Ascorbic acid ameliorates seizures and brain damage in rats through inhibiting autophagy

brain research 1535 (2013) 115–123

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Research Report

Ascorbic acid ameliorates seizures and brain damage in rats through inhibiting autophagy Yan Donga,1, Shengjun Wangb, Tongxia Zhangb, Xiuhe Zhaob, Xuewu Liub, Lili Caob,n, Zhaofu Chib a

Department of Nuclear Medicine, Qilu Hospital of Shandong University, Jinan 250012, China Department of Neurology, Qilu Hospital of Shandong University, Jinan 250012, China

b

art i cle i nfo

ab st rac t

Article history:

Oxidative stress is a mechanism of cell death induced by seizures. Antioxidant compounds

Accepted 20 August 2013

have neuroprotective effects due to their ability to inhibit free radical production. Autophagy is a

Available online 28 August 2013

process in which cytoplasmic components such as organelles and proteins are delivered to the

Keywords: Seizures Oxidative stress Ascorbic acid Autophagy Beclin 1

lysosomal compartment for degradation, and plays an essential role in the maintenance of cellular homeostasis. The activity of autophagy is enhanced during oxidative stress. The objectives of this work were first to study the inhibitory action of antioxidant ascorbic acid on behavioral changes and brain damage induced by high doses of pilocarpine, then to study the effect of ascorbic acid on oxidative stress (MDA and SOD were used to estimate oxidative stress) and activated autophagy (beclin 1 was used to estimate autophagy) induced by seizures, aiming to further clarify the mechanism of action of this antioxidant compound. In order to determinate neuroprotective effects, we studied the effects of ascorbic acid (500 mg/kg, i.p.) on the behavior and brain lesions observed after seizures induced by pilocarpine (340 mg/kg, i.p., P340 model) in rats. Ascorbic acid injections prior to pilocarpine suppressed behavioral seizure episodes by increasing the latency to the first myoclonic, clonic and tonic seizure and decreasing the percentage of incidence of clonic and tonic seizures as well as the mortality rate. These findings suggested that oxidative stress can be produced and autophagy is increased during brain damage induced by seizures. In the P340 model, ascorbic acid significantly decreased cerebral damage, reduced oxidative stress and inhibited autophagy by reducing de novo synthesis of beclin 1. Antioxidant compound can exert neuroprotective effects associated with inhibition of free radical production and autophagy. These results highlighted the promising therapeutic potential of ascorbic acid in treatment for seizures. & 2013 Elsevier B.V. All rights reserved.

Abbreviations: SE, status epilepticus; ROS, reactive oxygen species; MDA, Malondialdehyde; LPO, peroxidation; SOD, superoxide dismutase; AA, ascorbic acid; PI3K, phoshatidylinositol-3-kinase n Corresponding author. Fax: þ86 5318 602 6772. E-mail address: [email protected] (L. Cao). 1 Chief technician. 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.08.039

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1.

