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Akt pathway in epileptic rat
Neuroscience Letters 495 (2011) 130–134
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Diazoxide preconditioning against seizure-induced oxidative injury is via the PI3K/Akt pathway in epileptic rat Yuan Xue a , Nanchang Xie a , Lili Cao a , Xiuhe Zhao a , Hong Jiang b , Zhaofu Chi a,∗ a b
Department of Neurology, Qilu Hospital, Shandong University, 44#, Wenhua Xi Road, Jinan 250012, China Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Shandong University, Jinan, China
a r t i c l e
i n f o
Article history: Received 15 January 2011 Received in revised form 8 March 2011 Accepted 18 March 2011 Keywords: Diazoxide PI3K Oxidative stress Seizure Pilocarpine
a b s t r a c t Diazoxide (DZ), a highly selective opener of the mitochondrial ATP-sensitive potassium (mitoKATP ) channel, has neuroprotective effects against neuronal cell death by reducing oxidative stress. However, the mechanism of DZ protecting hippocampal neurons against seizure-induced oxidative injury is unknown. In this study, we investigated DZ attenuating neuronal loss caused by pilocarpine-induced seizures in rat hippocampus. DZ attenuated oxidative stress injury by upregulating superoxide dismutase (SOD) activity and downregulating malondialdehyde (MDA) level, which could be abolished with 5-hydroxydecanoic acid, an inhibitor of mitoKATP . In addition, wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), attenuated the changes in MDA and SOD levels after seizures. DZ could reduce oxidative injury induced by seizures by suppressing the activity of MDA and increasing the level of SOD in part by the PI3K/Akt pathway. © 2011 Elsevier Ireland Ltd. All rights reserved.
Accumulating laboratory and clinical data have indicated that seizures can cause neuronal damage in certain brain regions. Excessive production of reactive oxygen species (ROS) produces an imbalance in the redox system of cells. This situation results in potential damage of macromolecules, including proteins, lipids, and nucleic acids [2], collectively referred to as oxidative stress. Brain tissue is particularly vulnerable to oxidative damage because of its high consumption of oxygen and the consequent generation of high quantities of free radicals. Growing data suggest that injury resulting from oxidative stress may have an important role in pathophysiologic features after acute neurological insults such as stroke and seizures [21]. Malondialdehyde (MDA) is the final product of lipid peroxidation (LPO). Concentrations of MDA reflect the state of the free radical system [17]. Free radical scavengers, such as superoxide dismutase (SOD) and reduced glutathione, are protective against seizure-induced oxidative damage [14]. SOD, considered an important antioxidant enzyme, can remove superoxide anions from cells
Abbreviations: DZ, diazoxide; MitoKATP , mitochondrial ATP-sensitive potassium; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin; PI3K, phosphatidylinositol-3kinase; MDA, malondialdehyde; SOD, superoxide dismutase; CA3, cornu ammonis region 3. ∗ Corresponding author at: Department of Neurology, Qilu Hospital of Shandong University, 107#, Wenhua Xi Road, Jinan 250012, China. Tel.: +86 531 82169428; fax: +86 531 86927544. E-mail addresses: [email protected], [email protected] (Z. Chi). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.054
[15]. The recognition of the relationship between oxidative stress and neuronal loss in epilepsy has sparked an intensive interest in developing an antioxidation strategy to protect neurons against oxidative damage after such seizures [24]. Mitochondrial ATP-sensitive potassium (mitoKATP ) channel openers, particularly diazoxide (DZ), protects against status epilepticus (SE)-induced neuron damage during diabetic hyperglycemia [9]. Furthermore, preconditioning with mitoKATP channel openers protects against neuronal cell death by reducing oxidative stress [11,19]. As well, DZ could protect primary mesencephalic neurons by inhibiting ROS overproduction and preventing the loss of mitochondrial membrane potential and the release of mitochondrial cytochrome c [32]. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway plays a central role in intracellular processes such as cell survival and proliferation. PI3K/Akt regulates the survival response to oxidative stress-associated neuronal apoptosis [8]. Furthermore, mitoKATP channel openers such as DZ may activate the PI3K/Akt pathway in anoxia-reoxygenation injury [16]. We thus investigated a potential target for preventing hippocampal neuronal damage caused by SE. We examined whether DZ might upregulate SOD activity and downregulate MDA quantity by activating a PI3K/Akt signaling pathway in pilocarpine-induced seizures in rat. To test our hypothesis, we examined the effect of wortmannin (WTN) on activity of MDA and SOD. Specifically, we tested whether a neuroprotective effect of DZ was due to the
