraformaldehyde in 0.1 M PB, pH 7.4). Brains were removed immediately after perfusion, postfixed in the same fixative one night at 4 °C and impregnated with 20% sucrose diluted in PB for cryoprotection. Brains were subsequently cut into 10 um thick coronal sections on a freezing microtome. The specimens were pretreated with 0.3% hydrogen peroxide in PB for 15 min at room temperature and then rinsed several times in PB and incubated for 2 h with blocking buffer (2% horse serum/1% BSA/0.1% Triton X-100 in PBS, pH 7.5), incubated overnight at 4 °C with primary antibodies, and then incubated for 1 h at room temperature with secondary antibodies. Mouse anti-rat NeuN (1:2000, Chemicon) monoclonal antibody was used as markers for neuron. Biotinylated goat antimouse IgG (1:500, Sigma) and FITC-conjugated anti-mouse (1:500, Sigma) were used for the secondary antibodies. For qualitative analysis, stained sections were analyzed with standard light and a Leica DMR fluorescence microscope. 2.6. Western blot analysis Homogenized samples of hippocampal tissues were loaded and the proteins were size-separated in 12% SDS–polyacrylamide gel electrophoresis. Then, proteins were transferred to polyvinylidene difluoride transfer membranes (Schleicher and Schuell, Germany). The membranes were blocked with 4% nonfatty dry milk in Tris-buffered saline containing Tween-20 (TTBS), then incubated at 4 °C overnight with rabbit polyclonal anti-BDNF antibodies (1:1000, Santa Cruz, USA) and rabbit polyclonal anti-TrkB antibodies (1:500, Santa Cruz, USA). After three washing steps with TTBS, the membranes were incubated with horseradish peroxidase-conjugated
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goat anti-rabbit secondary antibodies (1:2000, Sigma) for 1 h at room temperature. Proteins recognized by antibody labeling were detected using enhanced chemiluminescence according to the manufacturer’s instructions. Pre-stained protein markers were used for molecular weight determinations. The bands intensities were quantified by densitometry of immunoblots using NIH Image, version 1.61.
2.7. ELISA for BDNF Levels of BDNF of hippocampal tissues 24 h after SE were determined using an ELISA assay. Firstly, Samples of hippocampal tissues were homogenizedin 0.5 ml lysis buffer. Then, after centrifugation (10,000g for 10 min), 50 ll of the supernatant of each sample was used for the Promega ELISA kit (Koyama et al., 2004). All the procedures were performed following the manufacturer’s instruction manual.
2.8. Cell quantification Quantitative analysis of neuro-positive cells was performed as previously reported (Jung et al., 2006) in a predefined field of the hippocampal regions with optimal magnification to discriminate the cell outline. Selected corresponding coronal sections for comparisons were determined using the rat atlas of Paxinos and Watson (1997)). Neuro-positive cells in the CA1 and CA3 areas, hilus and dentate gyrus of the hippocampus (6 sections per animal; n = 4 for each group) were counted. The cell numbers were expressed as average number of cells within a predefined surface area of microscopic field. Values were expressed as mean ± standard error of mean (SEM).
2.9. Statistical analysis of data All values were shown as mean ± SEM. Statistical significance between groups was assessed by using one-way ANOVA, followed by post hoc Dunnett tests for multiple comparisons with the SPSS 10.0 program (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
3. Results 3.1. SE increases BDNF and TrkB expression in the hippocampus We examined BDNF expression using different groups of rats at 1, 2, 3, 5, 7, 14, 21, and 28 days after SE in the hippocampus. BDNF protein levels were determined by western blot analysis and ELISA from extracts obtained from the total protein content of each hippocampus in rats from the different groups. A long-lasting increase in BDNF levels was observed in the SE group compared with the control group during all time points studied (Fig. 1B). In the SE groups, BDNF levels dramatically increased at 24 h after SE, reached a peak at 7 days after SE (Fig. 1A and B), and decreased at 14 days, but BDNF levels remained significantly higher at all time points compared with the control group (Fig. 1B). Moreover, to determine if SE also modifies TrkB level TrkB proteins from the hippocampus of rats in the different groups were subjected to western blot analysis. A gradual increase in the fulllength protein (TrkB-fl) was observed at 12, 24 and 72 h after SE (Fig. 2A–D). However, the level of truncated (TrkB-tr) version was largely unchanged (Fig. 2A and B), indicating that SE specifically up-regulates TrkB-fl (Fig. 2A–D).
Fig. 1. BDNF expression in the different times is shown that from the different groups. (A) Western blot analysis of BDNF levels dramatically increase at 24 h after SE, reach a peak at 7 days after SE. (B) Using EILSA ana lysis is shown that a longlasting increase in BDNF levels is observed in the SE group. ⁄P < 0.05; ⁄⁄P < 0.01 vs. control animals. (n = 4 per group, ANOVA with Dunnett’s post hoc test).
3.2. Rosiglitazone inhibits the expression of BDNF and TrkB-fl after SE We investigated the effect of rosiglitazone on BDNF expression levels at 7 days after SE. Rosiglitazone significantly inhibited the expression of BDNF in the hippocampus (Fig. 3A). Quantitative analysis of BDNF levels revealed that BDNF levels were decreased by 42% in the rosiglitazone-treated group compared with the vehicle-treated SE group (Fig. 3B). Additionally, SE induced up-regulation of TrkB-fl protein was attenuated by rosiglitazone (Fig. 3C and D). Meanwhile, results also showed that the effects of rosiglitazone were significantly reversed by K252a. There was no statistically significant difference in expression of BDNF and TrkB-fl between the K252a with rosiglitazone group and the vehicle-treated SE group (Fig. 3B and D, P > 0.05 by ANOVA with Dunnett’s post test). 3.3. Rosiglitazone prevents neuronal loss after SE by acting on BDNF/ TrkB signaling We have previously shown that rosiglitazone prevented neuronal loss in the hippocampus after SE, and suggested that rosiglitazone exerts a neuroprotective effect by inhibiting inflammation and oxidative stress after SE (Sun et al., 2008; Yu et al., 2008). However, there is extensive evidence that demonstrates a direct relationship between BDNF/TrkB signaling and neuronal injury after brain injury (Unsain et al., 2008, 2009). To determine if PPARc agonists influence BDNF/TrkB signaling and exert a neuroprotective effect after SE, we examined the effect of the rosiglitazone on neuronal loss in the CA1 area of hippocampus after SE. Similar to our previous results, rosiglitazone significantly prevented neuronal loss in
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Fig. 2. TrkB level in the hippocampus is detected by western blot analysis. (A) A gradually increase TrkB-fl is observed at 12, 24 and 72 h after SE, however, the level of TrkB-tr is largely unchanged. (B) Densitometer analysis of the western blotting in A. ⁄P < 0.05, ⁄⁄P < 0.01 vs. at 12 h after SE. (C) The level of TrkB-fl is gradually increase compared with the control; (D) Densitometer analysis of the western blotting in C. ⁄P < 0.05, NP > 0.05 vs. control (n = 4 ANOVA with Dunnett’s post hoc test).
