**2. Research review and discussion**

#### **2.1. Epileptogenesis**

Epileptogenesis is the main challenge for contemporary epileptology, still. Any nonspecific brain damage may result in process of epileptogenesis which leads to seizure occurrence and eventually to chronic epilepsy. Brain damage may be followed by immediate (with latency seconds to 1 hr) and/or early (minutes to 1 week) seizure and late (week to dozen of years) recurring seizures. Possible pathomechanism of these seizures, triggering factors, risk factors for development of epilepsy, onset after brain damage, age depending, type of clinical course of seizures, and responsiveness to AEDs are different in these groups (Majkowski, 1990).

consisted of 41 children divided into two subgroups: 1-with fever without convulsions and 2 – without fever and without convulsions. Both fever and convulsions had a significant effect on the oxidation mechanisms. Febrile and afebrile convulsions differed in their generation of oxygen stress. According to the authors, in afebrile convulsion higher levels of oxygen stress might affect prognosis adversely. This is interpreted in terms of fever as a protective factor preventing neuronal lesion during convulsions. Recently, relation between febrile convulsion and oxygen stress was studied in 32 pediatric patients who within the preceding 8 hrs had experienced respiratory tract infection and had been diagnosed with simple febrile convul‐ sions (Abuhandan et al., 2013). Total oxidant level (TOL) and total anti-oxidant level (TAL) were measured 8 hrs after seizure. The TOL and oxidative stress index were found to be significantly high (p<0.01 and p<0.01, respectively), and TAL was significantly lower (p< 0.03) compared to control group 30 healthy children. The authors conclude that increased oxidative

Since oxygen stress worsen the course of epilepsy, consistently with those observations, use of anticonvulsants in conventional epilepsy therapy and hence attenuation of oxygen stress could have a positive effect on the course of epilepsy (Costello and Delanty, 2004). Many authors share this opinion. However, these results are not used to inform and guide everyday

The chapter is intended to present and discuss current state of knowledge in various animal experimental models of seizures and human epilepsy research related to epileptogenesis and complex interaction between epilepsy, AEDs, and oxygen stress. The effects of various nutritional factors which restore balance in the oxidant/anti-oxidant system, and prevent epileptic attacks and AEDs from causing brain neuronal damages in experimental and human

A literature review was conduced to November 2013. The following search terms were used: oxygen stress, oxidants and antioxidants, animal seizure models, epilepsy and AEDs. It was searched data bases PubMed-line, the Cochrane Epilepsy Group's Specialized Register, indexed and non-indexed citation and relevant papers related to beneficial of antioxidants, and possible harmful effects AEDs and epileptic seizures in animal models and in epileptic

Epileptogenesis is the main challenge for contemporary epileptology, still. Any nonspecific brain damage may result in process of epileptogenesis which leads to seizure occurrence and

stress may increase the risk of occurrence febrile seizures.

6 Pharmacology and Nutritional Intervention in the Treatment of Disease

epilepsy medication.

epilepsies will be up-dated.

**2. Research review and discussion**

**1.4. Search method**

**2.1. Epileptogenesis**

patients.