Y. Xue et al. / Neuroscience Letters 495 (2011) 130–134
activation of the mitoKATP channel, by using 5-hydroxydecanoic acid (5-HD), an inhibitor of mitoKATP . Adult male Wistar rats (Experimental Animal Center of Shandong University, China) weighing 250–300 g were used. Animals were maintained at room temperature (20 ± 2 ◦ C) with a 12-h light/12-h dark cycle and had free access to food and water. The experimental procedures were approved by the Shandong University Commission for Ethics of Experiments on Animals in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996). All efforts were made to minimize the number of animals used and their suffering. The experimental animals were given lithium chloride intraperitoneally (i.p., 3 mEq/kg), then 20 h later, pilocarpine (30 mg/kg, i.p.; Sigma, St. Louis, MO, USA); control rats received the same volume of 0.9% saline instead of pilocarpine. Scopolamine methyl nitrate (1 mg/kg) was injected subcutaneously 30 min before pilocarpine to prevent peripheral cholinergic effects. Experimental rats showed stage 4 or 5 convulsive seizures [22]. Seizures were allowed to last for 60 min for all rats and then were terminated by the administration of diazepam (10 mg/kg, i.p.). Rats were killed by decapitation at 2, 24, 72 h and 7 days after seizures. Rats were randomly divided into 7 groups for treatment: (1) control, as described previously; (2) pilocarpine (pilo); lithium chloride–pilocarpine as described previously; (3) pilo + DZ, 10 mg/kg (Sigma), i.p. injected 30 min before pilocarpine; (4) pilo + DZ + 5-hydroxydecanoic acid (5-HD), 5 mg/kg (Sigma), i.p. injected 15 min before DZ; (5) pilo + DZ + wortmannin (WTN), 15 g/kg (Sigma), i.p. injected 15 min before DZ; (6) 5-HD; and (7) WTN. For groups 4 and 5, pilocarpine was given 30 min after DZ injection and for groups 6 and 7, pilocarpine was given 45 min after 5-HD or WTN injection. The solvent for DZ was dimethylsulfoxide (25 mg/ml) and for 5-HD salt serum at a final concentration of 1 mg/ml [28]. We recorded latency to stage IV seizures and percentage of animals with seizure episodes. Rats were killed by decapitation 24 h after seizures. Rats were anesthetized and intracardially perfused with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). Paraffinfixed brains were cut coronally in 5-m-thick sections for staining with hematoxylin and eosin (H&E). Surviving cells were defined as round-shaped, cytoplasmic membrane-intact cells, without any nuclear condensation or distorted aspect. The surviving pyramidal cells in the hippocampal CA3 region were observed under a microscope (400×). Hippocampi were homogenized in 0.9% saline (1:9, tissue:saline, w/v) on ice, by use of a homogenizer (10,000–15,000 rpm, 10 s). The tubes with homogenates were kept in ice water for 30 min and centrifuged at 4 ◦ C (3000 × g, 10 min) according to the commercial assay kits. The supernatants were separated and stored at −80 ◦ C. Protein content was determined by the use of BCA protein assay kits (Beyotime, Jiangsu, China). LPO was determined by measuring the accumulation of thiobarbituric acid-reactive substance in homogenates and expressed as MDA content. MDA and SOD referents were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), the contents of which were measured by the use of an UV/visible-120-2 Spectrophotometer (Shimadzu Corp.) as described by commercial assay kits. The concentrations of MDA and SOD were expressed as n mol/mg protein and U/mg protein respectively. All values are expressed as means ± standard error of the mean (SEM). The data were analyzed by one-way ANOVA, then Newman–Keuls test for multiple group comparisons. Comparisons of animals with stage 4 and 5 seizures were analyzed by Chi-square test. P < 0.05 was considered statistically significant. Pilocarpine-induced behavioral episodes typically increased in intensity and duration, gradually progressing towards SE. The latency period with DZ injection was significantly longer and the