Fig. 3. Rosiglitazone inhibites the expression of BDNF and TrkB-fl after SE. TrkB antagonist K252a attenuates the effects of rosiglitazone. (A) Western blot analysis of BDNF in the hippocampus. (B) BDNF level mean band density ratios to b-actin ± S.E.M. (C) Western blot analysis of TrkB-fl in the hippocampus. (D) TrkB-fl level mean band density ratios to b-actin ± S.E.M. ⁄⁄P < 0.01, NP > 0.05 vs. vehicle-treated group; P < 0.01 vs. control; #P < 0.05 vs. rosiglitazone-treated group.
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the vehicle and rosiglitazone treated groups (Fig. 5). From 1 to 6 h after SE, the number of epileptiform waves and the amplitude progressively reduced in the two groups, and were not significantly different between the two groups (Fig. 5). 3.5. Rosiglitazone attenuates development of SRS in the chronic phase
Fig. 4. Rosiglitazone prevents hippocampal neuronal loss in the CA1 area at 14 days after SE. (A) Display of neuronal cells in the CA1 area without SE. (B) Vehicle-treated SE group shows a severe neuronal loss. (C) Rosiglitazone administration reverses the neuronal loss induced by SE. (D) K252a counteracts the effect of rosiglitazone when the group was co-treated with K252a. (E) Quantitative analysis of neuronal loss demonstrates that the rosiglitazone prevents the neuronal loss after SE. Data are expressed as mean ± S.E.M.; ⁄⁄P < 0.01 vs. vehicle-treated SE group; ##P < 0.01 vs. control; P < 0.05 vs. rosiglitazone-treated group. (n = 4, per group, ANOVA with Dunnett’s post hoc test). Scale bar (B) 50 mm; inset (B) 20 mm.
the CA1 area of the hippocampus 14 days after SE (Fig. 4B and C). However, the neuroprotective effect of rosiglitazone was significantly reversed by K252a (Fig. 4C and D). Intracerebroventricular administration of K252a 1 h before rosiglitazone aggravated neuronal loss compared with the rosiglitazone only treated group (Fig. 4C and D). There was no significant difference between the vehicle-treated SE group and the K252a group (P > 0.05 ANOVA with Dunnett’s post hoc test, Fig. 4E).
3.4. Rosiglitazone does not affect development and severity of SE in the acute phase We assessed the effect of rosiglitazone on the development and severity of SE by analyzing behavior and EEG recordings. The number of rats that developed SE (stage 4/5) was 77% (37/48) in the rosiglitazone-treated group, and 71% (34/48) in the vehicle-treated SE group (Table 1). The time to development of SE was 32.5 ± 2.9 min in the rosiglitazone-treated group and 36.8 ± 3.6 min in the vehicle-treated SE group (Table 1). Rosiglitazone did not affect the severity of seizures during the 1 h of SE, assessed by the frequency of stage 4/5 seizures and the mean seizure duration. There was no significant difference between the vehicletreated SE group and the rosiglitazone-treated group (7.30 ± 0.48 versus 6.56 ± 0.32, p > 0.05; 13.60 ± 2.80 versus 11.98 ± 2.15, p > 0.05, Table 1). In addition, rosiglitazone did not affect EEG pattern changes in the acute phase (Fig. 5). At 30 min of SE, a large number of high-voltage spikes and polyspikes were recorded in
To determine the effect of rosiglitazone on the development of SRS in the chronic phase, SRS were observed from a 6 week video recording and long-term EEG monitoring, which started 2 weeks after SE. A total of 83% (10/12) of rats in the vehicle-treated SE group had SRS, whereas 50% (6/12) of the rats in the rosiglitazone-treated group had SRS (Table 2). The time to development of SRS was longer in the rosiglitazone-treated group compared with the vehicle-treated SE group (SE + Vehicle group: 17.8 ± 7.5 days; SE + Ros group: 33.6 ± 11.2 days; SE + Ros + K252a group: 20.3 ± 6.9 days, Table 2). The mean number of seizures (total number of seizures/number of recording days, calculated separately for each animal) was 2.67 ± 0.43 seizures/day in the vehicle-treated SE group and 1.24 ± 0.21 in the rosiglitazone-treated group. The mean seizure duration (seconds/seizure) was 13.60 ± 2.80 s in the vehicle-treated SE group and 7.98 ± 2.15 s in the rosiglitazone-treated group. The differences between the two groups were significant (p < 0.05 by ANOVA with Dunnett’s post hoc test, Table 2). In addition, the number of epileptiform wave forms was less in the rosiglitazone-treated group compared with the vehicle-treated SE group, and paroxysmal discharges in the chronic phase were reduced in the rosiglitazone-treated group (Fig. 6A-C). 3.6. TrkB antagonist K252a attenuates antiepileptic effect of rosiglitazone after SE K252a was administered 1 h before administration of rosiglitazone, and rosiglitazone with K252a were administered every day after SE until the animal was sacrificed. K252a administration attenuated the antiepileptic effect of rosiglitazone in the chronic phase. The number of rats that developed SRS was 50% (6/12) in the rosiglitazone-treated group, and 75% (9/12) in the rosiglitazone-added K252a treated group (Table 2). The time to development and duration of SRS were significantly different between the two groups (Table 2). K252a administration also altered the number of paroxysmal discharges and epileptiform waves compared with the group solely treated with rosiglitazone (Fig. 6C and D). 4. Discussion In the present study, we have demonstrated that the PPARc agonist rosiglitazone exerted a neuroprotective effect after SE, and that this effect was reversed by K252a, an antagonist of TrkB, suggesting that neuroprotection by rosiglitazone involves BDNF/ TrkB signaling. More importantly, our study showed that rosiglitazone suppressed SRS development, and that K252a attenuated this antiepileptic effect of rosiglitazone. In addition, rosiglitazone did not affect the development and severity of SE. Taken together, these findings show that rosiglitazone exerts a neuroprotective and antiepileptic effect involve BDNF/TrkB signaling. In the present, we found increase BDNF exacerbate neuronal loss after SE. Contrarily, several reports have shown that BDNF can prevent neuronal loss in the hippocampus (Kuramoto et al., 2011), these studies have shown only a temporary effect of BDNF in acute models of epilepsy (Kuramoto et al., 2011; Bovolenta et al., 2010). BDNF supports neuronal survival in the acute phase after SE, but long-lasting increases in BDNF may contribute to damage in the chronic phase after SE. Increases of BDNF may be
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Table 1 Parameters describing the development and severity of SE in acute phase.
