reperfusion injury in gerbils via activating GABAergic signaling and inhibiting astrogliosis

Neurochemistry International 60 (2012) 39–46

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Topiramate attenuates cerebral ischemia/reperfusion injury in gerbils via activating GABAergic signaling and inhibiting astrogliosis Xiaoyuan Mao a,1, Changwei Ji b,1, Chunyan Sun c, Danfeng Cao d, Ping Ma b, Zhong Ji e, Fangyuan Cao f, Dongyu Min a, Shuzhi Li f, Jiqun Cai a,⇑, Yonggang Cao f,⇑ a

Department of Pharmaceutical Toxicology, School of Pharmaceutical Science, China Medical University, Shenyang 110001, China Department of Anatomy, Daqing Campus of Harbin Medical University, Daqing 163319, China Department of Clinical Nursing, Daqing Campus of Harbin Medical University, Daqing 163319, China d Department of Genetics, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China e Department of Physiology, Daqing Campus of Harbin Medical University, Daqing 163319, China f Department of Pharmacology, Daqing Campus of Harbin Medical University, Daqing 163319, China b c

a r t i c l e

i n f o

Article history: Received 20 August 2011 Received in revised form 12 October 2011 Accepted 28 October 2011 Available online 7 November 2011 Keywords: Topiramate GABA receptor KCC2 NKCC1 GFAP Global cerebral ischemia

a b s t r a c t Impaired GABAergic inhibitory synaptic transmission plays an essential role in the pathogenesis of selective neuronal cell death following transient global ischemia. GABAA receptor (GABAAR), K+–Cl co-transporter 2 (KCC2), Na+–K+–Cl co-transporter 1 (NKCC1) and astrocytes are of particular importance to GABAergic transmission. The present study was designed to explore whether the neuroprotective effect of topiramate (TPM) was linked with the alterations of GABAergic signaling and astrocytes. The bilateral carotid arteries were occluded, and TPM (80 mg/kg/day (divided twice daily), i.p.) was injected into gerbils. At day 1, 3 and 7 post-ischemia, neurological deficit was scored and changes in hippocampal neuronal cell death were evaluated by Nissl staining. The apoptosis-related regulatory proteins (procaspase-3, caspase-3, Bax and Bcl-2) and GABAergic signal molecules (GABAAR a1, GABAAR c2, KCC2 and NKCC1) were also detected using western blot assay. In addition, the fluorescent intensity and protein level of glial fibrillary acidic protein (GFAP), a major component of astrocyte, were examined by confocal and immunoblot analysis. Our results showed that TPM treatment significantly decreased neurological deficit scores, attenuated the ischemia-induced neuronal loss and remarkably decreased the expression levels of procaspase-3, caspase-3 as well as the ratio of Bax/Bcl-2. Besides, treatment with TPM also resulted in the increased protein expressions of GABAAR a1, GABAAR c2 and KCC2 together with the decreased protein level of NKCC1 in gerbils hippocampus. Furthermore, fluorescent intensity and protein level of GFAP were evidently reduced in TPM-treated gerbils. These findings suggest that the therapeutic effect of TPM on global ischemia/reperfusion injury appears to be associated with the enhancement of GABAergic signaling and the inhibition of astrogliosis in gerbils. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cerebral ischemia forms the third leading cause of mortality and long-term disability in the modern world (Hong and Saver, 2009). Since 2011, ischemic stroke has ranked second to cancer as a leading cause of death worldwide (Wang et al., 2011). It is reported that more than 5 million patients die from ischemic stroke worldwide every year (Schwaninger et al., 2006). Currently, the only treatment available for acute ischemic stroke remains thrombolysis by administrating tissue-plasminogen activator (t-PA). ⇑ Corresponding authors. Tel./fax: +86 24 23255471 (J. Cai), +86 24 23255266 (Y. Cao). E-mail addresses: [email protected] (J. Cai), [email protected] (Y. Cao). 1 These authors contributed equally to this work. 0197-0186/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2011.10.015

However, only 1–2% of patients have the chance to receive thrombolytic therapy mainly as a result of the short time window for administration (Goldstein and Rothwell, 2008). Moreover, the reperfusion accompanied with thrombolysis may exacerbate the damage caused by cerebral ischemia due to activating the inflammatory cascades (Sacchetti, 2008). Therefore, it is of desperate need to develop the new therapeutic strategies in this field. Cumulative evidence has demonstrated that transient cerebral ischemia, induced by deprivation of blood flow to the brain for a short period of time and the hippocampal neurons model with oxygen–glucose deprivation, causes delayed neuronal cell death in the selectively vulnerable hippocampal CA1 region of rodent brain. Most interestingly, the damage produced by global ischemia may result from the imbalance between excitatory and inhibitory synaptic transmission (Schmidt-Kastner and Freund, 1991). Direct or indirect reduction of neuronal excitability hampers neuronal