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Introduction

Epilepsy is one of the most common serious neurological condition and approximately 50 million people worldwide have it, which is characterized by spontaneous recurrent seizures. Status epilepticus (SE) is a severe form of continuous seizure attacks and a medical emergency associated with brain damage and significant mortality (Aminoff and Simon, 1980). The common sequels of SE include continuing recurrent seizures, permanent neurological deficit and brain injury. Systemic injection of pilocarpine induces SE in rodents associated to histopathological alterations, which are most prominent in the limbic structures. Seizures and SE induced by pilocarpine are similar to human temporal lobe epilepsy in semiology and electrographic appearance (Martinez and N’Gouemo, 2010). Pilocarpine administration induces seizures with distinct phases. The first phase is an acute period lasting for 1–2 days, which is associated to repetitive seizures and SE, and usually was used as acute epileptic model. Histopathological examinations during the acute phase of seizures induced by pilocarpine are characterized by hippocampal brain damage including neuronal loss, gliosis and vacuolation (Scorza et al., 2009; Wieser, 2004). It has been reported that free radicals and oxidants systems may be responsible for propagating the brain damage induced by seizures, experimental evidence indicates that antioxidants compounds can protect against the neuronal damage. Reactive oxygen species (ROS) are involved in neurodegeneration in the pilocarpine model of temporal lobe epilepsy (Xue et al., 2011; Freitas, 2009). Malondialdehyde (MDA) is the final product of lipid peroxidation (LPO). Concentrations of MDA reflect the state of the free radical system (Mehla et al., 2010). Free radical scavengers, such as superoxide dismutase (SOD) and reduced glutathione, are protective against seizure-induced oxidative damage. SOD, considered as an important antioxidant enzyme, can remove superoxide anions from cells (Liu et al., 2010). The recognition of the relationship between oxidative stress and neuronal loss in epilepsy has raised an intensive interest in developing an antioxidation strategy to protect neurons against oxidative damage after seizures. Ascorbic acid (AA) has many nonenzymatic actions and is a powerful water-soluble antioxidant. Studies have demonstrated that AA can protect low density lipoproteins from oxidation and reduces harmful oxidants to ameliorate oxidative stress in the hippocampus during seizures, and reduced hippocampus lesion produced by seizures (Santos et al., 2008). However, the mechanism of ascorbic acid against the hippocampus lesion is not still established. Autophagy is a catalytic process of the bulk degradation of long-lived cellular components, ultimately resulting in lysosomal digestion within mature cytoplasmic compartments known as autophagolysosomes. Autophagy serves many functions in the cell, including maintaining cellular homeostasis, a means of cell survival during stress (e.g., nutrient deprivation or starvation) or conversely as a mechanism for cell death (type II progressed cell death) (Essick and Sam, 2010; Levine and Kroemer, 2008). Increased ROS production and the resulting oxidative cell stress that occurs in many disease states has been shown to induce autophagy. ROS have been ascribed as positive regulators of autophagy. Elevated ROS causing autophagy promotes either cell survival or cell death, the fate of

which depends upon the severity of the stress occurring with a particular disease (Cherra et al., 2010; Pivtoraiko et al., 2009). Studies show that autophagic activity may be rapidly increased in response to oxidative stress that occurs during seizures (Ceru et al., 2010; Tizon et al., 2010). Beclin 1, a Bcl-2interacting protein, is the mammalian homolog of yeast VPS30p/Apg6p, forms a complex with phoshatidylinositol-3kinase (PI3K), and participates in the early stages of autophagosome formation, promoting the nucleation of autophagosome. It has been shown that beclin 1 is essential for autophagy as well as for lysosomal enzyme transport (Liang et al., 2001; Suzuki et al., 2001). The level of beclin 1 is associated with the activity of autophagy. In this study, pilocarpine was used to elicit SE. We sought to determine whether autophagy was activated after SE in rats in vivo. In addition, whether pretreatment with antioxidant AA influenced behavioral changes, brain damage and autophagy after SE were determined. Beclin 1 was used to estimate autophagy. MDA and SOD were used to reflect the state of the free radical system and oxidative stress respectively. Neurons in CA3 region are very vulnerable to SE stress, so the extent of neuron loss in hippocampus CA3 after pilocarpine-induced seizures was used to evaluate brain damage.

2.

