Lipoic acid blocks seizures induced by pilocarpine via increases in δ-aminolevulinic dehydratase and Na+, K+-ATPase activity in rat brain

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Abstract

In the present study we investigated the effects of lipoic acid (LA) on δ-aminolevulinic dehydratase (δ-ALA-D) and Na+, K+-ATPase activities in rat brain after seizures induction by pilocarpine. Wistar rats were treated with 0.9% saline (i.p., control group), lipoic acid (10 mg/kg, i.p., LA group), pilocarpine (400 mg/kg, i.p., pilocarpine group), or the combination of LA (10 mg/kg, i.p.) with pilocarpine (400 mg/kg, i.p.), 30 min before administration of LA (LA plus pilocarpine group). After the treatments all groups were observed for 1 h. The enzyme activities (δ-ALA-D and Na+, K+-ATPase) were measured using spectrophotometric methods, and the results were compared with that obtained from saline and pilocarpine-treated animals. Neuroprotective effects of LA against seizures were evaluated based on those enzyme activities. The pilocarpine group showed a reduction in δ-ALA-D and Na+, K+-ATPase activities after seizures. In turn, LA plus pilocarpine abolished the appearance of seizures and reversed the decreased in δ-ALA-D and Na+, K+-ATPase activities produced by seizures, when compared to the pilocarpine seizing group. The results from the present study demonstrate that preadministration of LA abolished seizure episodes induced by pilocarpine in rat, probably by increasing δ-ALA-D and Na+, K+-ATPase activities in rat brain during seizures.

Introduction

The cholinergic system has been implicated in a variety of behavioral functions, including learning, control of cognition and memory, circadian cycle's synchronization and control of the body temperature. It is also involved in some electroencephalographic wave generation and regulation of the vigilance states (sleep–waking control) (Crouzier et al., 2006). Muscarinic cholinergic agonists, such as pilocarpine, have effects on slow wave sleep and rapid eye movement induction (Gamundí et al., 2003). Moreover, a high dose of pilocarpine is used to induce a pattern of repetitive limbic seizures and a status epilepticus state in rodents, which can last for several hours (Turski et al., 1989). The epileptic model induced by pilocarpine is a useful animal model to study the development and understanding of the neuropathology of temporal lobe epilepsy. This status epilepticus model is interesting because it reproduces behavioral and electroencephalographic alterations which are similar to those of human temporal lobe epilepsy (Turski et al., 1989).

Reactive oxygen species (ROS) have been implicated in the development of seizures and status epilepticus induced by pilocarpine (Freitas et al., 2004, Shin et al., 2008). For example, there is a temporal correlation between ROS formation and seizure development (Kim et al., 2002). In previous studies, we have demonstrated that pilocarpine induced seizure episodes in adult rats. Importantly, the seizure episodes induced by pilocarpine are related to oxidative stress, with increases on lipid peroxidation level and nitrite formation (Erakovié et al., 2000, Freitas et al., 2005). However, the effects of seizures related oxidative stress on δ-ALA-D and Na+, K+-ATPase activities have not been studied yet.

The brain is more susceptible to ROS damage due to the large lipid content of myelin sheaths and the high rate of brain oxidative metabolism (Rauca et al., 1999). In this context, the thiol redox state is an essential parameter associated with major biologic processes such as oxidative stress, intracellular redox homeostasis and gene expression (Sies, 1999). Lipoic acid (LA) which plays an essential role in mitochondrial dehydrogenase reactions, has recently gained considerable attention as an antioxidant. Lipoate, or its reduced form, dihydrolipoate, reacts with ROS such as superoxide radicals, hydroxyl radicals, hypochlorous acid, peroxyl radicals, and singlet oxygen. It also protects membranes by interacting with vitamin C and glutathione reduced, which may in turn recycle vitamin E (Packer et al., 1995). Recent studies showed that lipoic acid exerts anticonvulsant effects in an epilepsy model induced by pilocarpine (Freitas, 2009, Freitas et al., 2010).

In addition to its antioxidant activities, dihydrolipoate may exert prooxidant actions through reduction of iron. LA administration has been shown to be beneficial in a number of oxidative stress models such as ischemia, diabetes, and neurodegenerative diseases (Packer et al., 1997). Furthermore, LA can function as a redox regulator of proteins in neurodegenerative diseases (Genestra, 1997). Brain regions were chosen according to the neuropathology associated with status epilepticus that has been described in the hippocampus, striatum and frontal cortex. The aim of this study was to evaluate the effects of lipoic acid on δ-aminolevulinic dehydratase (δ-ALA-D) and Na+, K+-ATPase activities in rat brain after administration of high dose of pilocarpine.

Section snippets

Material and methods

The protocols for the animals experiments described in this study were performed in accordance with international (EEC Directive of 1986, 86/609/EEC) and national rules and institutional guidelines as prescribed by the ethical committee for animal experiments of the Federal University of Piaui (UFPI).

Effects of lipoic acid on pilocarpine-induced seizures

All the animals studied showed generalized tonic–clonic convulsions with status epilepticus, and 61% survived the seizures. Pilocarpine induced the first seizure at 34.93 ± 0.65 min. All animals pretreated with the lipoic acid selected for this study were observed for 1 h before pilocarpine injection for manifested alterations in behavior, such as peripheral cholinergic signs (100%), tremors (50%), staring spells, facial automatisms, wet dog shakes, rearing and motor seizures (25%), which develop

Discussion

Previous studies have demonstrated that δ-ALA-D and Na+, K+-ATPase activities were targets of free radical attack during oxidative stress, and these activities were decreased in oxidative stress environment (Lima et al., 2008). LA is an antioxidant that is both fat and water soluble. This important characteristic enables LA to easily cross the blood brain barrier and protect neurons in the brain (Freitas, 2009). In the present study, we investigated the effects of LA on brain oxidative stress

Acknowledgements

We would like to thank Stenio Gardel Maia for her technical assistance. This work was supported in part by grants from CNPq, Brazil. The I.M.S.S., C.M. F. and R.M.F. are fellows from CNPq.

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