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degeneration. c-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the adult brain (Pan et al., 2008). A disruption of the inhibition mediated by GABA is a major reason for the occurrence of neuronal hyperexcitability. It is reported that enhancement of GABA-mediated inhibitory neurotransmission with a benzodiazepine following an ischemic insult could prevent neuronal degeneration in the hippocampus and striatum of gerbils and rats (Schwartz et al., 1994, 1995). This study suggests that loss of GABAergic neurotransmission may lead to neuronal injury following global cerebral ischemia. The majority of the GABAmediated fast inhibitory action in the central nervous system (CNS) is modulated via GABAA receptors (GABAARs). Mammalian pentameric GABAARs are ligand-gated ion channels assembled from a variety of polypeptide subtypes (a1–a6, b1–b3, c1–c3, d, e, p, h and q1–q3) (Korpi et al., 2002). The a family is the largest with six different subunits and contributes significantly to the functional characterization of the GABAARs. GABAAR a1 is the most widely expressed of all the a subtypes and shows high expression in most of the brain areas (McKernan and Whiting, 1996). GABAAR c2 subunit is also a major component of the GABAAR (over 80% of GABAARs contain c2 subunit) which is colocalized extensively with GABAAR a1 throughout the brain. In the rat, their colocalization is specifically observed in the hippocampal pyramidal cell layer and in the granule cell layer of the dentate gyrus (Gambarana et al., 1991; Wisden et al., 1992). In mature neurons, GABAAR-mediated fast-hyperpolarizing inhibitory responses are dependent upon the intracellular Cl concentration that is maintained by members of the Na+–K+–Cl (NKCC) and K+–Cl (KCC) families of cation chloride co-transporters. The neuron-specific KCC2 operates in active Cl extrusion, while NKCC2 functions to import Cl into the neurons and glial cells in the adult CNS. The balance between this Cl extrusion system and Cl accumulation system may bear important consequences in the regulation of intracellular Cl concentration, and hence in the control of GABAergic functions (Payne et al., 2003). Alterations in the balance of KCC2 and NKCC1 activity may convert GABAARs from a hyperpolarizing to a depolarizing response and thereby contribute to neuronal hyperexcitability, finally causing many neurological disorders, such as epilepsy and cerebral ischemia. A recent report revealed that the excitatory effects of GABAARs in cell membranes acquired from temporal lobe epilepsy patients were attributable to decreased KCC2 and increased NKCC1 expression (Palma et al., 2006). Additionally, a substantial decrease of KCC2 mRNA and protein levels was found in a focal ischemia model (Jaenisch et al., 2010). Moreover, Kang et al. reported a remarkable elevation in NKCC1 immunoreactivity in the gerbil hippocampus following transient ischemia (Kang et al., 2002). These data suggest that the expressional changes of KCC2 and NKCC1 might be important in the occurrence of ischemia. They point towards KCC2 and NKCC1 as interesting therapeutic targets for the treatment of cerebral ischemic insult. Astrocytes, one class of glial cells, also play a leading role in the modulation of synaptic transmission throughout the brain (Pascual et al., 2005). Previous investigation has demonstrated that hippocampal astrocytes also respond to GABA, finally regulating GABAergic inhibitory neurotransmission (Verkhratsky et al., 1998). Glial fibrillary acidic protein (GFAP) is a major component of neurofilaments. Its overexpression is closely related to morphological alterations of astrocytes in response to neuronal damage. The accumulation of GFAP also involves in neuronal excitotoxic injury in perinatal rat brain (Burtrum and Silverstein, 1993). Kindy et al. reported the obvious upregulation of GFAP in a transient gerbil ischemia model, indicating an important role in ischemic brain injury (Kindy et al., 1992). Topiramate (2,3:4,5-bis-o-(1-methylethylidene) b-D-fructo-pyranose sulfamate; TPM) is a structurally novel drug with a broad spectrum of antiepileptic activities in both experimental and clinical studies (Borowicz