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percentage of animals reaching stage V SE was considerably lower than with pilocarpine alone. In addition, 5-HD or WTN treatment reversed the DZ-mediated protection of seizures as compared with DZ treatment alone (Table 1). Control animals did not exhibit any behavioral seizure activity. We examined neuronal loss in the hippocampal CA3 region after pilocarpine-induced seizures by H&E staining; seizures led to significantly increased cell death at 24 h (Fig. 1) as compared with controls. Moreover, DZ pretreatment significantly attenuated the neuronal loss induced by seizures, and the protective effect was attenuated by preconditioning with 5-HD. MDA level started to increase at 2 h after seizures and peaked at 7 days (Fig. 2A and B). SOD activity was significantly decreased at 24 h after SE, with no difference in levels from controls at 2 h, 72 h and 7 d. DZ pretreatment markedly decreased MDA level and increased SOD activation as compared with no pretreatment. 5-HD, when administered with DZ, completely prevented the DZ-mediated decrease in MDA activity and increase in SOD level. Similarly, 5-HD treatment alone could aggravate seizure-induced oxidative damage (Fig. 2C and D). Inhibiting PI3K activation by WTN increased MDA activity and decreased SOD level as compared with DZ treatment alone. Corresponding, WTN treatment alone aggravated seizure-induced oxidative damage (Fig. 2C and D). Epilepsy is one of the most common neurological disorders. Although great progress has been made in elucidating cell death after seizures, the mechanisms underlying neuronal death have not been studied well. Moreover, strategies to protect against neuron death are still limited. Recently, DZ, a highly selective opener of the mitoKATP channel, was reported to reduce SE neuron damage by reducing oxidative stress [9]. Our research in rats demonstrated massive neuronal loss in the vulnerable CA3 hippocampus area of rat at 24 h after seizures, and DZ could significantly attenuate neuronal loss induced by seizures. DZ attenuated oxidative stress injury through the mitoKATP channel as was previously found [9] by upregulating SOD activity and downregulating MDA level, because these alterations could be partially abolished by 5-HD, an inhibitor of mitoKATP . In addition, WTN, an inhibitor of PI3K, attenuated the changes in MDA and SOD levels after seizures. Thus, DZ could reduce oxidative injury induced by seizures by suppressing the activity of MDA and increasing the level of SOD in part by the PI3K/Akt pathway. Oxidative stress has been implicated in many human degenerative diseases, including epilepsy. It is one of the important mechanisms that play a role in the etiology of seizure-induced neuronal death after the first hour of the acute phase of seizures [21]. The most important effect of free radicals is LPO. A growing body of evidence suggests that elevated levels of LPO and/or its metabolites are potentially neurotoxic [4,25]. Seizures alter membrane lipid composition can affect membrane fluidity permeability and consequently the function of membrane-bound enzymes, which in turn, may have serious consequences on neuronal functioning [10]. We demonstrated that the level of MDA, a measure of LPO, increased significantly after SE, from 2 h to a peak on day 7, which agrees with previous reports [23]. The increased MDA level indicates that the existing LPO could be responsible for neuronal damage after SE and the existing antioxidant capacity is not enough to protect brain cells against oxidative damage. Free radical scavengers, considered the primary antioxidant defense system, are a protective pathway. We observed no alteration in hippocampal SOD activity during 2 h, 72 h or 7 d after seizures, which suggests that a high amount of H2 O2 , released during O2 – dismutation can inhibit SOD activity during this phase of pilocarpine-induced seizures [1,30]. In addition, the decreased SOD activity 24 h after SE agrees with earlier reports [1], so LPO could depend on decreased SOD activity. Furthermore, other antioxidant
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Table 1 Effect of diazoxide on behavioral episodes in rat hippocampus after seizures. Group
n
Latency period to stage IV (mean ± SD)
Pilo Pilo + DZ Pilo + DZ+ 5-HD Pilo + DZ+ WTN
20 20 20 20
42.3 67.8 48.9 50.7
± ± ± ±
6.2 9.6* 9.1** 6.3**
Proportion of stage IV (%)
Proportion of stage V (%)
38.9 80.1* 41.2** 43.4**
661.1 119.9* 548.8** 556.6**
Data are mean ± SD (n = 20/group). Pilo, pilocarpine; DZ, diazoxide; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin. * p < 0.05 vs. Pilo. ** p < 0.05 vs. Pilo + DZ.