Li–Pilo + Vehicle Li–Pilo + Ros Li–Pilo + Ros + K252a
The number of rats developed SE
The time to development of SRS (min)
The frequency of state 4/5 seizures (1 h)
The mean seizure duration (sec/seizure)
37 (48) 34 (48)* 39 (48)*
36.8 ± 3.6 32.5 ± 2.9* 35.3 ± 2.6*
7.30 ± 0.48 6.56 ± 0.32* 6.98 ± 0.45*
13.60 ± 2.80 11.98 ± 2.15* 12.48 ± 1.35*
Note: rosiglitazone or rosiglitazone added K252a did not affect development and severity of SE in acute phase. Data are means ± S.E.M. * P > 0.05, there were not statistically significant differences from Li-Pilo + Vehicle rats.
Fig. 5. EEG patterns are shown that in the vehicle and rosiglitazone treated groups. Rosiglitazone treated rats exhibit an EEG pattern close to that of the vehicle-treated groups. Rosiglitazone not affects EEG pattern change in acute phase.
part of an endogenous neuroprotective mechanism in pathological insults of epilepsy. Moreover, TrkB-tr was unchanged in the present study, and is also consistent with previous studies (Unsain et al., 2008). This suggests that TrkB-tr is not recycled to the cell surface and targeted to the lysosomal degradation system, as suggested previously (Rose et al., 2003). Recently, studies have shown that up-regulation of BDNF could participate in the establishment and persistence of neuronal hyperexcitability in hippocampal networks during the chronic phase after SE, thus promoting epileptogenesis (Heinrich et al., 2011; Unsain et al., 2008; Koyama et al., 2004). The present study further confirmed that SE induces alterations in BDNF/TrkB signaling and worsens neuronal damage and promotes epileptogenesis. Data have shown that PPARc agonists have a neuroprotective effect in CNS diseases (Bordet et al., 2006; Kapadia et al., 2008), but the mechanisms of action are not clear. Most studies have shown that PPARc action in the CNS is anti-inflammatory and anti-oxidative. However, emerging evidence suggests that PPARc could regulate the expression of many genes in the brain including neurotransmitters and BDNF (Yu et al., 2012; Wang et al., 2011; Okada et al., 2006). Further study showed that PPARc could correct disturbances in neurotransmitter concentrations after an insult to the brain (Abdallah, 2010). Studies showed that activated PPARc can prevent the binding of transcription factors AP-1 and NF-kB proteins to their target sequences (Gao et al., 2011) blocking of NF-kB signaling inhibited the increased activities of BDNF promoters I and IV (Kairisalo et al., 2009). The current study showed that rosiglitazone can modify the expression level BDNF/TrkB after SE. Thus, we presume that rosiglitazone may modulate BDNF/TrkB pathway through NF-KB signaling in the
Fig. 6. EEG patterns are shown that in the different groups in the chronic phase. (A) Normal state of alpha and beta waves in the control group. (B) The number of epileptiform wave forms are shown that in vehicle-treated group. (C) Minor spikes and epileptiform wave forms are presented in the rosiglitazone-treated group. (D) Quite a few spikes and epileptiform wave forms are presented in the rosiglitazoneadded K252a rats.
epilepsy. In addition, our previous study showed that rosiglitazone attenuated seizure-induced neuronal loss and suggested that rosiglitazone may exert antiepileptogenic effect (Sun et al., 2008, 2012). Thus, rosiglitazone exerts antiepileptogenic effect possibly through different pathways. Previous studies have shown that the PPARc agonist pioglitazone has an anticonvulsant effect in different models of epilepsy (Okada et al., 2006; Abdallah, 2010). Studies have also demonstrated that the PPARc ligand, FMOC-l-leucine possesses an antiepileptic effect against audiogenic seizure induced in adult magnesium-deficient mice (Maurois et al., 2008). Although evidence indicated that activation of PPARc exerts antiepileptic properties, the mechanisms of action were mainly considered to be regulation of inflammatory and oxidative pathways (Adabi et al., 2012). In contrast, the present study shows that the antiepileptic effect of rosiglitazone is related to its regulation of BDNF/TrkB signaling. And we found that rosiglitazone did not affect development and severity of SE, but it significantly suppressed SRS development, assessed by behavior recordings and EEG monitoring. It is difficult to explain this discrepancy because there is a paucity of studies examining changes in behavior or EEG after rosiglitazone treatment in the lithium pilocarpine-induced model of epilepsy. However, it is likely due to the model of epilepsy used, the choice of agonist, and other factors. Previous studies have mostly used the pentylenetetrazol (PTZ)-kindling model, which is considered an acute model of epilepsy, and use of pioglitazone in the acute phase of epilepsy inhibited the severity of SE and was considered an anticonvulsant effect (Okada et al., 2006; Abdallah, 2010; Adabi et al., 2012). In contrast, the lithium pilocarpine model is a chronic epileptic model. It is considered close to being the ideal homologous
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S. Hong et al. / Neurochemistry International 63 (2013) 405–412 Table 2 Parameters describing the development and severity of SRS in chronic phase.
Li–Pilo + Vehicle Li–Pilo + Ros Li–Pilo + Ros + K252a
The number of rats developed SRS
The time to development of SRS (days)
The mean number of seizures (seizures/day)
The mean seizure duration (sec/seizure)
10 (12) 6 (12)a,c 9 (12)b
17.8 ± 7.5 33.6 ± 11.2a,c 20.3 ± 6.9b
2.67 ± 0.43 1.24 ± 0.21a,c 2.56 ± 0.37b
13.60 ± 2.80 7.98 ± 2.15a,c 12.48 ± 1.35b
Note: rosiglitazone attenuated development and severity of SRS, but added K252a reversed this effect in chronic phase. Data are means ± S.E.M. P < 0.01. b P > 0.05 vs. vehicle treated rats. c P < 0.05 vs. rosiglitazone added K252a treated rats.