Results

1. Pilocarpine induced the first myoclonic, clonic and tonic seizure at 39.38717.75 min (n ¼16). Eighty percent of the animals in pilocarpine group presented generalized tonic– clonic convulsions (80%) with SE, and 50% of them survived the seizures at 24 h after pilocarpine injection. All animals pretreated with the ascorbic acid selected for this study were observed for 24 h after pilocarpine injection. Half of them manifested motor seizures, which develop progressively within 1–2 h into a long-lasting SE (50%). Results have shown that when administered before pilocarpine, AA (500 mg/kg) reduced seizures significantly (po0.05), increased a latency to the first myoclonic, clonic and tonic seizure (58.14723.95 min, n ¼10) (po0.01) and augmented survival percentage (90%) (po0.05) when compared to the pilocarpine-treated group. None of the control animals (isotonic saline or AA) showed seizure activity (Table 1). 2. MDA level increased significantly at 24 h after seizures, but SOD significantly decreased at 24 h after SE. AA pretreatment markedly decreased MDA and increased SOD as compared with pilocarpine group (Table 2). 3. Seizures led to severe cell death in CA3 at 24 h after seizures. The surviving neuron numbers were sharply decreased in pilocarpine group compared with control group. Moreover, AA significantly attenuated the neuron loss induced by seizures (Table 3 and Fig. 1). 4. Beclin 1 mRNA level increased significantly at 24 h after seizures, the same was to beclin 1 protein, which indicated that autophagy was activated in hippocampus neurons by seizures. Pretreatment with AA inhibited activated autophagy in rats with seizures by reducing both mRNA and protein levels of beclin 1 (Fig. 2).

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Table 1 – Effect of AA on behavioral episodes in adult rats after seizures. Data are mean 7 s.d. (n ¼5) for latency to first seizures. Groups

Latency to SE (min)

Percentage of incidence of SE

Survival percentage

Number of animals per group

Control Pilo AAþpilo AA

0 36.90714.46 58.62723.62n 0

0 80 50nn 0

100 50 90nnn 100

15 20 20 20

n

po0.01 as compared with pilo group (ANOVA and Student-Newman-Keuls test). po0.05 as compared with pilo group (Chi-square test). nnn po0.05 as compared with pilo group (Chi-square test). Pilo:pilocarpine; AA: ascorbic acid. nn

Table 2 – Effect of AA on MDA and SOD in rat hippocampus after seizures. Data are mean 7 s.d. (n ¼5). Groups

MDA (n mol/mg protein)

SOD (U/mg protein)

Control Pilo AAþpilo AA

2.0570.48 4.9971.14n 3.1570.65nn 1.8270.55

219.46763.48 94.82727.53n 169.64756.50nn 234.28768.24

n

po0.01 as compared with control group (ANOVA and Student-Newman-Keuls test). po0.05 as compared with pilo group (ANOVA and Student-Newman-Keuls test); pilo:pilocarpine; AA: ascorbic acid.

nn

3.

Dicussion

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. There is considerable evidence that neuronal damage after generalized SE is due to generation of reactive oxygen species. Oxidative stress occurs as a consequence of prolonged seizures and may contribute to seizure-induced brain damage and to the generation of the epileptic state, in which there are spontaneous seizures (Aguiar et al., 2012). The brain is uniquely vulnerable to oxidative stress-induced damage because of processing a large quantity of mitochondria, a high degree of oxidizable lipids and metals, high oxygen consumption, and less antioxidant capacity than other tissues making oxidative stress a likely contributor to neurological disorders such as the epilepsies (Waldbaum and Patel, 2010). Such a depressed defense system may be adequate under normal circumstances. However, under pro-oxidative conditions, such as during seizures, these low antioxidant defenses can predispose the brain to oxidative stress. In addition, hippocampus may be particularly sensitive to oxidative stress because of their low endogenous levels of vitamin E, an important biochemical antioxidant, relatively to other brain regions. The neuron death and loss caused by seizures were mainly located in CA1 and CA3 of hippocampus (Freitas et al., 2005). In present study, the number of surviving neuron in CA3 decreased significantly in rats with seizures, indicating that significant neuron death and hippocampus damage are present in rats with seizures induced by pilocarpine. During seizures, the oxygen consumption of brain increases sharply. Due to hardly any reservation of oxygen and glucose in brain, hypoglycemia and ischemia lead to lower ATP generation and energy depletion, which trigger ion pump

Table 3 – Effect of AA on surviving neurons in rats CA3 after seizures. Neuron number is expressed as the number of surviving pyramidal cells/0.5 mm length of the CA3 subfield of the hippocampus counted under light microscopy. Data are mean 7 s.d. (n¼ 5). Groups