et al., 2003; Yen et al., 2000). In general, TPM is considered to produce its anticonvulsant effects through enhancement of GABAergic activity, inhibition of AMPA receptors, voltage-sensitive sodium and calcium channels (Guerrini and Parmeggiani, 2006). These potential mechanisms may provide pharmacological basis on the treatment of ischemia/reperfusion injury. Recent study has illustrated that TPM can exert neuroprotective effects on embolic focal cerebral ischemia (Yang et al., 1998). Besides, Noh et al. (2006) reported that TPM treatment markedly reduced neuronal damage in a cultured neuron ischemia model caused by oxygen-glucose deprivation. However, whether TPM protects against ischemiainduced neuronal cell death via the regulation of GABAergic signaling and astrocytes remains to be elucidated. Furthermore, there are some differences in pathophysiological processes between focal cerebral ischemia and global cerebral ischemic injury. So far, whether TPM provides a protective effect on the global cerebral ischemia in gerbils via GABAergic pathway and astrocytic modulation is elusive. Therefore, the present study was conducted to explore whether the neuroprotective potential of TPM was linked with the alteration of GABAergic signaling pathway and astrocytes. 2. Materials and methods 2.1. Ethical statement Great efforts were made to reduce animal suffering and minimize the number of animals used. All experiments were approved by Animal Care Committee of China Medical University and performed strictly according to its related guidelines. 2.2. Animals and treatment Adult male Mongolian gerbils weighing between 55 and 80 g were used in the present study. They were kept under a controlled environment (12:12 h light/dark cycle, 50–70% humidity, 24 °C) and allowed free access to food and water. Transient cerebral ischemia was performed as previously described with minor modification (Gupta and Sharma, 2006). In brief, gerbils were anesthetized intraperitoneally (i.p.) with chloral hydrate (300 mg/kg). In the supine position, a 2 cm midline ventral neck incision was made. The bilateral common carotid arteries were exposed and separated carefully from the vagus nerves and occluded bilaterally for 10 min using non-traumatic aneurysm clips. After 10 min of occlusion, the aneurysm clips were removed to restore cerebral blood flow. Complete blood flow was confirmed visually and the neck incision area was then sutured. The rectal temperature was monitored and maintained at 37 ± 0.5 °C with a heating pad and lamp during and after surgery until gerbils completely recovered from anesthesia. TPM (80 mg/kg/day (divided twice daily), i.p., Sigma) or vehicle (0.01 M phosphate-buffered saline (PBS) with 0.01% (v/v) DMSO, Sigma) was injected immediately after ischemia. The dosage and dosing frequency of TPM were chosen on the basis of the previous studies (Cha et al., 2002; Chen et al., 2010; Shatskikh et al., 2009). Sham-operated animals received identical surgical procedures, except that the carotid arteries were not occluded after the neck incisions. Efforts were made to reduce the number of animals used and their suffering. 2.3. Neurological deficit evaluation The neurologic deficit score was assessed for neurological symptoms at day 1, 3 and 7 post-ischemia, according to McGraw’s method with minor modification (McGraw, 1977), which was as follows: 0, no neurological deficit; 1, hunched posture; 2, ptosis; 3, circling behavior; 4, splayed-out hind limb; 5, seizures. The average score was used as the degree of neurological impairment. The

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higher the score, the higher was the ischemic insult. The neurobehavioral assessment was carried out by an investigator blinded to the treatment groups. 2.4. Nissl staining Ischemia-induced neuronal cell death was assessed by Nissl staining. In brief, at 1, 3 and 7 d post-surgery, gerbils from each group (n = 6) were transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Brains were then removed and frozen sections (8 lm) were collected. The slides were immersed in 0.1% cresyl violet for 10 min at room temperature. Sections were then dehydrated in graded alcohol, coverslipped with neutral balsam and analyzed with a light microscope. To evaluate the neuronal survival in hippocampal CA1 subfield, neurons with round and palely stained nuclei were regarded as surviving cells, while shrunken neurons with pyknotic nuclei were regarded as damaged cells. Six brain sections were selected from each group (n = 6) and the hippocampal neuronal damage in CA1 region was determined by counting the surviving neurons. Data were presented as the number of surviving neurons/field. 2.5. Immunofluorescence and confocal analysis For immunofluorescent staining and confocal microscopic analysis, brain sections were preincubated with 5% bovine serum albumin (BSA) for 1 h and then incubated overnight at 4 °C with the primary antibody anti-GFAP (1:50, Santa Cruz, USA). After overnight incubation, the slices were rinsed with PBS and incubated with FITC-conjugated goat anti-rabbit IgG (1:200, Zhongshan Golden Bridge Biotechnology, China) for 2 h at room temperature. After rinsing, the sections were counterstained with DAPI (1:500, Jackson Immunoresearch, USA) for 5 min, mounted using an anti-fading mounting medium and observed under a confocal laser scanning microscope (C1, Nikon, Japan). Excitation filters for DAPI (408 nm) and FITC (488 nm) were selected. The sections were photographed using confocal imaging program (C1, Nikon, Japan). In order to assess nonspecific staining, negative controls were performed with normal serum instead of the primary antibody followed by all subsequent incubations as mentioned above. 2.6. Western blot analysis Western blot analysis was carried out on the whole hippocampus isolated from gerbil brains. For short, samples were homogenized at a ratio of 1:5 (w/v) in cold RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride). After centrifugation at 13,200g for 20 min at 4 °C, the supernatant was collected and total protein levels were quantified by a BCA protein assay kit (Beyotime Institute of Biotechnology, China). An equal amount of protein (30 lg) was separated by means of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) and transferred onto nitrocellulose membranes (Millipore, MA). The membranes were blocked with 5% defatted milk for 1 h at room temperature and then probed respectively with the following primary antibodies: rabbit anti-procaspase-3 (1:300, Santa Cruz, USA), rabbit anti-caspase-3 (1:300, Santa Cruz, USA), rabbit anti-Bax (1:200, Santa Cruz, USA), rabbit anti-Bcl-2 (1:200, Santa Cruz, USA), rabbit anti-GABAAR a1 (1:300, Santa Cruz, USA), goat anti-GABAAR c2 (1:200, Santa Cruz, USA), rabbit anti-KCC2 (1:1500, Upstate Biotechnology, NY), rabbit anti-NKCC1 (1:300, Santa Cruz, USA), rabbit anti-GFAP (1:200, Santa Cruz, USA), mouse anti-b-actin (1:2000, Santa Cruz, USA) and mouse anti-GAPDH (1:2000, Kang Chen, China), overnight at 4 °C. The membranes were washed with three changes of PBS, followed by the