a
model of temporal lobe epilepsy (TLE). In fact, previous studies have indicated that the antiepileptogenic and anticonvulsant effects were dissociative (Suchomelova et al., 2006). Partial antiepileptic drugs (AEDs) may have antiepileptogenic properties, but it is uncertain whether they have anticonvulsant properties (Suchomelova et al., 2006). For example, a study showed that topiramate could not modify the course of SE, yet completely prevented SRS (Suchomelova et al., 2006; Rigoulot et al., 2004). In addition, an NMDA receptor antagonist did not block seizures, but effectively prevented the epileptogenic process (Khalilov et al., 2003). In studies of epilepsy, there are many pathological mechanisms related to SRS in the latent phase. These events include neuronal loss, glial cell activation, and hippocampal circuit reorganization (Siebzehnrubl and Blumcke, 2008; Holopainen, 2008; Walker, 2007). Although there is no definitive evidence that any of these pathological changes are critical in the development of SRS (Kobow et al., 2012), some studies have suggested that neuronal loss and network reorganization participate in the initiation of SRS, and lead to chronic epilepsy (Ben-Ari and Dudek, 2010; Zhang et al., 2002). More importantly, data has indicated that BDNF/TrkB signaling appears to be vital for modulating neuronal network reorganization (Zheng et al., 2011). This suggests that SE-induced BDNF increases might contribute to lasting structural and functional changes in the hippocampus contributing to SRS and epileptogenesis. Our study found that rosiglitazone attenuates the development and severity of SRS after SE. And these effects were reversed when the TrkB antagonist K252a was administrated. K252a, as antagonist of TrkB (a non-selective kinase inhibitor with affinity TrkB), and it is widely used for blocking BDNF/TrkB signaling and inhibiting TrkB activation. Thus, this further supports the notion that inhibiting BDNF/trkB signaling attenuates the risk of SRS, and also confirmed that rosiglitazone can exert antiepileptogenic effects through BDNF/TrkB signaling. In addition, this study has shown that rosiglitazone does not affect the development and severity of SE, and suggests that the key role of PPARc is mainly in the chronic phase. Specifically, PPARc modulates cytokine and microglia mediated inflammation (Sun et al., 2008; Yu et al., 2008), while seizure-induced expression of cytokines mostly occurs during the chronic phase alongside increases in BDNF. We found that BDNF level peaked at 7 days, and microglia began to be activated at 12 h and peaked in number at 6–7 days after SE (Sun et al., 2008). BDNF is mostly produced by activated microglia (Zhou et al., 2011; Miao et al., 2012). Thus, this evidence also indirectly supports the finding here that rosiglitazone affects the development of SRS through BDNF signal pathways, but has no effect on the development of SE. In summary, this study provides additional evidence concerning the neuroprotective effects of the PPARc agonist rosiglitazone, and offers new perspectives for the treatment of epilepsy. Importantly, a direct antiepileptic effect of rosiglitazone was demonstrated in this study. In addition, we showed that the neuroprotective and antiepileptic effects of rosiglitazone are derived, at least in part, by regulation of BDNF/TrkB signaling. These results suggest that alteration of BDNF/TrkB signaling may inhibit the development of SRS, and exert an antiepileptic effect.
Acknowledgment This study was supported by the ‘‘Xi’an Jiaotong University Campus Fund’’. We thank Dr. Dong Yan (Department of Immunology, Xijing Hospital) for his valuable technical advice.
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Neurochemistry International journal homepage: www.elsevier.com/locate/nci
The PPARc agonist rosiglitazone prevents neuronal loss and attenuates development of spontaneous recurrent seizures through BDNF/TrkB signaling following pilocarpine-induced status epilepticus Hong Sun a,⇑, Xin Yu b, HaiQin Wu a, GuiLian Zhang a, Ru Zhang a, ShuQin Zhan a, HuQing Wang a, Li Yao a, Ning Bu a, YongNan Li c a
Department of Neurology, The Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004, Shaanxi Province, China Department of Neurology, People’s Liberation Army 401 Hospital, Qingdao, Shandong 266071, China c Department of Neurology, The Fourth Affiliated Hospital, Harbin Medical University, Harbin 150001, Heilongjiang Province, China b
a r t i c l e
i n f o
Article history: Received 1 February 2013 Received in revised form 14 July 2013 Accepted 22 July 2013 Available online 6 August 2013 Keywords: Epilepsy BDNF TrkB PPARc Rosiglitazone Spontaneous recurrent seizures K252a
a b s t r a c t Hippocampal neuronal loss plays an important role in epileptogenesis, and it is considered a trigger of repeated spontaneous recurrent seizures (SRS). The BDNF/TrkB signaling pathway regulates neuronal plasticity in the CNS, and promotes epileptogenesis. Previous studies have shown that Peroxisome proliferator-activated receptor gamma (PPARc) agonists exert neuroprotective effects by inhibiting oxidative stress and inflammation in epilepsy. In the present study, the PPARc agonist rosiglitazone inhibited increases in BDNF and TrkB after status epilepticus (SE), and also prevented hippocampal neuronal loss. More importantly, our study showed that rosiglitazone suppressed SRS. However, the effects of rosiglitazone were significantly reversed by cotreatment with K252a, an antagonist of TrkB. Additionally, rosiglitazone did not affect the development and severity of SE. Thus, our data provide evidence that rosiglitazone exerts neuroprotective and antiepileptic effects involve BDNF/TrkB signaling. Our study also offers new perspectives for the treatment of epilepsy. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Hippocampal neuronal loss is a feature of the neuropathology of epilepsy. Specifically, it is characterized by profound neuronal loss in the CA1, CA3 and the hilus regions of the hippocampus, and glial cell activation and neurogenesis in different animal models of epilepsy (Siebzehnrubl and Blumcke, 2008; Holopainen, 2008). These pathological changes may contribute to alterations in neuronal circuits of the epileptic brain, and may play a role in epileptogenesis (Walker, 2007). Growing evidence indicates that neuronal degeneration is critical for plastic changes in hippocampal neuronal networks, and it is considered a powerful trigger for repeated spontaneous recurrent seizures (SRS) (Ben-Ari and Dudek, 2010; Zhang et al., 2002), but the molecular mechanisms are still poorly understood.
⇑ Corresponding author. Address: Department of Neurology, The Second Affiliated Hospital, Medical School of Xi’an Jiaotong University, No.157 West 5 Road, Xi’an 710004, Shaanxi Province, China. Tel./fax: +86 29 87679249. E-mail addresses: [email protected], [email protected] (S. Hong). 0197-0186/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuint.2013.07.010
Brain-derived neurotrophic factor (BDNF) is mediated activation of the TrkB signal transduction pathway regulates neuronal plasticity in the CNS (Autry and Monteggia, 2012). In recent years, studies have suggested that BDNF can enhance neurogenesis and regulate neuronal circuits in the hippocampus in an epileptic model (Kuramoto et al., 2011). Previous studies have demonstrated that BDNF and TrkB mRNA and protein are up-regulated in the hippocampus after status epilepticus (SE), suggesting that BDNF may have a protective role against seizure-mediated excitotoxicity (Danzer et al., 2004; Paradiso et al., 2009; Kuramoto et al., 2011). Chronic intrahippocampal infusion of BDNF delayed amygdala kindling-induced seizure development, and reduced the increase in after discharge duration (Osehobo et al., 1999). However, other studies have suggested that increased BDNF could contribute to lasting structural and functional changes, and establish neuronal hyperexcitability in the hippocampus, thus promoting epileptogenesis (Kotloski and McNamara., 2010; Scharfman, 2005). Similarly, it has been demonstrated that transgenic mice overexpressing BDNF show increased SE severity in a KA-induced model of epilepsy (Barton and Shannon, 2005). In addition, BDNF up-regulation also accelerated neuronal apoptosis in the hippocampus and increased SRS (Heinrich et al., 2011). Thus, the role
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of BDNF/TrkB signaling in the development of epilepsy is controversial. Moreover, it is still uncertain whether BDNF upregulation and TrkB activation are a result of initial seizure induction and play a primary role in the epileptogenic process, or are a secondary consequence of excitotoxicity from repeated SRS. Peroxisome proliferator-activated receptor gamma (PPARc) is a nuclear membrane-associated transcription factor. In the past several years, a number of studies have shown that PPARc agonists exert neuroprotective effects in models of neurological damage and disease (Bordet et al., 2006; Kapadia et al., 2008), and some clinical data also confirming these experimental results (Gamboa et al., 2010). Importantly, our previous study showed that the PPARc agonist rosiglitazone attenuated seizure-induced neuronal loss and improved cognitive impairment by inhibiting inflammation and oxidative stress in lithium pilocarpine-induced SE in rats (Sun et al., 2008, 2012). Indeed, most studies have focused on the role of PPARc in anti-inflammatory and anti-oxidative actions, but no studies have attempted to address whether PPARc can influence BDNF/TrkB signaling in diseases of the CNS. In addition, it is not clear if PPARc agonists can influence SRS and SE in epilepsy. Thus, the aim of the present study was to determine whether rosiglitazone inhibits neuronal loss via-regulation BDNF/TrkB signaling in the epilepsy. And further to observe influence of rosiglitazone in the SRS. First, we examined BDNF and TrkB expression after SE, and the role of rosiglitazone in modulation of BDNF and TrkB expression. Next, we evaluated the effect of rosiglitazone on SRS and SE through observation of behavior and EEG in the lithium–pilocarpine model of epilepsy.