Surviving neuron

Control Pilo AAþpilo AA

129.40717.56 88.60712.89n 113.20712.49nn 128.80716.43

n

po0.01 as compared with control group (ANOVA and StudentNewman-Keuls test). nn po0.05 as compare with pilo group (ANOVA and StudentNewman-Keuls test ). Pilo: pilocarpine, AA: ascorbic acid.

dysfunction, Ca2þ influx and release of excitatory amino acid. Glutamate-mediated excitotoxicity is the principal mechanism driving neuronal death after SE, whereby excessive glutamate release leads to intracellular calcium overload, oxidative stress, organelle swelling and rupture of intracellular membranes, activation of proteases and necrosis. Apoptosis plays an important role in neuron death induced by seizures (Henshall and Simon, 2005; Engel and Henshall, 2009; Meldrum, 2000). 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 (Golden et al., 2009; Júnior et al., 2009). We demonstrated that the level of MDA, a measure of LPO, increased significantly at 24 h after SE, which agrees with previous reports (Freitas, 2009; Freitas et al., 2004). 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

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Fig. 1 – High power (400  ) photomicrographs showing Nissl staining with toluidine blue of the hippocampal CA3 pyramidal neurons in rats with seizures (B, C) and non-seizure rats (A, D). (A) Control rat, showing normal CA3 pyramidal neurons (round and palely stained nuclei); (B) pilo group, showing CA3 pyramidal neurons death (shrunken neurons with pyknotic nuclei); (C) AAþPilo group, showing the effect of AA against neuron loss of CA3; (D) AA group, showing normal CA3 pyramidal neurons (round and palely stained nuclei). Bar¼ 20 μm, n ¼ 4; Pilo: pilocarpine, AA: ascorbic acid.

Fig. 2 – Beclin 1 protein and mRNA levels in hippocampus of rats. (A) Immunoblot analysis of beclin 1 in rat hippocampus in four groups, (C) quantitative representation of the protein levels of beclin 1 in all groups, (B) semi-quantitative RT-PCR analysis of beclin 1 mRNA in rat hippocampus in four groups, and (D) quantitative representation of the mRNA levels of beclin 1 in all groups. Data are expressed as mean7s.d. of at least five independent animals. *Po0.01 vs. control group, #Po0.05 vs. pilo group. aa, ascorbic acid; pilo, pilocarpine.

to protect brain cells against oxidative damage. These lipid metabolites along with abnormal ion homeostasis and lack of energy generation may contribute to cell injury and death (Arzimanoglou et al., 2002). Our results showed that pilocarpine administration produced increased lipid peroxidation content in hippocampus of adult animals, and, therefore,

demonstrated and confirmed the possible involvement of free radical injury in pilocarpine induced brain damage. Cells contain an elaborate antioxidant defense system consisting of enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GP) and glutathione reductase (GR), and numerous non-enzymatic antioxidants