incubation with horseradish peroxidase-conjugated goat antirabbit antibody (1:5000, Santa Cruz, USA) or rabbit antigoat antibody (1:5000, Santa Cruz, USA) or goat antimouse antibody (1:5000, Santa Cruz, USA) for 2 h at room temperature. Immunodetection was carried out using an enhanced chemiluminescence (ECL) kit (Pierce, CA). Films were digitized by a scanner and the gray value of the protein bands was analyzed using Quantity One software (BioRad, USA).

2.7. Data expression and statistical analysis All values were presented as means ± SD. Differences between vehicle control and TPM treatment groups at each time point were compared using Student’s t-test. All statistical analyses were carried out by SPSS 13.0 software. Value of p < 0.05 was deemed statistically significant.

3. Results 3.1. Effects of TPM on neurological deficit scores in gerbils after global cerebral ischemia The effects of TPM on neurological scores were evaluated at day 1, 3 and 7 post-ischemia. As illustrated in Table 1, there were no abnormal neurological symptoms in sham gerbils. Ischemic gerbils at 1 d, 3 d and 7 d all exhibited characteristics of ischemia/reperfusion injury with high neurological deficit score. Treatment with TPM by the daily dose of 80 mg/kg resulted in a significant decrease of deficit score in comparison to vehicle-treated group (p < 0.05–0.01, Table 1).

3.2. TPM protected against neuronal loss and apoptosis in gerbils hippocampus after ischemia The neuronal loss in gerbil’s hippocampal CA1 region after 10 min of global cerebral ischemia was histopathologically evaluated by Nissl staining. As indicated in Fig. 1A, gerbils in sham control group did not show any histopathological abnormalities while massive damaged neurons with pycnotic nucleus were observed in CA1 region of vehicle-treated ischemic gerbils. TPM treatment significantly increased the number of surviving neurons with palely stained nuclei and intact Nissl substance in comparison to the vehicle-treated ischemic group. The quantitative analysis demonstrated that treatment with TPM in ischemic gerbils caused a marked elevation in the average number of viable CA1 pyramidal neurons compared with vehicle-treated ischemic group (p < 0.01, Fig. 1B).

Table 1 The neuroprotective effects of TPM on neurological deficit scores in gerbils after global ischemia (mean ± S.D., n = 8)

1d

3d 7d

Groups

Gerbil number (n)

Neurological deficit scores

Sham Vehicle TPM Vehicle TPM Vehicle TPM

8 8 8 8 8 8 8

0.00 ± 0.00 2.75 ± 1.04 1.50 ± 0.76* 3.25 ± 1.16 1.25 ± 0.71** 3.63 ± 0.92 2.13 ± 1.36*

Gerbils were received TPM (80 mg/kg/day, divided twice daily, i.p.) or vehicle at 1 d, 3 d and 7 d after ischemia. Neurological deficit scores were assessed at day 1, 3 and 7 post-ischemia according to the McGraw’s method. * p < 0.05 vs. vehicle control. ** p < 0.01 vs. vehicle control.