systems such as glutathione peroxidase may be involved by their inhibiting neurotoxicity induced by seizures [18]. Pilocarpine thus caused an imbalance between the antioxidant and oxidant defense systems, which may be partly responsible for seizures. Oxidative stress is recognized as a fundamental pathway leading to cellular death and dysfunction [31]. Therefore, antioxidants are protective against neuronal loss in the hippocampus following oxidative damage [26,27]. In agreement with an earlier report showing that DZ preconditioning has protective effects by attenuating oxidative stress injury through upregulating SOD activity
and downregulating MDA quantity in hypothermic preservationinduced renal injury [33], we demonstrated that DZ has an antioxidative stress effect in this epilepsy model by decreasing MDA level and enhancing antioxidant enzymes activities such as SOD in rats subjected to SE. This finding extends the protection profile of DZ, which has shown a beneficial effect in other models such as ischemia/reperfusion [7,20]. Our histological and biochemical findings were related to the primary neuroprotective effects of DZ [9,33], and the ability of DZ to increase the activity of antioxidant enzymes and decrease free radical formation could lead to a signif-
Fig. 1. Hematoxylin and eosin staining of pyramidal neurons of the rat hippocampus CA3 (Panels A–E) 24 h after pilocarpine-induced seizures (magnification 400×). (A). A, control group, showing normal pyramidal neurons; B, pilocarpine group, showing neuronal death; C, diazoxide (DZ) treatment protected neurons against cell death; D, 5hydroxydecanoic acid (5-HD) treatment attenuated the DZ-mediated effect; E, wortmannin (WTN) treatment partially blocked the neuroprotective effect of DZ. Bar = 50 m, n = 6/group. (A ). The number of surviving cells after seizures. Bars indicate mean ± SD. *p < 0.05 vs. control, # p < 0.05 vs. Pilo and **p < 0.05 vs. DZ. Pilo, pilocarpine; DZ, diazoxide; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin; DZ = pilo + DZ. 5-HD = pilo + DZ + 5-HD. WTN = pilo + DZ + WTN.
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Fig. 2. Changes in malondialdehyde (MDA) level and superoxide dismutase (SOD) activity after seizures. (A) Time course of MDA and (B) SOD expression in hippocampus after seizures of pilo group. (C) MDA and (D) SOD activity at 24 h after seizures. Data are mean ± SD (n = 10/group). *p < 0.05 vs. control, # p < 0.05 vs. Pilo and **p < 0.05 vs. DZ. Pilo, pilocarpine; DZ, diazoxide; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin; DZ = pilo + DZ. DZ + 5-HD = pilo + DZ + 5-HD. DZ + WTN = pilo + DZ + WTN.
icant decrease in susceptibility to seizures induced by pilocarpine. These observations suggest that DZ has promising anticonvulsant effects on pilocarpine-induced seizures. We found that 5-HD, when administered with DZ, reversed the protective effect of DZ on oxidative damage, which was consistent with previous results [29]. However, the mechanism by which mitoKATP channel opening leads to reduced MDA and increased SOD levels remains for further investigation. The PI3K pathway plays an important role in cell survival by both enhancing the expression of anti-apoptotic proteins and inhibiting the activity of pro-apoptotic ones [12]. Neuroprotection against oxidative stress may be via activation of Akt survival signaling [5,6]. Thus, we hypothesized that direct PI3K/Akt activation might protect neurons against oxidative stress-induced neurotoxicity after seizures. Inhibition of PI3K with WTN greatly attenuated the protective effects of DZ, including decreasing SOD level and enhancing MDA activity, which suggested that some of the protective effects of DZ can be through a PI3K/Akt pathway and agrees with previous conclusions [13]. Recently Cui et al. found that DZ could activate Akt signaling pathway to improve the survival of cells under oxidative stress in myocardial infarction [3]. However, the upregulation of SOD activity was not completely reestablished by WTN, so activation of PI3K/Akt may not be the only mechanism connecting the opening of the mitoKATP channel, inhibition of oxidative stress and neuroprotection. In summary, we used a rat model of epileptic seizures induced by pilocarpine to show that DZ may protect hippocampal neurons against cell death through the mitoKATP channel and PI3K/Akt to downregulate MDA and upregulate SOD, thus preventing oxidative
stress. DZ could be a potential treatment for hippocampal neuron demise caused by seizures.
Acknowledgments This study was supported by a grant from the National Natural Science Foundation of China (no. 30870838).