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such as Vitamin E and glutathione. Cellular antioxidant systems have demonstrated a great adaptation to oxidative stress in order to avoid the oxidative damage caused by ROS overproduction. Antioxidant enzymes are regulated by ROS and cytokines, along with other factors (Shull et al., 1991; White et al., 1989). SOD, free radical scavengers, considered as the primary antioxidant defense system, especially is important part of the antioxidant system of neurons. We observed a significant decrease in SOD in hippocampus at 24 h after seizures, which agree with earlier reports. It is proposed that a high amount of H2O2, released during O2 dismutation can inhibit SOD during this phase of pilocarpine-induced seizures (Xue et al., 2011). The reduced SOD may weaken the protective function of brain and aggravate neuron damage induced by seizures. The completely opposite change of LPO to SOD indicates that increased LPO could depend on decreased SOD. However, some report showed that seizures induce oxidative stress, which is accompanied by an immediate compensatory increase in antioxidant enzymes including SOD that may be protective (Tejada et al., 2007). Furthermore, other antioxidant systems such as glutathione peroxidase may be involved by their inhibiting neurotoxicity induced by seizures. Pilocarpine thus impaired the balance 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. Therefore, antioxidants are protective against neuronal loss in the hippocampus following oxidative damage (Santos et al., 2011). Vitamins C (ascorbic acid, AA) and E (α-tocopherol) are exogenous powerful antioxidant molecules that act together with other endogenous antioxidant systems within tissue cells in order to scavenge the formed ROS (Tomé et al., 2010). AA can enter mitochondria by means of facilitative glucose transporter and confers mitochondrial protection against oxidative injury. AA has been implicated in many biological processes. It is a cofactor for several enzymatic steps in the synthesis of collagen, monoamines, amino acids, peptide hormones, and carnitine, and plays an important role in antioxidant defense at a number of levels. It can directly metabolize reactive oxygen species, acts to maintain α-tocopherol in its reduced form, and mediates electron transfer to ascorbate-dependent peroxidases. The protective capacity of ascorbic acid against seizures-induced cerebral damage in adult rats has been reported (Santos et al., 2008; Xavier et al., 2007). By biochemical test, we found that AA pretreatment significantly reduced the level of MDA and increased SOD at 24 h after seizures, suggesting that this drug acts positively in reduction lipid peroxidation levels. The present study also showed that antioxidant AA produced a significant reduction in both SE mobility and seizures mortality, and an increase in seizure latency in the pilocarpine model of acute seizure. Furthermore, AA also produced a significant increase in numbers of surviving neuron in CA3 in rats with seizures. It is proposed that AA play neuroprotective function by inhibiting oxidative stress in rats with seizures. It has been also shown that vitamin C can block the efflux, rather than influx, of calcium, and therefore, it can interfere with the mechanisms of neurotransmitter release and/or uptake from neuronal terminals. The neuronal protective effect of vitamin C can be responsible for the decrease of excitotoxicity that has been related to an over production of free radicals during

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seizures and SE, but the exact mechanisms of action are not fully established (Miura et al., 2009). Although studies have shown that antioxidants AA produced a significant reduction in seizure severity and an increase in seizure latency in the pilocarpine model of acute seizure. However, the antioxidant effect in pentylenetetrazol (PTZ) is still argumentative. It has been reported that there was no significant effect of AA in the kainic acid model. Thus, the antioxidant effect on seizure onset could be specific for the mechanism behind the seizures induced by pilocarpine. Pilocarpine is a potent muscarinic agonist, but the exact mechanism(s) underlying seizure onset in this model is not known (Rubio et al., 2010; Cavalheiro, 1995). The present study would suggest that early after administration of pilocarpine there is a generation of free radicals which contribute somewhat to the onset of the seizures. Autophagy is responsible for the clearance of long-lived proteins, organelles and protein aggregates. Autophagic degradation leads to the recycling of biological macromolecules, which are released for use in biosynthetic pathways (Yang et al., 2006). While autophagy is required for homeostasis in all cell types, non-dividing cells such as neurons, are particularly sensitive to changes in autophagic degradation (Cherra and Chu, 2008; Chu, 2006). The integrity of postmitotic neurons is heavily dependent on high basal autophagy compared to nonneuronal cells because misfolded proteins and damaged organelles cannot be diluted through cell division. Moreover, neurons contain the specialized structures for intercellular communication, such as axons, dendrites and synapses, which require the reciprocal transport of proteins, organelles and autophagosomes over significant distances from the soma (Son et al., 2012). As most neurons must survive for the lifetime of the organism, maintenance of organelle function and clearance of aberrant or damaged proteins are critical processes regulated by autophagy. It has been shown that autophagy play an important role in many neurodegenerative diseases such as Parkinson, Huntington and Alzheimer disease. It was reported that conditional autophagy-deficient mice rapidly develop neurodegenerative phenotypes in selected neuronal populations early in post-natal life (Hara et al., 2006; Komatsu et al., 2006). Neuronal cell loss in these mice supports a neuroprotective role for autophagy. At the other end of the spectrum, reduced levels of beclin 1, which is essential for autophagy pathway, have been found in aged humans and in brain tissues from Huntington and Alzheimer disease patients (Marino and Lopez-Otin, 2004; Xie and Klionsky, 2007). In some scenarios, autophagy may be initially induced as a protective response; however, in other scenarios, it may also contribute to the demise of the cell. Inhibiting the injury-induced elevations in autophagy can also protect against certain types of neuronal insults, particularly in the setting of acute neuronal injuries. For example, the parkinsonian neurotoxin MPPþ was found to cause a form of autophagic neuronal cell death, which was prevented using RNAi based inhibition of autophagy (Zhu et al., 2007). Similar observations have been reported in vivo during neonatal hypoxic-ischemic injury in mice, suggesting that autophagy induction elicited by certain neuronal injuries contributes to cell death (Koike et al., 2008). A recent paper proposed that autophagy might also alter the mode of cell death after hypoxic-ischemic injury in rats, changing the cell death pathway from apoptotic to necrotic