2.3. EEG recording and behavioral observation All undergoing Li–Pilo-induced SE was used for cortical electroencephalographic (EEG) recording. The bilateral EEG cortical activity was recorded during the acute (SE and 12 h after), and the chronic (14 days after SE) phases. In the chronic phase, a 3 h EEG recording was obtained every day until convulsive SRS at stages 3–5 was observed. SRS occurrence was evaluated through a 6 week video recording started 2 weeks after SE. All recordings for SRS were done during the light period. Epileptic rats were video recording for at least 12 h daily for 6 week from starting 2 week after SE. The frequency and duration of stage 4/5 seizures were recorded, and the severity of SRS was scored according to the Racine’s scale (Racine, 1972). The recordings were analyzed by observers who were blind to the treatment of each rat. 2.4. Drug administration Animals were randomly assigned into 4 groups: Control group, SE group, SE + rosiglitazone group and SE + rosiglitazone + K252a group. All drug solutions were prepared fresh daily. Rosiglitazone (Cayman Chemical, USA) was diluted in a 30% DMSO and 70% isotonic saline solution, and intracerebroventricularly injected (i.c.v) at doses of 100 lg/kg at 1 h before the injections of lithium chloride. K252a (10 lg in PBS containing 1% DMSO, Calbiochem, San Diego, CA) was administered i.c.v at 1 h before rosiglitazone, this dose was found that it blocked the effect of exogenously administered BDNF on serotonin clearance (Benmansour et al., 2008). For long-term survival studies (12–42 days), rosiglitazone and/or K252a were administered everyday as described above until every animal was sacrificed.
2. Materials and methods 2.5. Immunohistochemistry and histology 2.1. Lithium pilocarpine-induced model of epilepsy Male Sprague–Dawley rats (weight 200–220 g; 12 weeks old) were used for all experiments. The detailed procedure was performed as previously described (Sun et al., 2008). Lithium chloride (127 mg/kg, i.p., Sigma) was injected 24 h prior to the administration of pilocarpine. One single dose of pilocarpine hydrochloride (30 mg/kg, i.p., Sigma) was administered. Then, the rats received repeated injections of pilocarpine (10 mg/kg, i.p.) every 30 min until they developed seizures which were scored by Racine (1972))’s scale. Control rats (n = 4) received all treatments with saline instead of pilocarpine. In order to reduce the mortality of rats, we require that the duration of the SE is the seizure lasted for 1 h (Glien et al., 2001). All animal protocol conformed to the Guide for the Care and Use of Laboratory Animals (Declaration of Helsinki of the World Medical Association).
2.2. Electrodes and cannula implantation Intracerebroventriclar administration was performed as described previously (Rigoulot et al., 2004; Yananli et al., 2008). Briefly, the rats were fixed in a stereotaxic frame, anesthetized, and a midline incision was made along the scalp. The skull was exposed and a burr hole was made in the location over the left sensorimotor cortex, 2 mm posterior and 2 mm lateral to bregma. The guide cannula was implanted 1.5 mm ventral to the superior surface of the skull. Then six single-contact electrodes were placed epidural 2 mm lateral to sagittal suture on both sides: 2 mm anterior, 2 mm posterior, 6 mm posterior to the bregma. And the electrodes and guide cannula fixed with jeweler acrylic cement. Animals were allowed to recover for one week prior to EEG recordings.
At each time point, rats were deeply anesthetized with pentobarbital (1.8 g/kg, Sigma) and perfused transcardially with ice-cold phosphate buffer (PB, 0.1 M, pH 7.4) followed by ice-cold fixative (4% paraformaldehyde in 0.1 M PB, pH 7.4). Brains were removed immediately after perfusion, postfixed in the same fixative one night at 4 °C and impregnated with 20% sucrose diluted in PB for cryoprotection. Brains were subsequently cut into 10 um thick coronal sections on a freezing microtome. The specimens were pretreated with 0.3% hydrogen peroxide in PB for 15 min at room temperature and then rinsed several times in PB and incubated for 2 h with blocking buffer (2% horse serum/1% BSA/0.1% Triton X-100 in PBS, pH 7.5), incubated overnight at 4 °C with primary antibodies, and then incubated for 1 h at room temperature with secondary antibodies. Mouse anti-rat NeuN (1:2000, Chemicon) monoclonal antibody was used as markers for neuron. Biotinylated goat antimouse IgG (1:500, Sigma) and FITC-conjugated anti-mouse (1:500, Sigma) were used for the secondary antibodies. For qualitative analysis, stained sections were analyzed with standard light and a Leica DMR fluorescence microscope. 2.6. Western blot analysis Homogenized samples of hippocampal tissues were loaded and the proteins were size-separated in 12% SDS–polyacrylamide gel electrophoresis. Then, proteins were transferred to polyvinylidene difluoride transfer membranes (Schleicher and Schuell, Germany). The membranes were blocked with 4% nonfatty dry milk in Tris-buffered saline containing Tween-20 (TTBS), then incubated at 4 °C overnight with rabbit polyclonal anti-BDNF antibodies (1:1000, Santa Cruz, USA) and rabbit polyclonal anti-TrkB antibodies (1:500, Santa Cruz, USA). After three washing steps with TTBS, the membranes were incubated with horseradish peroxidase-conjugated
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goat anti-rabbit secondary antibodies (1:2000, Sigma) for 1 h at room temperature. Proteins recognized by antibody labeling were detected using enhanced chemiluminescence according to the manufacturer’s instructions. Pre-stained protein markers were used for molecular weight determinations. The bands intensities were quantified by densitometry of immunoblots using NIH Image, version 1.61.