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a1, GABAAR c2, KCC2 and NKCC1 proteins were detected by immunoblot analysis. As shown in Fig. 3A, immunoblotting for GABAAR

a1 detected the expected band at 51 KDa in the gerbil hippocampus. Statistical analysis illustrated that the protein expression of GABAAR a1 was significantly elevated in TPM-treated ischemic hippocampi at the time points of 1 d, 3 d and 7 d, in comparison to the vehicle-treated control (p < 0.01, Fig. 3E). Fig. 3B indicated that the western blot with GABAAR c2 exhibited a single band of 54 KDa. Following t-test analysis, a drastically elevated level of GABAAR c2 protein was detected in ischemia-induced gerbils after administration of TPM at 1 d, 3 d and 7 d, compared with the vehicle group (p < 0.05–0.01, Fig. 3F). In the mean time, KCC2 and NKCC1, the major Cl extruder and Cl intruder, respectively, which were responsible for regulating GABAAR-mediated hyperpolarized responses, were also investigated by immunoblotting. Our western blot results showed that KCC2 and NKCC1 proteins were detected in the anticipated band located at 140 KDa and 135 kDa, respectively (Fig. 3C and D). The results of t-test analyses demonstrated that TPM treatment remarkably increased the protein expression of KCC2 and evidently decreased the protein level of NKCC1 in ischemic gerbils at the time points of 1 d, 3 d and 7 d, in comparison with the vehicle control (p < 0.05–0.01, Fig. 3G and H). Taken together, these findings depicted that treatment with TPM could significantly enhance the protein levels of GABAAR a1, GABAAR c2 and KCC2 but reduce NKCC1 protein expression following an insult caused by transient global ischemia in gerbils.

Fig. 1. TPM exerted neuroprotective effects on the pyramidal neurons in gerbils hippocampal CA1 region after global cerebral ischemia with Nissl staining (mean ± S.D., n = 6). (A) The representative images of hippocampus from Sham, Vehicle and TPM groups at day 1, 3 and 7 post-ischemia were established. The shrunk dark damaged neurons (arrows) were marked. Scale bars: 100 lm. (B) was the Statistical analysis of the surviving neurons in each group at day 1, 3 and 7 postischemia. ⁄⁄p < 0.01 vs. vehicle-treated group.

3.3. TPM suppressed the expressions of apoptosis-regulatory proteins in gerbils hippocampus after ischemia To explore whether TPM modulated the expression of apoptosis-related proteins in gerbils hippocampus after 10 min of cerebral ischemia, western blot assay was carried out to detect the protein levels of procaspase-3, caspase-3, Bax and Bcl-2 on hippocampal samples from vehicle-treated and TPM-treated ischemic gerbils at different time points. Fig. 2A illustrated that the detected protein bands were located at 32 kDa for procaspase-3 and 20 kDa for caspase-3. With the resultant analysis of t-test, statistically significant reduction in the protein expression of both procaspase-3 (inactive) and cleaved caspase-3 (active) were found in the hippocampus of gerbils treated with TPM at 1 d, 3 d and 7 d, respectively, compared to the ischemia-subjected group (p < 0.01, Fig. 2C and D). In the mean time, western blot with Bax and Bcl-2 antibodies exhibited the anticipated bands of 23 kDa and 26 kDa, respectively, as shown in Fig. 2B. Statistical analyses showed that the ischemic gerbils with administration of TPM resulted in a lower expression of Bax and a higher expression of Bcl-2 in the hippocampus. Thus, the ratio of Bax/Bcl-2 was drastically reduced in TPM-treated gerbils hippocampus at the time points of 1 d, 3 d and 7 d, respectively, than that in the vehicle controls (p < 0.01, Fig. 2E). 3.4. TPM altered the protein expressions of GABAAR a1, GABAAR c2, KCC2 and NKCC1 in gerbils hippocampus after ischemia We further examined whether TPM exerted neuroprotective effects on gerbils global cerebral ischemia/reperfusion injury via regulation of GABAergic pathways. The expression levels of GABAAR

3.5. TPM inhibited the astrogliosis in gerbils hippocampus after global cerebral ischemia Astrocyte activity is mainly characterized by the expression of GFAP, the principal intermediate filament protein. GFAP overexpression is involved in the morphological changes of astrocytes after brain ischemic injuries. Thus, in the present study, we made use of the immunoreaction of GFAP to represent the activity of astrocytes. Our confocal analyses indicated that TPM treatment significantly attenuated the fluorescent intensity of GFAP in ischemic gerbils hippocampal CA1 area at 1 d, 3 d and 7 d, respectively, compared with the vehicle control (Fig. 4A). In order to quantify the therapeutic effect of TPM on the protein expression of GFAP in gerbils hippocampus, western blot analysis was further carried out. Fig. 4B showed that GFAP protein was detected in the band located at 50 kDa. It was noted that the protein expression level of GFAP in TPM-treated ischemic hippocampi was remarkably decreased at day 3 and 7 post-ischemia, compared to the vehicletreated control (p < 0.01, Fig. 4C). Taken together, these findings illustrated that TPM treatment suppressed ischemia-induced astrocyte activation in CA1 pyramidal neurons. 4. Discussion The data in the present study provided further support for the hypothesis that TPM could display neuroprotective effects on cerebral ischemic reperfusion damage in a gerbil model by enhancing GABAAR-mediated inhibitory transmission and inhibiting astrogliosis. An obvious improvement of neurological function was observed in TPM-treated ischemic gerbils at day 1, 3 and 7 postischemia. Nissl staining results showed a marked suppression of neuronal cell death in the hippocampal CA1 area of ischemic gerbils after treatment with TPM, suggesting the inhibitory effect of TPM on neuronal apoptosis. Procaspase-3, caspase-3, Bax and Bcl-2, the key apoptotic regulatory proteins, were also found to be remarkably changed in the TPM-treated ischemic gerbils. Further examinations showed that the increased expression levels of GABAAR a1, GABAAR c2, KCC2 and the decreased expression of