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when autophagy is inhibited (Carloni et al., 2008). Determinants of whether autophagy promotes cell survival or cell death depend upon the severity of the stress occurring with a particular disease. The present study showed that beclin 1 increased significantly in rats with seizures and decreased in AA-pretreated rats with seizures. It is indicated that autophagy was activated in acute neuronal injuries induced by seizures, and antioxidant AA could increase surviving neurons in CA3 by inhibiting autophagy, which suggests that autophagy induction elicited by seizures contributes to cell death and AA exerts neuroprotective function by inhibiting autophagy. It is shown recently that autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death, and suppression of beclin 1 prevents cell death suggesting it has a complex role regulating both apoptosis and autophagy, which further supports our study (Higgins et al., 2011). Elimination of damaged mitochondria and other damaged organelles by autophagy may act as rescue mechanism that the cell uses to escape from cell death. It is proposed that acitivated autophagy is helpful to remove the damaged mitochondria and abnormal protein produced during seizures, but as type II programmed cell death, overactivated autophagy can promote cell death. It is recently tested that excessive autophagy induced by ischemic stress contributes to neuron death, which also supports our study (Shi et al., 2012). Although what remains unclear is whether oxidative stress of SE provokes autophagy, contributes to secondary damage, or initiates cell death, which provokes autophagy setting the stage for recovery and/or repair. Regardless, it is clear that autophagy and oxidative stress are connected during SE, similar to autophagy and starvation in vitro, and that one effect of antioxidant therapy is a reduction in autophagy (Cao et al., 2009). The autophagic process is highly regulated and is stimulated by several factors. Cellular oxidative stress and increased generation of ROS have been reported to serve as important stimuli of autophagy during periods of nutrient deprivation, ischemia/reperfusion, hypoxia, and in response to cell stress (Scherz-Shouval et al., 2007; Azad et al., 2009). ROS induce autophagy through a beclin 1 dependent pathway that is associated with autophagic induced cell-death (De Meyer and Martinet, 2009). Beclin 1 is negatively regulated by its interaction with the anti-apoptotic protein Bcl-2 under normal conditions (Pattingre et al., 2005). However, increased ROS activates the ubiquitin-proteosome system, which functions to degrade Bcl-2. This allows for beclin 1 activation subsequently resulting in autophagic cell death (Meyer and Martinet, 2009). Additionally, oxidized low density lipoproteins may enhance autophagy by upregulating beclin-1 gene expression. siRNA against beclin 1 attenuates ROS-mediated ganglioside induced autophagic cell death (Hwang et al., 2010; Turski et al., 1983). In rats with seizures induced by pilocarpine, we found that MDA, beclin 1 and neuron death increased highly, but SOD decreased significantly. AA pretreatment lowered MDA, beclin 1 and neural damage, and raised SOD at the same time. So, it is proposed that during oxidative stress caused by seizures, ROS and LPO not only cause cell loss in CA3 but also induce autophagy through a beclin 1 dependent pathway, which aggravates the neuron death in hippocampus. Antioxidant AA exerts neuroprotective function by inhibiting oxidative stress and autophagy. But another study showed that antioxidants inhibited