2.7. ELISA for BDNF Levels of BDNF of hippocampal tissues 24 h after SE were determined using an ELISA assay. Firstly, Samples of hippocampal tissues were homogenizedin 0.5 ml lysis buffer. Then, after centrifugation (10,000g for 10 min), 50 ll of the supernatant of each sample was used for the Promega ELISA kit (Koyama et al., 2004). All the procedures were performed following the manufacturer’s instruction manual.
2.8. Cell quantification Quantitative analysis of neuro-positive cells was performed as previously reported (Jung et al., 2006) in a predefined field of the hippocampal regions with optimal magnification to discriminate the cell outline. Selected corresponding coronal sections for comparisons were determined using the rat atlas of Paxinos and Watson (1997)). Neuro-positive cells in the CA1 and CA3 areas, hilus and dentate gyrus of the hippocampus (6 sections per animal; n = 4 for each group) were counted. The cell numbers were expressed as average number of cells within a predefined surface area of microscopic field. Values were expressed as mean ± standard error of mean (SEM).
2.9. Statistical analysis of data All values were shown as mean ± SEM. Statistical significance between groups was assessed by using one-way ANOVA, followed by post hoc Dunnett tests for multiple comparisons with the SPSS 10.0 program (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered statistically significant.
3. Results 3.1. SE increases BDNF and TrkB expression in the hippocampus We examined BDNF expression using different groups of rats at 1, 2, 3, 5, 7, 14, 21, and 28 days after SE in the hippocampus. BDNF protein levels were determined by western blot analysis and ELISA from extracts obtained from the total protein content of each hippocampus in rats from the different groups. A long-lasting increase in BDNF levels was observed in the SE group compared with the control group during all time points studied (Fig. 1B). In the SE groups, BDNF levels dramatically increased at 24 h after SE, reached a peak at 7 days after SE (Fig. 1A and B), and decreased at 14 days, but BDNF levels remained significantly higher at all time points compared with the control group (Fig. 1B). Moreover, to determine if SE also modifies TrkB level TrkB proteins from the hippocampus of rats in the different groups were subjected to western blot analysis. A gradual increase in the fulllength protein (TrkB-fl) was observed at 12, 24 and 72 h after SE (Fig. 2A–D). However, the level of truncated (TrkB-tr) version was largely unchanged (Fig. 2A and B), indicating that SE specifically up-regulates TrkB-fl (Fig. 2A–D).
Fig. 1. BDNF expression in the different times is shown that from the different groups. (A) Western blot analysis of BDNF levels dramatically increase at 24 h after SE, reach a peak at 7 days after SE. (B) Using EILSA ana lysis is shown that a longlasting increase in BDNF levels is observed in the SE group. ⁄P < 0.05; ⁄⁄P < 0.01 vs. control animals. (n = 4 per group, ANOVA with Dunnett’s post hoc test).
3.2. Rosiglitazone inhibits the expression of BDNF and TrkB-fl after SE We investigated the effect of rosiglitazone on BDNF expression levels at 7 days after SE. Rosiglitazone significantly inhibited the expression of BDNF in the hippocampus (Fig. 3A). Quantitative analysis of BDNF levels revealed that BDNF levels were decreased by 42% in the rosiglitazone-treated group compared with the vehicle-treated SE group (Fig. 3B). Additionally, SE induced up-regulation of TrkB-fl protein was attenuated by rosiglitazone (Fig. 3C and D). Meanwhile, results also showed that the effects of rosiglitazone were significantly reversed by K252a. There was no statistically significant difference in expression of BDNF and TrkB-fl between the K252a with rosiglitazone group and the vehicle-treated SE group (Fig. 3B and D, P > 0.05 by ANOVA with Dunnett’s post test). 3.3. Rosiglitazone prevents neuronal loss after SE by acting on BDNF/ TrkB signaling We have previously shown that rosiglitazone prevented neuronal loss in the hippocampus after SE, and suggested that rosiglitazone exerts a neuroprotective effect by inhibiting inflammation and oxidative stress after SE (Sun et al., 2008; Yu et al., 2008). However, there is extensive evidence that demonstrates a direct relationship between BDNF/TrkB signaling and neuronal injury after brain injury (Unsain et al., 2008, 2009). To determine if PPARc agonists influence BDNF/TrkB signaling and exert a neuroprotective effect after SE, we examined the effect of the rosiglitazone on neuronal loss in the CA1 area of hippocampus after SE. Similar to our previous results, rosiglitazone significantly prevented neuronal loss in
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Fig. 2. TrkB level in the hippocampus is detected by western blot analysis. (A) A gradually increase TrkB-fl is observed at 12, 24 and 72 h after SE, however, the level of TrkB-tr is largely unchanged. (B) Densitometer analysis of the western blotting in A. ⁄P < 0.05, ⁄⁄P < 0.01 vs. at 12 h after SE. (C) The level of TrkB-fl is gradually increase compared with the control; (D) Densitometer analysis of the western blotting in C. ⁄P < 0.05, NP > 0.05 vs. control (n = 4 ANOVA with Dunnett’s post hoc test).
Fig. 3. Rosiglitazone inhibites the expression of BDNF and TrkB-fl after SE. TrkB antagonist K252a attenuates the effects of rosiglitazone. (A) Western blot analysis of BDNF in the hippocampus. (B) BDNF level mean band density ratios to b-actin ± S.E.M. (C) Western blot analysis of TrkB-fl in the hippocampus. (D) TrkB-fl level mean band density ratios to b-actin ± S.E.M. ⁄⁄P < 0.01, NP > 0.05 vs. vehicle-treated group; P < 0.01 vs. control; #P < 0.05 vs. rosiglitazone-treated group.
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the vehicle and rosiglitazone treated groups (Fig. 5). From 1 to 6 h after SE, the number of epileptiform waves and the amplitude progressively reduced in the two groups, and were not significantly different between the two groups (Fig. 5). 3.5. Rosiglitazone attenuates development of SRS in the chronic phase
Fig. 4. Rosiglitazone prevents hippocampal neuronal loss in the CA1 area at 14 days after SE. (A) Display of neuronal cells in the CA1 area without SE. (B) Vehicle-treated SE group shows a severe neuronal loss. (C) Rosiglitazone administration reverses the neuronal loss induced by SE. (D) K252a counteracts the effect of rosiglitazone when the group was co-treated with K252a. (E) Quantitative analysis of neuronal loss demonstrates that the rosiglitazone prevents the neuronal loss after SE. Data are expressed as mean ± S.E.M.; ⁄⁄P < 0.01 vs. vehicle-treated SE group; ##P < 0.01 vs. control; P < 0.05 vs. rosiglitazone-treated group. (n = 4, per group, ANOVA with Dunnett’s post hoc test). Scale bar (B) 50 mm; inset (B) 20 mm.
the CA1 area of the hippocampus 14 days after SE (Fig. 4B and C). However, the neuroprotective effect of rosiglitazone was significantly reversed by K252a (Fig. 4C and D). Intracerebroventricular administration of K252a 1 h before rosiglitazone aggravated neuronal loss compared with the rosiglitazone only treated group (Fig. 4C and D). There was no significant difference between the vehicle-treated SE group and the K252a group (P > 0.05 ANOVA with Dunnett’s post hoc test, Fig. 4E).