**1.3. Objective**

Epileptogenic process can be arbitrarily divided into two − overlapping to some extent − stages: cascade of biochemical processes followed by better known electrophysiological stage which precedes epilepsy occurrence. Brain damage initiates a series of non-specific, complex biochemical changes at the neural, synaptic and molecular levels. Among these biochemical changes, oxygen stress resulting in disequilibrium between oxidants and antio-xidants has been postulated in pathogenesis of seizures by many authors (Ueda et al., 1998; Jacobson et al., 1999; Dal-Pizzol et al., 2000, Patel, 2002). Role of oxygen stress has been shown and discussed in experimental animal model of epileptic seizures (Mori et al., 1990; Dakin and Weaver, 1993; de la Pena and Porta-Etessam, 1998; Majkowski, 2007; Rowley and Patel, 2013; Ryan et al., 2012). The latter provided evidence for the occurrence of specific and irreversible oxidative modification of an important mitochondrial enzyme of a protein complex I. The complex is critical for cellular bioenergetics during the process of epileptogenesis. Mechanism of epileptogenesis is not known. However, data from animal models and from patients with temporal lobe epilepsy suggest that steady-state mitochondrial ROS and resulting oxidative damage of neurons occurs during different phases of epileptogenesis (Rowley and Patel, 2013). Epileptogenic substances produce, before seizure occurrence, an increase of free radicals, lipid peroxidation and decrease of GSH-Px – the most important anti-oxidant in brain. Lipid peroxidase correlates with an increase of seizure susceptibility. In turn, occurs dysfunc‐ tion of the mitochondria, neuronal membrane permeability, disturbance of the balance between excessive neuronal activation and inhibition of neuronal transmission which may be due to an increase of glutaminergic or decrease of gabaergic transmitters (Murashima et al., 2005; Narkilati and Pitkanen 2005; Lasoń, 2006). These biochemical changes decrease seizure threshold and lead to epileptic neuronal discharges which may initiate kindling process (electro-epileptogenesis) leading to epileptic attacks (Tayarani et al., 1987; Majkowski, 1993; Frantseva et al., 1998; Frantseva et al., 2000; Liang and Patel, 2004; Waldbaum and Patel; 2010a). These biochemical study, indicating formation of specific protein in process of epileptogenesis, corresponds well with long-lasting synaptic plasticity which is seen in brain modification of sensory evoked potentials and epileptiform potentials development during kindling process of epileptogenesis (Majkowski and Kwast, 1981; Majkowski, 1989). Electrical kindling with its gradual development of epileptiform discharges is the most elegant model of chronic epilepsy which allows to study electrophysiological stage of epileptogenesis. Kindling phenomenon − produced by weak repeated electrical stimulation − described by Goddard et al., (1969), is characterized by widespread and long-lasting neuronal plasticity changes which can be seen in modification of behavior (seizure) and sensory evoked potential changes expressing long-lasting synaptic modification (Majkowski, 1989; 1993). This evoked potentals' modification is of the same kind as during learning processes (Majkowski and Kwast, 1981), what suggests new protein formation in the neurons is involved in epileptogen‐ esis (Hyden, 1980).

It seems that oxygen stress may play essential role in the earliest biochemical stage of epilep‐ togenesis − understood as formation of epileptogenic focus resulting in focal onset seizures. However, antiepileptogenic effects of various anti-oxidants used in different animal acute models of seizures is equivocal; the same anti-oxidant, and its dose, may have different effect in different models. Prevention or inhibition of epileptogenesis in different animal models, at best, shows delayed seizure occurrence in some seizure models (Zhao et at., 2006) and in some animals (Mori et al., 1990; Willmore et al., 1986; Suzer et al., 2000) but not prevention of epileptogenesis. A delay in seizure occurrence is understood as antiepileptogenic effects. In fact, it is not antiepileptogenic but anti-ictal effect. AEDs which increase seizure threshold, in the animal models and humans, may delay the first seizure occurrence e.g. in prevention of epileptogenesis of posttraumatic epilepsy, but not have antiepileptogenic effect. This delayed or diminished severity of seizures due to anticovulsants or anti-oxidants is also seen in chronic animal model of epilepsy (like kindling) with developed epileptiform neuronal discharges. This misunderstanding was shown in elegant hippocampal slice cultures as a model of traumatic brain injury, during acute and chronic (8 weeks) electrical recordings (Berdichevsky et al., 2011) Characteristic evolution of spontaneous epileptiform discharges, interictal spikes, seizure activity and electrical status epileptious was recorded. Peak cell death occurred immediately following slicing, and later secondary peak was associated with the peak of seizure-like activity. The secondary peak in neural death was abolished by either blockade of glutaminergic transmission by kynurenic acid or by elimination of ictal activity and status epilepticus by PHT. Withdrawal of these inhibitors was followed by spontaneous seizure activity recurrence. PHT anticonvulsant and neuroprotective effect disapeared after four weeks of continous administration. These interesting results show that AED may prevent seizures but not epileptogenesis. The authors conclude that in this vitro model secondary neuronal death is correlated with ictal but not interictal electrical activity.

**2.2. Preventing oxygen stress due to epileptic seizures in animal models**

*2.2.1. DL-homocysteic acid-induced seizures in immature rats*

cytoskeleton, surviving and protecting neurons from Hcy damage.

*2.2.2. Lithium-pilocarpine (Li-PIL) model of seizures*

DEX for preventing the induction of seizures

*2.2.3. The pentylenetetrazol seizure model (PTZ) and anti-oxidants*

Research on animal experimental models (and clinical observation) has shown that epileptic seizures lead to a number of harmful activities in the brain: disturbed blood circulation, increased cerebrospinal fluid pressure, brain oedema, hypoxia, all of which lead to the sudden reduction of energy carriers (ADP, ATP, phosphocreatinine) and neuronal pH reduction. During seizures, arachidonic acid is released in the postsynaptic membranes. This has an activating effect on the presynaptic neuronal endings and leads to increased glutamate release. Arachidonic acid also increases the production of free oxygen radicals, leading to increased lipid peroxidation. These in turn may activate phospholipase C and then lead to the release of arachidonic acid from the cellular membranes, setting a vicious circle in motion (Bartosz, 2006).