autophagy and enhanced neurodegeneration in models of polyglutamine disease, which is opposite to the present study (Underwood et al., 2010). It may be due to the different role of autophagy in different diseases. Even in the same disease, the role of autophagy is controversial. It is generally believed that oxidative stress is a strong proautophagic stimulus. However, some evidence coming from neurobiology as well as from other fields indicates an inhibitory role of reactive oxygen species and reactive nitrogen species on the autophagic machinery. Furthermore, experimental evidence suggests a complex and ambiguous role of autophagy in PD since either impaired or abnormally upregulated autophagic flux has been shown to cause neuronal loss (Janda et al., 2012; Tung et al., 2012). The same is also to AD (Barnett and Brewer, 2011). So the role of autophagy in oxidative stress is complex and further research is needed.

4.

Conclusion

In summary, the major finding of this study is that both oxidative stress, detected by increased MDA and decreased SOD, and autophagy, as detected by increases beclin 1, are induced in injured brain after seizures in rats. An additional finding is that oxidative stress and autophagy are partially inhibited by the antioxidant AA. Furthermore, oxidative stress and autophagy induced by seizures can lead to hippocampus damage which is inhibited partly by AA. The results of our present study suggest that vitamin C displays anticonvulsive and antioxidant activities that might explain, at least in part, the drug ability to behave as a possible neuronal protective agent, increased the latency of first seizures, reducing mortality rate and increasing hippocampus SOD levels of adult rats.

5.

Experimental procedures

Adult male Wistar rats (Experimental Animal Center of Shandong University, China) weighing (250–280 g) were maintained at room temperature (2072 1C) 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 substances used were pilocarpine hydrochloride (Sigma Chemical, USA), ascorbic acid (Sigma Chemical, USA) and atropine (Sigma Chemical, USA). All doses are expressed in milligrams per kilogram (mg/kg) and were intraperitoneally administered. The experimental animals were given pilocarpine (340 mg/kg, i.p.; Sigma, St. Louis, MO, USA); control rats received the same volume of 0.9% saline instead of pilocarpine. Atropine methylnitrate (1 mg/kg) was injected subcutaneously 30 min before pilocarpine to prevent peripheral cholinergic effects. Rats were randomly divided into 4 groups for treatment: (1) control, as described previously (n¼ 15); (2) pilocarpine (pilo), pilocarpine as described previously (n¼20); (3) AAþpilo, 500 mg/kg (Sigma), i.p. injected 30 min before pilocarpine