3.4. Rosiglitazone does not affect development and severity of SE in the acute phase We assessed the effect of rosiglitazone on the development and severity of SE by analyzing behavior and EEG recordings. The number of rats that developed SE (stage 4/5) was 77% (37/48) in the rosiglitazone-treated group, and 71% (34/48) in the vehicle-treated SE group (Table 1). The time to development of SE was 32.5 ± 2.9 min in the rosiglitazone-treated group and 36.8 ± 3.6 min in the vehicle-treated SE group (Table 1). Rosiglitazone did not affect the severity of seizures during the 1 h of SE, assessed by the frequency of stage 4/5 seizures and the mean seizure duration. There was no significant difference between the vehicletreated SE group and the rosiglitazone-treated group (7.30 ± 0.48 versus 6.56 ± 0.32, p > 0.05; 13.60 ± 2.80 versus 11.98 ± 2.15, p > 0.05, Table 1). In addition, rosiglitazone did not affect EEG pattern changes in the acute phase (Fig. 5). At 30 min of SE, a large number of high-voltage spikes and polyspikes were recorded in
To determine the effect of rosiglitazone on the development of SRS in the chronic phase, SRS were observed from a 6 week video recording and long-term EEG monitoring, which started 2 weeks after SE. A total of 83% (10/12) of rats in the vehicle-treated SE group had SRS, whereas 50% (6/12) of the rats in the rosiglitazone-treated group had SRS (Table 2). The time to development of SRS was longer in the rosiglitazone-treated group compared with the vehicle-treated SE group (SE + Vehicle group: 17.8 ± 7.5 days; SE + Ros group: 33.6 ± 11.2 days; SE + Ros + K252a group: 20.3 ± 6.9 days, Table 2). The mean number of seizures (total number of seizures/number of recording days, calculated separately for each animal) was 2.67 ± 0.43 seizures/day in the vehicle-treated SE group and 1.24 ± 0.21 in the rosiglitazone-treated group. The mean seizure duration (seconds/seizure) was 13.60 ± 2.80 s in the vehicle-treated SE group and 7.98 ± 2.15 s in the rosiglitazone-treated group. The differences between the two groups were significant (p < 0.05 by ANOVA with Dunnett’s post hoc test, Table 2). In addition, the number of epileptiform wave forms was less in the rosiglitazone-treated group compared with the vehicle-treated SE group, and paroxysmal discharges in the chronic phase were reduced in the rosiglitazone-treated group (Fig. 6A-C). 3.6. TrkB antagonist K252a attenuates antiepileptic effect of rosiglitazone after SE K252a was administered 1 h before administration of rosiglitazone, and rosiglitazone with K252a were administered every day after SE until the animal was sacrificed. K252a administration attenuated the antiepileptic effect of rosiglitazone in the chronic phase. The number of rats that developed SRS was 50% (6/12) in the rosiglitazone-treated group, and 75% (9/12) in the rosiglitazone-added K252a treated group (Table 2). The time to development and duration of SRS were significantly different between the two groups (Table 2). K252a administration also altered the number of paroxysmal discharges and epileptiform waves compared with the group solely treated with rosiglitazone (Fig. 6C and D). 4. Discussion In the present study, we have demonstrated that the PPARc agonist rosiglitazone exerted a neuroprotective effect after SE, and that this effect was reversed by K252a, an antagonist of TrkB, suggesting that neuroprotection by rosiglitazone involves BDNF/ TrkB signaling. More importantly, our study showed that rosiglitazone suppressed SRS development, and that K252a attenuated this antiepileptic effect of rosiglitazone. In addition, rosiglitazone did not affect the development and severity of SE. Taken together, these findings show that rosiglitazone exerts a neuroprotective and antiepileptic effect involve BDNF/TrkB signaling. In the present, we found increase BDNF exacerbate neuronal loss after SE. Contrarily, several reports have shown that BDNF can prevent neuronal loss in the hippocampus (Kuramoto et al., 2011), these studies have shown only a temporary effect of BDNF in acute models of epilepsy (Kuramoto et al., 2011; Bovolenta et al., 2010). BDNF supports neuronal survival in the acute phase after SE, but long-lasting increases in BDNF may contribute to damage in the chronic phase after SE. Increases of BDNF may be
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Table 1 Parameters describing the development and severity of SE in acute phase.
Li–Pilo + Vehicle Li–Pilo + Ros Li–Pilo + Ros + K252a
The number of rats developed SE
The time to development of SRS (min)
The frequency of state 4/5 seizures (1 h)
The mean seizure duration (sec/seizure)
37 (48) 34 (48)* 39 (48)*
36.8 ± 3.6 32.5 ± 2.9* 35.3 ± 2.6*
7.30 ± 0.48 6.56 ± 0.32* 6.98 ± 0.45*
13.60 ± 2.80 11.98 ± 2.15* 12.48 ± 1.35*
Note: rosiglitazone or rosiglitazone added K252a did not affect development and severity of SE in acute phase. Data are means ± S.E.M. * P > 0.05, there were not statistically significant differences from Li-Pilo + Vehicle rats.
Fig. 5. EEG patterns are shown that in the vehicle and rosiglitazone treated groups. Rosiglitazone treated rats exhibit an EEG pattern close to that of the vehicle-treated groups. Rosiglitazone not affects EEG pattern change in acute phase.
part of an endogenous neuroprotective mechanism in pathological insults of epilepsy. Moreover, TrkB-tr was unchanged in the present study, and is also consistent with previous studies (Unsain et al., 2008). This suggests that TrkB-tr is not recycled to the cell surface and targeted to the lysosomal degradation system, as suggested previously (Rose et al., 2003). Recently, studies have shown that up-regulation of BDNF could participate in the establishment and persistence of neuronal hyperexcitability in hippocampal networks during the chronic phase after SE, thus promoting epileptogenesis (Heinrich et al., 2011; Unsain et al., 2008; Koyama et al., 2004). The present study further confirmed that SE induces alterations in BDNF/TrkB signaling and worsens neuronal damage and promotes epileptogenesis. Data have shown that PPARc agonists have a neuroprotective effect in CNS diseases (Bordet et al., 2006; Kapadia et al., 2008), but the mechanisms of action are not clear. Most studies have shown that PPARc action in the CNS is anti-inflammatory and anti-oxidative. However, emerging evidence suggests that PPARc could regulate the expression of many genes in the brain including neurotransmitters and BDNF (Yu et al., 2012; Wang et al., 2011; Okada et al., 2006). Further study showed that PPARc could correct disturbances in neurotransmitter concentrations after an insult to the brain (Abdallah, 2010). Studies showed that activated PPARc can prevent the binding of transcription factors AP-1 and NF-kB proteins to their target sequences (Gao et al., 2011) blocking of NF-kB signaling inhibited the increased activities of BDNF promoters I and IV (Kairisalo et al., 2009). The current study showed that rosiglitazone can modify the expression level BDNF/TrkB after SE. Thus, we presume that rosiglitazone may modulate BDNF/TrkB pathway through NF-KB signaling in the
Fig. 6. EEG patterns are shown that in the different groups in the chronic phase. (A) Normal state of alpha and beta waves in the control group. (B) The number of epileptiform wave forms are shown that in vehicle-treated group. (C) Minor spikes and epileptiform wave forms are presented in the rosiglitazone-treated group. (D) Quite a few spikes and epileptiform wave forms are presented in the rosiglitazoneadded K252a rats.