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Fig. 2. TPM treatment inhibited the expression levels of apoptosis-regulatory proteins in ischemic gerbils hippocampus (mean ± S.D., n = 6). (A and B) Showed the representative images of immunoblots with antibodies against procaspase-3, caspase-3, Bax and Bcl-2, respectively. Procaspase-3: 32 kDa; caspase-3: 20 kDa; Bax: 23 kDa; Bcl-2: 26 kDa; b-actin: 43 kDa. (C–E) were the Quantitative analysis of the protein levels of procaspase-3, caspase-3, Bax/ Bcl-2 between TPM-treated and vehicle-treated control groups at the time points of 1 d, 3 d and 7 d. The data were normalized to the loading control b-actin. ⁄⁄p < 0.01 vs. vehicle control.

NKCC1 and GFAP in ischemic gerbils hippocampus were noticed after administration of TPM. These findings indicated that TPM might exert beneficial effects on ischemia/reperfusion injury in stroke therapy. The mechanism underlying the neuroprotection of TPM likely involved enhancement of GABAAR-mediated inhibitory transmission and inhibition of astrogliosis. Recent studies have examined the neuroprotective potential of many new antiepileptic drugs against brain ischemia (Chan et al., 1998; Lee et al., 1999; Minato et al., 1997). TPM, a sulfamate-substituted monosaccharide, is a recently marketed broad-spectrum antiepileptic drug which is particularly used to treat the partial seizures with or without secondary generalization in clinic (Sander, 1997). Most importantly, TPM was reported to be neuroprotective in an embolic focal cerebral ischemia model of rats and in a cultured ischemic model with oxygen-glucose deprivation (Noh et al., 2006; Yang et al., 1998). However, whether TPM is effective against global ischemic reperfusion damage remains obscure. Furthermore, the underlying mechanism for the neuroprotection of TPM in a gerbil global ischemia model is still elusive. Our current data showed that TPM exerted neuroprotective role in a gerbil model of transient forebrain ischemia, which was consistent with previous reports (Noh et al., 2006; Yang et al., 1998). Mongolian gerbils were selected in the present study for inducing transient global ischemia because they possessed characteristics of global brain ischemia just after brief occlusion of common carotid arteries due to an incomplete circle of Willis (Kirby and Shaw, 2004). Nissl staining illustrated that

TPM treatment significantly reduced the neuronal death by the dose of 80 mg/kg compared with the vehicle-treated group. It is generally accepted that global cerebral ischemia leads to delayed neuronal cell death in the selectively vulnerable CA1 subfield of hippocampus (Zhang et al., 2009). Our investigation confirmed that a 10-min occlusion of common bilateral carotid arteries in gerbils caused remarkable neuronal damage in hippocampal CA1 region as shown histologically. Neuronal damage after ischemic injury could contribute to mitochondrial dysfunction and consequently activate an apoptotic cascade. In order to further examine the improvement of ischemia/ reperfusion damage in gerbils with the administration of TPM, we also measured the expression levels of apoptosis-related proteins in gerbils hippocampus. Caspase-3 is regarded as an executioner molecule and is the most abundant caspase under normal and pathological conditions (Lamkanfi et al., 2007). Previous studies reported that caspase-3 could activate caspase-activated DNAse, consequently leading to DNA fragmentation and cell death (Chan, 2004; Manabat et al., 2003). Cumulative evidence has indicated that the up-regulation of caspase-3 is observed after cerebral ischemia. Results from the current study displayed that administration of TPM significantly caused the down-regulation of procaspase-3 and caspase-3 in ischemic gerbils, suggesting that TPM treatment might directly decreased procaspase-3 expression in ischemic gerbils. In addition to caspases, Bcl-2 family proteins have been also shown to play a critical role in the modulation of