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9

Homocysteine (Hcy) is an exscitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitatory and oxidative injury. Recently, it has been shown that oxidative stress, occurring in the brain of immature 12-day-old rats during and following the seizures, induced by *DL-homocysteic acid*, is apparently due to the increased free radicals production (SOD, CuZn SOD, MnSOD and GPX) and the limited anti-oxidant defense (catalase activity decrease). The pronounced and selective upregulation of SOD2 (MnSOD) indicates to in‐ creased ROS in the mitochondrial matrix. This may be associated with inhibiton of respiratory chain complex I (Folbergrová et al., 2013). The authors suggest that in addition to AED, substances with anti-oxidant properties might provide beneficial effects in treatment of epilepsy in children. Protective role of astrocytes in neuronal survival in response to the damage induced by Hcy was studied (Loureiro et al., 2010). The authors found that the cytoskeleton of cortical astrocytes (but not of neurons in culture) is a target to Hcy and effects are mediated by redox signaling. Astrocytes were able to respond to Hcy reorganizing their

The modulatory effect of *dexamethasone (DEX)* using 5, 10, 20 mg/kg body mass of male Wistar rats was studied in Li-PIL epilepsy model (Al-Shorbagy et al., 2012). The authors found that effective anticonvulsant activity was only observed with 10 mg DEX/kg which reduced seizure production and incidence, as well as neuronal cell loss in the CA3 region of the hippocampus. It was associated with enhancements in the anti-oxidant system and interleukin as well as suppression of altered inflammatory markers. Dose 20 mg/kg DEX showed a tendency to shorten seizure latency, and neither affected seizure incidences nor CA3 neuronal loss. There was a lack of protection at 5 mg DEX/kg. The study indicates that there is an optimal dose of

*Astragalus mongholicus.* The root extract of Astragalus mongholicus (AM), a traditional medicinal herbas, has powerful anticonvulsant effects in the mouse PTZ-induced seizure

Epilepsies in humans and in animal models of seizures or epilepsy are extremely heterogenous in their etiology, pathomechanisms and diversity of their behavioural menifestations. This heterogeneity corresponds to diversities of results in using anti-oxidants, various seizure models and animals. At present, there are no well documented − on evidence base medicine − studies with anti-oxidants in prophylaxis of epileptogenesis. The same may be said about AEDs (Beghi, 2003). The prophylactic use of AEDs should be short lasting and may be effective in immediate and early seizures. Recently, prophylatic effect for post-craniotomy seizures in patients without epilepsy was reported (Pulman et al., 2013). The authors reviewed the relevant literature and found that there is little evidence to suggest that PHT, CBZ, PB, VPA or ZNS administered prophylactically is effective or not effective.

The well documented beneficial effects of anti-oxidants are related to diminishing neurode‐ generation produced by induced seizures and epileptic discharges. However, research on the role of oxygen stress opens a new chapter in epileptology with a hope of prevention biochem‐ ical processes leading to epileptiform neuronal discharges, epileptogenesis and cognitive function impairments in epileptic patients.

#### **2.2. Preventing oxygen stress due to epileptic seizures in animal models**

Research on animal experimental models (and clinical observation) has shown that epileptic seizures lead to a number of harmful activities in the brain: disturbed blood circulation, increased cerebrospinal fluid pressure, brain oedema, hypoxia, all of which lead to the sudden reduction of energy carriers (ADP, ATP, phosphocreatinine) and neuronal pH reduction. During seizures, arachidonic acid is released in the postsynaptic membranes. This has an activating effect on the presynaptic neuronal endings and leads to increased glutamate release. Arachidonic acid also increases the production of free oxygen radicals, leading to increased lipid peroxidation. These in turn may activate phospholipase C and then lead to the release of arachidonic acid from the cellular membranes, setting a vicious circle in motion (Bartosz, 2006).