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Diazoxide preconditioning against seizure-induced oxidative injury is via the PI3K/Akt pathway in epileptic rat Yuan Xue a , Nanchang Xie a , Lili Cao a , Xiuhe Zhao a , Hong Jiang b , Zhaofu Chi a,∗ a b
Department of Neurology, Qilu Hospital, Shandong University, 44#, Wenhua Xi Road, Jinan 250012, China Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Shandong University, Jinan, China
a r t i c l e
i n f o
Article history: Received 15 January 2011 Received in revised form 8 March 2011 Accepted 18 March 2011 Keywords: Diazoxide PI3K Oxidative stress Seizure Pilocarpine
a b s t r a c t Diazoxide (DZ), a highly selective opener of the mitochondrial ATP-sensitive potassium (mitoKATP ) channel, has neuroprotective effects against neuronal cell death by reducing oxidative stress. However, the mechanism of DZ protecting hippocampal neurons against seizure-induced oxidative injury is unknown. In this study, we investigated DZ attenuating neuronal loss caused by pilocarpine-induced seizures in rat hippocampus. DZ attenuated oxidative stress injury by upregulating superoxide dismutase (SOD) activity and downregulating malondialdehyde (MDA) level, which could be abolished with 5-hydroxydecanoic acid, an inhibitor of mitoKATP . In addition, wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI3K), attenuated the changes in MDA and SOD levels after seizures. DZ could reduce oxidative injury induced by seizures by suppressing the activity of MDA and increasing the level of SOD in part by the PI3K/Akt pathway. © 2011 Elsevier Ireland Ltd. All rights reserved.
Accumulating laboratory and clinical data have indicated that seizures can cause neuronal damage in certain brain regions. Excessive production of reactive oxygen species (ROS) produces an imbalance in the redox system of cells. This situation results in potential damage of macromolecules, including proteins, lipids, and nucleic acids [2], collectively referred to as oxidative stress. Brain tissue is particularly vulnerable to oxidative damage because of its high consumption of oxygen and the consequent generation of high quantities of free radicals. Growing data suggest that injury resulting from oxidative stress may have an important role in pathophysiologic features after acute neurological insults such as stroke and seizures [21]. Malondialdehyde (MDA) is the final product of lipid peroxidation (LPO). Concentrations of MDA reflect the state of the free radical system [17]. Free radical scavengers, such as superoxide dismutase (SOD) and reduced glutathione, are protective against seizure-induced oxidative damage [14]. SOD, considered an important antioxidant enzyme, can remove superoxide anions from cells
Abbreviations: DZ, diazoxide; MitoKATP , mitochondrial ATP-sensitive potassium; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin; PI3K, phosphatidylinositol-3kinase; MDA, malondialdehyde; SOD, superoxide dismutase; CA3, cornu ammonis region 3. ∗ Corresponding author at: Department of Neurology, Qilu Hospital of Shandong University, 107#, Wenhua Xi Road, Jinan 250012, China. Tel.: +86 531 82169428; fax: +86 531 86927544. E-mail addresses: [email protected], [email protected] (Z. Chi). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.054
[15]. The recognition of the relationship between oxidative stress and neuronal loss in epilepsy has sparked an intensive interest in developing an antioxidation strategy to protect neurons against oxidative damage after such seizures [24]. Mitochondrial ATP-sensitive potassium (mitoKATP ) channel openers, particularly diazoxide (DZ), protects against status epilepticus (SE)-induced neuron damage during diabetic hyperglycemia [9]. Furthermore, preconditioning with mitoKATP channel openers protects against neuronal cell death by reducing oxidative stress [11,19]. As well, DZ could protect primary mesencephalic neurons by inhibiting ROS overproduction and preventing the loss of mitochondrial membrane potential and the release of mitochondrial cytochrome c [32]. The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway plays a central role in intracellular processes such as cell survival and proliferation. PI3K/Akt regulates the survival response to oxidative stress-associated neuronal apoptosis [8]. Furthermore, mitoKATP channel openers such as DZ may activate the PI3K/Akt pathway in anoxia-reoxygenation injury [16]. We thus investigated a potential target for preventing hippocampal neuronal damage caused by SE. We examined whether DZ might upregulate SOD activity and downregulate MDA quantity by activating a PI3K/Akt signaling pathway in pilocarpine-induced seizures in rat. To test our hypothesis, we examined the effect of wortmannin (WTN) on activity of MDA and SOD. Specifically, we tested whether a neuroprotective effect of DZ was due to the