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Fig. 3. TPM treatment upregulated the protein expressions of GABAAR a1, GABAAR c2 KCC2 and NKCC1 in ischemic gerbils hippocampus (mean ± S.D., n = 6). (A–D) Showed the representative images of immunoblots with antibodies against GABAAR a1, GABAAR c2, KCC2 and NKCC1, respectively. GABAAR a1: 51 kDa; GABAAR c2: 54 kDa; KCC2: 140 kDa; NKCC1: 135 kDa; b-actin: 43 kDa; GAPDH: 36 kDa. (E–H) were the Quantitative analysis of the protein levels of GABAAR a1, GABAAR c2, KCC2 and NKCC1 between TPM-treated and vehicle-treated control groups at the time points of 1 d, 3 d and 7 d. The data were normalized to the loading control b-actin or GAPDH. ⁄p < 0.05 vs. vehicle control; ⁄⁄p < 0.01 vs. vehicle control.

Fig. 4. TPM suppressed the astrocyte activation in gerbils hippocampus after global brain ischemia. (A) Displayed the representative confocal images of GFAP protein distribution between TPM-treated and vehicle-treated control groups in gerbils hippocampal CA1 region at day 1, 3 and 7 post-ischemia. The arrows indicated the positive reactions. Cell nucleus was counterlabeled with DAPI (in blue). Scale bar: 100 lm. (B) Showed representative bands of GFAP in ischemic gerbils hippocampus of TPM and vehicle groups by western blot (mean ± S.D., n = 6). GFAP: 50 kDa; GAPDH: 36 kDa. (C) was the Quantitative analysis of the protein level of GFAP between TPM-treated and vehicle-treated control groups in gerbils hippocampus at day 1, 3 and 7 post-ischemia. The data were normalized to the loading control GAPDH. ⁄⁄p < 0.01 vs. vehicle control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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neuronal apoptosis. Bcl-2 itself acts as an anti-apoptotic protein, whereas another member of the family, Bax, functions as a proapoptotic molecule (Shacka and Roth, 2005). Our data showed that evident reduction of Bax/Bcl-2 was found in the hippocampus of ischemia-induced gerbils treated with TPM. These findings implied that the neuroprotective action of TPM might involve the regulation of Bax/Bcl-2 and caspase-3 pathways in the vulnerable neurons of ischemic gerbils hippocampus. It is noted that neuronal apoptosis may result from the excitotoxicity during ischemia/reperfusion injury. An interruption of GABAergic synaptic transmission plays a fundamental role in the development of neuronal hyperexcitability. Specially, a lowering of GABAAR-mediated inhibitory responses contributes to the generation of ischemic insult. This notion was supported by the recent report that the binding of [35S] t-butylbicyclophosphorotionate ([35S] TBPS) to a site linked with the GABAA-gated chloride channel decreased in the CA1 region of gerbils hippocampus (Li et al., 1993). GABAAR a1 and c2 are two most widely expressed isoforms of GABAARs that gate a chloride channel to produce neuronal inhibition. Li et al. (1993) found that the expression of GABAAR a1 subunit mRNA was significantly reduced in a region selectively vulnerable to transient global ischemia at day 4 post-ischemia, CA1 pyramidal cell layer. It implies that the decreased expression of GABAARs may lead to ischemia-induced neuronal injury and preservation of normal GABAAR structure is of vital importance to maintaining normal GABAergic synaptic inhibitory neurotransmission. The current investigation demonstrated that TPM treatment significantly upregulated protein expressions of GABAAR a1 and c2 subunits in gerbils hippocampal CA1 subfield following ischemia/reperfusion injury. In fact, partly in agreement with our result, previous study illustrated the direct activation of GABAAR c2 subunit expressed in Xenopus laevis oocytes (Simeone et al., 2006), suggesting that modulation of GABAAR a1 and GABAAR c2 proteins expression was, at least in part, related to the TPM’s neuroprotection against global ischemic reperfusion injury in gerbils hippocampus. In the CNS, GABA-induced chloride flux through GABAAR-related chloride channels is determined by the intracellular Cl concentration which is set by the activity of KCCs and NKCCs. Alterations in the balance of KCC2 and NKCC1 activity may produce the GABA hyperpolarizing-to-depolarizing shift. A conspicuous downregulation of KCC2 expression was observed in a hippocampal slice model with oxygen-glucose deprivation and in vivo model of transient forebrain ischemia (Jaenisch et al., 2010; Papp et al., 2008). Previous investigation also illustrated that NKCC1 immunoreactivity was highly elevated in the gerbil hippocampus following ischemic insult (Kang et al., 2002). These findings appear to implicate that decrease of KCC2 and/or increase of NKCC1 activity may lower GABAAR-mediated synaptic transmission via elevating intracellular Cl concentration, finally making the animals more vulnerable to ischemia. Thus, regulating KCC2 and NKCC1 expressions might have a therapeutic effect on ischemic injury. In the present study, we found the increased protein level of KCC2 accompanied with the decreased NKCC1 protein expression in ischemic gerbils treated with TPM. It is further confirmed that TPM exerts the neuroprotective effects on gerbils global ischemia/reperfusion injury via modulating GABAergic signaling due to the fact that altered expressions of KCC2 and NKCC1 can influence GABAAR-mediated inhibitory responses. Further work will be required to elucidate how they affect GABAergic functions under ischemic conditions after treating with TPM. Reactive astrocytes activation in the hippocampus, including the increase of size and number, has been involved in the causation of ischemia. A recent report has elucidated that the activation of reactive astrocytes increases Src immunoreactivity and consequently exacerbates ischemic injury (Zan et al., 2011). It is firmly established that astrocytes also regulate synaptic function