epilepsy. In addition, our previous study showed that rosiglitazone attenuated seizure-induced neuronal loss and suggested that rosiglitazone may exert antiepileptogenic effect (Sun et al., 2008, 2012). Thus, rosiglitazone exerts antiepileptogenic effect possibly through different pathways. Previous studies have shown that the PPARc agonist pioglitazone has an anticonvulsant effect in different models of epilepsy (Okada et al., 2006; Abdallah, 2010). Studies have also demonstrated that the PPARc ligand, FMOC-l-leucine possesses an antiepileptic effect against audiogenic seizure induced in adult magnesium-deficient mice (Maurois et al., 2008). Although evidence indicated that activation of PPARc exerts antiepileptic properties, the mechanisms of action were mainly considered to be regulation of inflammatory and oxidative pathways (Adabi et al., 2012). In contrast, the present study shows that the antiepileptic effect of rosiglitazone is related to its regulation of BDNF/TrkB signaling. And we found that rosiglitazone did not affect development and severity of SE, but it significantly suppressed SRS development, assessed by behavior recordings and EEG monitoring. It is difficult to explain this discrepancy because there is a paucity of studies examining changes in behavior or EEG after rosiglitazone treatment in the lithium pilocarpine-induced model of epilepsy. However, it is likely due to the model of epilepsy used, the choice of agonist, and other factors. Previous studies have mostly used the pentylenetetrazol (PTZ)-kindling model, which is considered an acute model of epilepsy, and use of pioglitazone in the acute phase of epilepsy inhibited the severity of SE and was considered an anticonvulsant effect (Okada et al., 2006; Abdallah, 2010; Adabi et al., 2012). In contrast, the lithium pilocarpine model is a chronic epileptic model. It is considered close to being the ideal homologous
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Li–Pilo + Vehicle Li–Pilo + Ros Li–Pilo + Ros + K252a
The number of rats developed SRS
The time to development of SRS (days)
The mean number of seizures (seizures/day)
The mean seizure duration (sec/seizure)
10 (12) 6 (12)a,c 9 (12)b
17.8 ± 7.5 33.6 ± 11.2a,c 20.3 ± 6.9b
2.67 ± 0.43 1.24 ± 0.21a,c 2.56 ± 0.37b
13.60 ± 2.80 7.98 ± 2.15a,c 12.48 ± 1.35b
Note: rosiglitazone attenuated development and severity of SRS, but added K252a reversed this effect in chronic phase. Data are means ± S.E.M. P < 0.01. b P > 0.05 vs. vehicle treated rats. c P < 0.05 vs. rosiglitazone added K252a treated rats.
a
model of temporal lobe epilepsy (TLE). In fact, previous studies have indicated that the antiepileptogenic and anticonvulsant effects were dissociative (Suchomelova et al., 2006). Partial antiepileptic drugs (AEDs) may have antiepileptogenic properties, but it is uncertain whether they have anticonvulsant properties (Suchomelova et al., 2006). For example, a study showed that topiramate could not modify the course of SE, yet completely prevented SRS (Suchomelova et al., 2006; Rigoulot et al., 2004). In addition, an NMDA receptor antagonist did not block seizures, but effectively prevented the epileptogenic process (Khalilov et al., 2003). In studies of epilepsy, there are many pathological mechanisms related to SRS in the latent phase. These events include neuronal loss, glial cell activation, and hippocampal circuit reorganization (Siebzehnrubl and Blumcke, 2008; Holopainen, 2008; Walker, 2007). Although there is no definitive evidence that any of these pathological changes are critical in the development of SRS (Kobow et al., 2012), some studies have suggested that neuronal loss and network reorganization participate in the initiation of SRS, and lead to chronic epilepsy (Ben-Ari and Dudek, 2010; Zhang et al., 2002). More importantly, data has indicated that BDNF/TrkB signaling appears to be vital for modulating neuronal network reorganization (Zheng et al., 2011). This suggests that SE-induced BDNF increases might contribute to lasting structural and functional changes in the hippocampus contributing to SRS and epileptogenesis. Our study found that rosiglitazone attenuates the development and severity of SRS after SE. And these effects were reversed when the TrkB antagonist K252a was administrated. K252a, as antagonist of TrkB (a non-selective kinase inhibitor with affinity TrkB), and it is widely used for blocking BDNF/TrkB signaling and inhibiting TrkB activation. Thus, this further supports the notion that inhibiting BDNF/trkB signaling attenuates the risk of SRS, and also confirmed that rosiglitazone can exert antiepileptogenic effects through BDNF/TrkB signaling. In addition, this study has shown that rosiglitazone does not affect the development and severity of SE, and suggests that the key role of PPARc is mainly in the chronic phase. Specifically, PPARc modulates cytokine and microglia mediated inflammation (Sun et al., 2008; Yu et al., 2008), while seizure-induced expression of cytokines mostly occurs during the chronic phase alongside increases in BDNF. We found that BDNF level peaked at 7 days, and microglia began to be activated at 12 h and peaked in number at 6–7 days after SE (Sun et al., 2008). BDNF is mostly produced by activated microglia (Zhou et al., 2011; Miao et al., 2012). Thus, this evidence also indirectly supports the finding here that rosiglitazone affects the development of SRS through BDNF signal pathways, but has no effect on the development of SE. In summary, this study provides additional evidence concerning the neuroprotective effects of the PPARc agonist rosiglitazone, and offers new perspectives for the treatment of epilepsy. Importantly, a direct antiepileptic effect of rosiglitazone was demonstrated in this study. In addition, we showed that the neuroprotective and antiepileptic effects of rosiglitazone are derived, at least in part, by regulation of BDNF/TrkB signaling. These results suggest that alteration of BDNF/TrkB signaling may inhibit the development of SRS, and exert an antiepileptic effect.
Acknowledgment This study was supported by the ‘‘Xi’an Jiaotong University Campus Fund’’. We thank Dr. Dong Yan (Department of Immunology, Xijing Hospital) for his valuable technical advice.
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