Y. Xue et al. / Neuroscience Letters 495 (2011) 130–134
activation of the mitoKATP channel, by using 5-hydroxydecanoic acid (5-HD), an inhibitor of mitoKATP . Adult male Wistar rats (Experimental Animal Center of Shandong University, China) weighing 250–300 g were used. Animals were maintained at room temperature (20 ± 2 ◦ C) with a 12-h light/12-h dark cycle and had free access to food and water. The experimental procedures were approved by the Shandong University Commission for Ethics of Experiments on Animals in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, revised 1996). All efforts were made to minimize the number of animals used and their suffering. The experimental animals were given lithium chloride intraperitoneally (i.p., 3 mEq/kg), then 20 h later, pilocarpine (30 mg/kg, i.p.; Sigma, St. Louis, MO, USA); control rats received the same volume of 0.9% saline instead of pilocarpine. Scopolamine methyl nitrate (1 mg/kg) was injected subcutaneously 30 min before pilocarpine to prevent peripheral cholinergic effects. Experimental rats showed stage 4 or 5 convulsive seizures [22]. Seizures were allowed to last for 60 min for all rats and then were terminated by the administration of diazepam (10 mg/kg, i.p.). Rats were killed by decapitation at 2, 24, 72 h and 7 days after seizures. Rats were randomly divided into 7 groups for treatment: (1) control, as described previously; (2) pilocarpine (pilo); lithium chloride–pilocarpine as described previously; (3) pilo + DZ, 10 mg/kg (Sigma), i.p. injected 30 min before pilocarpine; (4) pilo + DZ + 5-hydroxydecanoic acid (5-HD), 5 mg/kg (Sigma), i.p. injected 15 min before DZ; (5) pilo + DZ + wortmannin (WTN), 15 g/kg (Sigma), i.p. injected 15 min before DZ; (6) 5-HD; and (7) WTN. For groups 4 and 5, pilocarpine was given 30 min after DZ injection and for groups 6 and 7, pilocarpine was given 45 min after 5-HD or WTN injection. The solvent for DZ was dimethylsulfoxide (25 mg/ml) and for 5-HD salt serum at a final concentration of 1 mg/ml [28]. We recorded latency to stage IV seizures and percentage of animals with seizure episodes. Rats were killed by decapitation 24 h after seizures. Rats were anesthetized and intracardially perfused with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). Paraffinfixed brains were cut coronally in 5-m-thick sections for staining with hematoxylin and eosin (H&E). Surviving cells were defined as round-shaped, cytoplasmic membrane-intact cells, without any nuclear condensation or distorted aspect. The surviving pyramidal cells in the hippocampal CA3 region were observed under a microscope (400×). Hippocampi were homogenized in 0.9% saline (1:9, tissue:saline, w/v) on ice, by use of a homogenizer (10,000–15,000 rpm, 10 s). The tubes with homogenates were kept in ice water for 30 min and centrifuged at 4 ◦ C (3000 × g, 10 min) according to the commercial assay kits. The supernatants were separated and stored at −80 ◦ C. Protein content was determined by the use of BCA protein assay kits (Beyotime, Jiangsu, China). LPO was determined by measuring the accumulation of thiobarbituric acid-reactive substance in homogenates and expressed as MDA content. MDA and SOD referents were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), the contents of which were measured by the use of an UV/visible-120-2 Spectrophotometer (Shimadzu Corp.) as described by commercial assay kits. The concentrations of MDA and SOD were expressed as n mol/mg protein and U/mg protein respectively. All values are expressed as means ± standard error of the mean (SEM). The data were analyzed by one-way ANOVA, then Newman–Keuls test for multiple group comparisons. Comparisons of animals with stage 4 and 5 seizures were analyzed by Chi-square test. P < 0.05 was considered statistically significant. Pilocarpine-induced behavioral episodes typically increased in intensity and duration, gradually progressing towards SE. The latency period with DZ injection was significantly longer and the