#### *2.2.1. DL-homocysteic acid-induced seizures in immature rats*

It seems that oxygen stress may play essential role in the earliest biochemical stage of epilep‐ togenesis − understood as formation of epileptogenic focus resulting in focal onset seizures. However, antiepileptogenic effects of various anti-oxidants used in different animal acute models of seizures is equivocal; the same anti-oxidant, and its dose, may have different effect in different models. Prevention or inhibition of epileptogenesis in different animal models, at best, shows delayed seizure occurrence in some seizure models (Zhao et at., 2006) and in some animals (Mori et al., 1990; Willmore et al., 1986; Suzer et al., 2000) but not prevention of epileptogenesis. A delay in seizure occurrence is understood as antiepileptogenic effects. In fact, it is not antiepileptogenic but anti-ictal effect. AEDs which increase seizure threshold, in the animal models and humans, may delay the first seizure occurrence e.g. in prevention of epileptogenesis of posttraumatic epilepsy, but not have antiepileptogenic effect. This delayed or diminished severity of seizures due to anticovulsants or anti-oxidants is also seen in chronic animal model of epilepsy (like kindling) with developed epileptiform neuronal discharges. This misunderstanding was shown in elegant hippocampal slice cultures as a model of traumatic brain injury, during acute and chronic (8 weeks) electrical recordings (Berdichevsky et al., 2011) Characteristic evolution of spontaneous epileptiform discharges, interictal spikes, seizure activity and electrical status epileptious was recorded. Peak cell death occurred immediately following slicing, and later secondary peak was associated with the peak of seizure-like activity. The secondary peak in neural death was abolished by either blockade of glutaminergic transmission by kynurenic acid or by elimination of ictal activity and status epilepticus by PHT. Withdrawal of these inhibitors was followed by spontaneous seizure activity recurrence. PHT anticonvulsant and neuroprotective effect disapeared after four weeks of continous administration. These interesting results show that AED may prevent seizures but not epileptogenesis. The authors conclude that in this vitro model secondary

8 Pharmacology and Nutritional Intervention in the Treatment of Disease

neuronal death is correlated with ictal but not interictal electrical activity.

or ZNS administered prophylactically is effective or not effective.

function impairments in epileptic patients.

Epilepsies in humans and in animal models of seizures or epilepsy are extremely heterogenous in their etiology, pathomechanisms and diversity of their behavioural menifestations. This heterogeneity corresponds to diversities of results in using anti-oxidants, various seizure models and animals. At present, there are no well documented − on evidence base medicine − studies with anti-oxidants in prophylaxis of epileptogenesis. The same may be said about AEDs (Beghi, 2003). The prophylactic use of AEDs should be short lasting and may be effective in immediate and early seizures. Recently, prophylatic effect for post-craniotomy seizures in patients without epilepsy was reported (Pulman et al., 2013). The authors reviewed the relevant literature and found that there is little evidence to suggest that PHT, CBZ, PB, VPA

The well documented beneficial effects of anti-oxidants are related to diminishing neurode‐ generation produced by induced seizures and epileptic discharges. However, research on the role of oxygen stress opens a new chapter in epileptology with a hope of prevention biochem‐ ical processes leading to epileptiform neuronal discharges, epileptogenesis and cognitive Homocysteine (Hcy) is an exscitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitatory and oxidative injury. Recently, it has been shown that oxidative stress, occurring in the brain of immature 12-day-old rats during and following the seizures, induced by *DL-homocysteic acid*, is apparently due to the increased free radicals production (SOD, CuZn SOD, MnSOD and GPX) and the limited anti-oxidant defense (catalase activity decrease). The pronounced and selective upregulation of SOD2 (MnSOD) indicates to in‐ creased ROS in the mitochondrial matrix. This may be associated with inhibiton of respiratory chain complex I (Folbergrová et al., 2013). The authors suggest that in addition to AED, substances with anti-oxidant properties might provide beneficial effects in treatment of epilepsy in children. Protective role of astrocytes in neuronal survival in response to the damage induced by Hcy was studied (Loureiro et al., 2010). The authors found that the cytoskeleton of cortical astrocytes (but not of neurons in culture) is a target to Hcy and effects are mediated by redox signaling. Astrocytes were able to respond to Hcy reorganizing their cytoskeleton, surviving and protecting neurons from Hcy damage.