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(n¼ 20); (4) AA, 500 mg/kg (Sigma), i.p. injected 30 min before saline (n¼20). After the last administration of the drug, the animals were placed in 30  30 cm2 chambers to observe their behavior and register the number of animals that had seizures, status epilepticus (SE) and deaths after pilocarpine administration. SE was defined as continuous seizures and characterized as stage 4 (rearing and falling), stage 5 (loss of balance, continuous rearing and falling), or stage 6 (severe tonic–clonic seizures) for a period longer than 30 min (Levy et al., 1992). We recorded latency to SE, percentage of animals with SE and the percentage of animals who survived at 24 h after seizures. Seizures were allowed to last for 120 min for all rats and then were terminated by the administration of diazepam (10 mg/kg, i.p.). Rats were killed by decapitation at 24 h after seizures. Rats were anesthetized and intracardially perfused with 4% paraformaldehyde in phosphate-buffered saline (pH 7.4). The brains were removed and kept in 4% paraformaldehyde for 12 h, then immersed in 25% sucrose for 3–4 days at 4 1C. The paraffinfixed brains were sectioned coronally in 5 μm thickness. Nissl staining with toluidine blue was performed. 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. The sections were examined with light microscopy (400  ) and number of surviving hippocampal CA3 (middle of CA3a) pyramidal cells per 0.5 mm length (selected one region of 0.5 mm length) was blindly counted. We used scale to select the range in which numbers of cells were counted. We did neuronal counts on CA3 in selected randomly every fifteenth section (6 sections of left or right hemisphere). Four rats from every group were used to obtain the cell counts. Detailed procedure was carried out as previously described (Hui et al., 2005). 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 1C (3000g, 10 min) according to the commercial assay kits. The supernatants were separated and stored at 80 1C. 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. Total RNA was prepared from hippocampus with Trizol reagent (Invitrogen, USA) and reversely transcribed to cDNA using AMV First Strand DNA Synthesis Kit (Biotech Company, China). Briefly, a 1 μg of the isolated RNA was reversely transcribed to cDNA at 37 1C for 1 h in a 20 μl of reaction mixture containing 1 μl AMV reverse transcriptase, 1 μl random hexamer, 4 μl 5  AMV buffer, 1 μl RNase inhibitor (20 U/μl), 1 μl dNTP (10 mM). The PCR amplification mixture (25 μl) consisted of 1 μl cDNA mixture, 1 μl Taq DNA polymerase, 5 μl of 5  PCR buffer, 5 mM dNTP mixture, and 50 pM sense and antisense primers each. Beta-actin was used as an internal control. Beclin 1 was

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analyzed by PCR and the used oligonucleotide primers included: for beclin 1 (316 bp) forward primer 5'-AGGAGCAGTGGACAAAGG-3' and reverse primer 5'-AGGGAAGAGGGAAAGGAC-3', for beta-actin (142 bp) forward primer 5'-GACAGGATGCAGAAGGAGATTACT-3' and reverse primer 5'-TGATCCACATCTGCTGGAAGGT-3'. The PCR conditions were as follows: for initial denaturing at 94 1C for 5 min, followed by 30 PCR cycles with temperatures below: 94 1C for 40 s, for beclin 1 54 1C for 40 s, or for beta-actin 52 1C for 40 s each, and then 72 1C for 40 s; a final extension at 72 1C for 10 min. The amplified products were subjected to electrophoresis at 120 V for 30 min on 1.5% agarose gels containing 0.5 mg/ml ethidium bromide, and quantified by respectively comparing luminosity of beclin 1 to that of b-actin using AlphaEase FC (FluorChem 9900) (Alpha Innotech). Equal amounts of protein were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), and then transferred to 0.45 lm nitrocellulose membrane using a Trans-Blot semidry system (Bio-Rad). After blocking in 5% fat-free milk in Tris-buffered saline with Tween buffer for 2 h, the membranes were incubated with a primary antibody such as anti-beclin 1 (1:500, AbAbcam, UK), anti-actin (1:500, Zhongshan Goldenbriodge Biotechnology CO.LTD, China), which served as a loading control, overnight at 4 1C. Then the membranes were washed and incubated with horseradish peroxidaseconjugated secondary antibody (goat antirabbit IgG, 1:10,000, Zhongshan Goldenbriodge Biotechnology CO.LTD, China) for 2 h under room temperature. Immunoreactivity was enhanced by chemiluminescence kit (Pierce, Rockford, Illinois, USA) and exposed to film. The bands on the film were scanned and analyzed with an image analyzer (Alpha Innotech, San Leandro, California, USA). All values are expressed as means7standard deviation (s.d.). The data were analyzed by one-way ANOVA, then Newman–Keuls test for multiple group comparisons. Comparisons of mortality and rate of Status epilepticus between pilo group and AA group were analyzed by χ2 test. Po0.05 was considered statistically significant.

Acknowledgment This work was supported by a grant from the Natural Sience Fundation of Shandong Province, China (No. Y2007C117), a grant from the National Nature Science Foundation of China (No. 81100971) and a grant from Independent Inovation Fundation of Shandong University (No. 2012TS169).

r e f e r e nc e s

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