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percentage of animals reaching stage V SE was considerably lower than with pilocarpine alone. In addition, 5-HD or WTN treatment reversed the DZ-mediated protection of seizures as compared with DZ treatment alone (Table 1). Control animals did not exhibit any behavioral seizure activity. We examined neuronal loss in the hippocampal CA3 region after pilocarpine-induced seizures by H&E staining; seizures led to significantly increased cell death at 24 h (Fig. 1) as compared with controls. Moreover, DZ pretreatment significantly attenuated the neuronal loss induced by seizures, and the protective effect was attenuated by preconditioning with 5-HD. MDA level started to increase at 2 h after seizures and peaked at 7 days (Fig. 2A and B). SOD activity was significantly decreased at 24 h after SE, with no difference in levels from controls at 2 h, 72 h and 7 d. DZ pretreatment markedly decreased MDA level and increased SOD activation as compared with no pretreatment. 5-HD, when administered with DZ, completely prevented the DZ-mediated decrease in MDA activity and increase in SOD level. Similarly, 5-HD treatment alone could aggravate seizure-induced oxidative damage (Fig. 2C and D). Inhibiting PI3K activation by WTN increased MDA activity and decreased SOD level as compared with DZ treatment alone. Corresponding, WTN treatment alone aggravated seizure-induced oxidative damage (Fig. 2C and D). Epilepsy is one of the most common neurological disorders. Although great progress has been made in elucidating cell death after seizures, the mechanisms underlying neuronal death have not been studied well. Moreover, strategies to protect against neuron death are still limited. Recently, DZ, a highly selective opener of the mitoKATP channel, was reported to reduce SE neuron damage by reducing oxidative stress [9]. Our research in rats demonstrated massive neuronal loss in the vulnerable CA3 hippocampus area of rat at 24 h after seizures, and DZ could significantly attenuate neuronal loss induced by seizures. DZ attenuated oxidative stress injury through the mitoKATP channel as was previously found [9] by upregulating SOD activity and downregulating MDA level, because these alterations could be partially abolished by 5-HD, an inhibitor of mitoKATP . In addition, WTN, an inhibitor of PI3K, attenuated the changes in MDA and SOD levels after seizures. Thus, DZ could reduce oxidative injury induced by seizures by suppressing the activity of MDA and increasing the level of SOD in part by the PI3K/Akt pathway. Oxidative stress has been implicated in many human degenerative diseases, including epilepsy. It is one of the important mechanisms that play a role in the etiology of seizure-induced neuronal death after the first hour of the acute phase of seizures [21]. The most important effect of free radicals is LPO. A growing body of evidence suggests that elevated levels of LPO and/or its metabolites are potentially neurotoxic [4,25]. Seizures alter membrane lipid composition can affect membrane fluidity permeability and consequently the function of membrane-bound enzymes, which in turn, may have serious consequences on neuronal functioning [10]. We demonstrated that the level of MDA, a measure of LPO, increased significantly after SE, from 2 h to a peak on day 7, which agrees with previous reports [23]. The increased MDA level indicates that the existing LPO could be responsible for neuronal damage after SE and the existing antioxidant capacity is not enough to protect brain cells against oxidative damage. Free radical scavengers, considered the primary antioxidant defense system, are a protective pathway. We observed no alteration in hippocampal SOD activity during 2 h, 72 h or 7 d after seizures, which suggests that a high amount of H2 O2 , released during O2 – dismutation can inhibit SOD activity during this phase of pilocarpine-induced seizures [1,30]. In addition, the decreased SOD activity 24 h after SE agrees with earlier reports [1], so LPO could depend on decreased SOD activity. Furthermore, other antioxidant
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Table 1 Effect of diazoxide on behavioral episodes in rat hippocampus after seizures. Group
n
Latency period to stage IV (mean ± SD)
Pilo Pilo + DZ Pilo + DZ+ 5-HD Pilo + DZ+ WTN
20 20 20 20
42.3 67.8 48.9 50.7
± ± ± ±
6.2 9.6* 9.1** 6.3**
Proportion of stage IV (%)
Proportion of stage V (%)
38.9 80.1* 41.2** 43.4**
661.1 119.9* 548.8** 556.6**
Data are mean ± SD (n = 20/group). Pilo, pilocarpine; DZ, diazoxide; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin. * p < 0.05 vs. Pilo. ** p < 0.05 vs. Pilo + DZ.