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throughout the brain (Ortinski et al., 2010). In various CNS neurological diseases, including epilepsy, ischemic stroke and traumatic brain injury, the downregulation of glutamine synthetase, which synthesizes glutamine selectively in astrocytes, is associated with reactive astrogliosis. Reduced glutamine synthetase expression results in a disruption of astrocytic glutamate/glutamine cycle, leading to impaired GABA-mediated inhibitory neurotransmission (Ortinski et al., 2010). As the predominant intermediate filament protein in astrocytes of mammalian CNS, GFAP has been widely used to evaluate the ‘reactive state’ of astrocytes. The dramatic elevation of GFAP mRNA and protein was reported in a focal cerebral ischemia model with ischemic injury confined to the cerebral cortex (Cheung et al., 1999). This indicates that pharmacological intervention with astrocytes may ameliorate the ischemic insult. Intriguingly, our present work demonstrated that the immunoreactivity of GFAP was remarkably diminished in the gerbils after administration of TPM, implying that the neuroprotection of TPM against global ischemic injury could be correlated with inhibiting astrogliosis. The underlying mechanism by which TPM suppresses astrocytic activation remains to be clarified. However, TPM also exerts the therapeutic effects by alternative pathways. It was reported that there existed an interaction with glutamate (Rogawski and Porter, 1990). Particularly, TPM inhibits the AMPA/kainite subtype of glutamate receptor without obviously affecting the N-methyl-D-aspartate (NMDA) receptor. Therefore, blockade of AMPA/kainite receptor may involve in TPM’s effectiveness on global ischemia. In addition, evidence indicates that the common mechanism of action by some antiepileptic drugs is due to suppression of neuronal voltage-dependent sodium (Na+) channels during seizure. Excessive Na+ influx into neurons activates voltage-dependent Ca2+ channels, triggering the significant Ca2+ influx, which can disrupt cellular homeostasis and ultimately lead to neuronal cell death following ischemic insult (Edmonds et al., 1996). As a well known anti-epileptic drug, TPM was reported to block voltage-dependent sodium channels in vitro (Zona et al., 1997). Thus, TPM may also reduce ischemic injury by the inhibition of Na+ influx. Collectively, it is likely that there are other mechanisms of TPM involved in neuroprotective action after ischemic insult. In summary, our current work demonstrated that TPM could attenuate global ischemia/reperfusion injury and improve neurological dysfunction in the gerbil hippocampus by enhancing GABAAR activation and inhibiting astrogliosis. The neuroprotective effect of TPM might be associated with the activation of GABAAR, inhibition of astrogliosis and its anti-apoptotic properties in global cerebral ischemia in gerbils. Acknowledgements This work was supported in part by Natural Science Foundation of China (30270535) and Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP-20060159003). References Borowicz, K.K., Luszczki, J.J., Duda, A.M., Czuczwar, S.J., 2003. Effect of topiramate on the anticonvulsant activity of conventional antiepileptic drugs in two models of experimental epilepsy. Epilepsia 44, 640–646. Burtrum, D., Silverstein, F.S., 1993. Excitotoxic injury stimulates glial fibrillary acidic protein mRNA expression in perinatal rat brain. Exp. Neurol. 121, 127–132. Cha, B.H., Silveira, D.C., Liu, X., Hu, Y., Holmes, G.L., 2002. Effect of topiramate following recurrent and prolonged seizures during early development. Epilepsy Res. 51, 217–232. Chan, P.H., 2004. Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia. Neurochem. Res. 29, 1943–1949. Chan, S.A., Reid, K.H., Schurr, A., Miller, J.J., Iyer, V., Tseng, M.T., 1998. Fosphenytoin reduces hippocampal neuronal damage in rat following transient global ischemia. Acta Neurochir. (Wien) 140, 175–180.

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