systems such as glutathione peroxidase may be involved by their inhibiting neurotoxicity induced by seizures [18]. Pilocarpine thus caused an imbalance between the antioxidant and oxidant defense systems, which may be partly responsible for seizures. Oxidative stress is recognized as a fundamental pathway leading to cellular death and dysfunction [31]. Therefore, antioxidants are protective against neuronal loss in the hippocampus following oxidative damage [26,27]. In agreement with an earlier report showing that DZ preconditioning has protective effects by attenuating oxidative stress injury through upregulating SOD activity
and downregulating MDA quantity in hypothermic preservationinduced renal injury [33], we demonstrated that DZ has an antioxidative stress effect in this epilepsy model by decreasing MDA level and enhancing antioxidant enzymes activities such as SOD in rats subjected to SE. This finding extends the protection profile of DZ, which has shown a beneficial effect in other models such as ischemia/reperfusion [7,20]. Our histological and biochemical findings were related to the primary neuroprotective effects of DZ [9,33], and the ability of DZ to increase the activity of antioxidant enzymes and decrease free radical formation could lead to a signif-
Fig. 1. Hematoxylin and eosin staining of pyramidal neurons of the rat hippocampus CA3 (Panels A–E) 24 h after pilocarpine-induced seizures (magnification 400×). (A). A, control group, showing normal pyramidal neurons; B, pilocarpine group, showing neuronal death; C, diazoxide (DZ) treatment protected neurons against cell death; D, 5hydroxydecanoic acid (5-HD) treatment attenuated the DZ-mediated effect; E, wortmannin (WTN) treatment partially blocked the neuroprotective effect of DZ. Bar = 50 m, n = 6/group. (A ). The number of surviving cells after seizures. Bars indicate mean ± SD. *p < 0.05 vs. control, # p < 0.05 vs. Pilo and **p < 0.05 vs. DZ. Pilo, pilocarpine; DZ, diazoxide; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin; DZ = pilo + DZ. 5-HD = pilo + DZ + 5-HD. WTN = pilo + DZ + WTN.
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Fig. 2. Changes in malondialdehyde (MDA) level and superoxide dismutase (SOD) activity after seizures. (A) Time course of MDA and (B) SOD expression in hippocampus after seizures of pilo group. (C) MDA and (D) SOD activity at 24 h after seizures. Data are mean ± SD (n = 10/group). *p < 0.05 vs. control, # p < 0.05 vs. Pilo and **p < 0.05 vs. DZ. Pilo, pilocarpine; DZ, diazoxide; 5-HD, 5-hydroxydecanoic acid; WTN, wortmannin; DZ = pilo + DZ. DZ + 5-HD = pilo + DZ + 5-HD. DZ + WTN = pilo + DZ + WTN.
icant decrease in susceptibility to seizures induced by pilocarpine. These observations suggest that DZ has promising anticonvulsant effects on pilocarpine-induced seizures. We found that 5-HD, when administered with DZ, reversed the protective effect of DZ on oxidative damage, which was consistent with previous results [29]. However, the mechanism by which mitoKATP channel opening leads to reduced MDA and increased SOD levels remains for further investigation. The PI3K pathway plays an important role in cell survival by both enhancing the expression of anti-apoptotic proteins and inhibiting the activity of pro-apoptotic ones [12]. Neuroprotection against oxidative stress may be via activation of Akt survival signaling [5,6]. Thus, we hypothesized that direct PI3K/Akt activation might protect neurons against oxidative stress-induced neurotoxicity after seizures. Inhibition of PI3K with WTN greatly attenuated the protective effects of DZ, including decreasing SOD level and enhancing MDA activity, which suggested that some of the protective effects of DZ can be through a PI3K/Akt pathway and agrees with previous conclusions [13]. Recently Cui et al. found that DZ could activate Akt signaling pathway to improve the survival of cells under oxidative stress in myocardial infarction [3]. However, the upregulation of SOD activity was not completely reestablished by WTN, so activation of PI3K/Akt may not be the only mechanism connecting the opening of the mitoKATP channel, inhibition of oxidative stress and neuroprotection. In summary, we used a rat model of epileptic seizures induced by pilocarpine to show that DZ may protect hippocampal neurons against cell death through the mitoKATP channel and PI3K/Akt to downregulate MDA and upregulate SOD, thus preventing oxidative
stress. DZ could be a potential treatment for hippocampal neuron demise caused by seizures.
Acknowledgments This study was supported by a grant from the National Natural Science Foundation of China (no. 30870838).
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