#### *2.2.2. Lithium-pilocarpine (Li-PIL) model of seizures*

The modulatory effect of *dexamethasone (DEX)* using 5, 10, 20 mg/kg body mass of male Wistar rats was studied in Li-PIL epilepsy model (Al-Shorbagy et al., 2012). The authors found that effective anticonvulsant activity was only observed with 10 mg DEX/kg which reduced seizure production and incidence, as well as neuronal cell loss in the CA3 region of the hippocampus. It was associated with enhancements in the anti-oxidant system and interleukin as well as suppression of altered inflammatory markers. Dose 20 mg/kg DEX showed a tendency to shorten seizure latency, and neither affected seizure incidences nor CA3 neuronal loss. There was a lack of protection at 5 mg DEX/kg. The study indicates that there is an optimal dose of DEX for preventing the induction of seizures

#### *2.2.3. The pentylenetetrazol seizure model (PTZ) and anti-oxidants*

*Astragalus mongholicus.* The root extract of Astragalus mongholicus (AM), a traditional medicinal herbas, has powerful anticonvulsant effects in the mouse PTZ-induced seizure model (Aldarmaa et al., 2010). Moreover, this effect was associated with an inhibition of PTZinduced increase of lipid and protein peroxidation and ROS. The authors suggest that anticonvulsant effect of AM may be mediated by its protective actions against oxidative damage and amelioration of mitochondrial dysfunction.

*Topiramate (TPM) and selenium*. TPM and selenium had protective effects on PTZ-induced brain damage in rats by inhibiting free radical production, regulating calcium-dependent processes, and supporting the antioxidant redox system (Naziroglu et al., 2008). Recent studies indicate that selenium with/without topiramate administration in human and animals decreased seizure levels, although anti-oxidant values were increased (Naziroglu and Yürekli, 2013).

Epilepsy Treatment and Nutritional Intervention

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11

*Vitamin E and selenium*. Intraperitoneal administration of PTZ − an antagonist of the GABA Areceptor − induced seizures and ruptured the blood-brain barrier in rats. This has been demonstrated by means of Evans dye, used to mark the permeability of this barrier (Oztas et al., 2001). It has been suggested that free radicals are involved in the permeability of the bloodbrain barrier; this permeability leads to albumin extravasation to the thalamic nuclei, brain stem, frontal cortex and occipital cortex. Animals that have been given vitamin E or selenium (Se) prior to seizure induction, had less extravasation in these structures. It has also been demonstrated that in young rats and in normotermic conditions, barrier permeability was

The KA model is used as a model substance in the assessment of neurotoxicity. It leads to excessive ROS production due to reduced antioxidant activity. When KA was administered to rats, lipid peroxidation of the neuronal membrane increased in proportion to seizure progres‐ sion (Ueda et al., 1997). In the same model, SOD and catalase activity increased significantly on day 5 following KA administration and returned to base level three weeks later; GSH-Px activity also increased significantly on day 5 but was still high three weeks later (Bruce and Baudry, 1995). Lipid and protein peroxidation, assessed by MDA concentration, increased significantly 8 and 16 hours later, then decreased on day 2 and day 5 following KA adminis‐ tration. The authors attribute the rapid increase in MDA and protein peroxidation to free radicals produced in this phase of the pathological KA effect; they conclude that the changes in enzymatic scavenger activity and the reduced MDA concentration may have been caused

*Gastrodia elata B1* (GE). Hsieh et al. (2001) tested a traditional Chinese herb GE, used to treat epilepsy, in a controlled study using the rat KA seizure model. They found that prior admin‐ istration of GE significantly reduced in vitro lipid peroxidation in the brain, an effect analogous to the effect of phenytoin (PHT) – 20 mg/kg. In the authors' opiniont GE has an antiepileptic effect and is a free radical scavenger. This antiepileptic effect may be, at least, partly attributable

*Melatonin*. In the mouse KA model, prior or simultaneous administration of melatonin (a powerful hydroxyl radical scavenger) (20 mg/kg i.p.) had an anti-oxidising effect and pre‐ vented lipid peroxidation, cerebral mitochondria DNA damage and seizures (Mohanan and

Since oxidative stress is thought to play a role in pathogenesis of hypertention and epilepto‐ genesis, it could be used as a tool for studying co-morbidity of both conditions (Atanasova et al., 2013). The authors studied efficacy of chronic pretreatment with *melatonin*, infused via

greater in males that in females (p<0.05) (Oztas et al., 2007).

*2.2.4. The kainic acid model (KA) and anti-oxidants*

by glia proliferation due to neuronal death.

to the GE's vanilla component (Hsieh et al., 2000).

Yamamoto, 2002).

*Erdosteine*. Prior administration of erdosteine (mucoliticum), which acts as an antioxidant, attenuated OS and delayed onset of PTZ-induced seizures in mice (p<0.05) (Ilhan et al., 2005b). The erdosteine pretreated mice had lower levels of MDA and xanthine oxidase (oxidisers) and a higher level of SOD than control animals (p<0.001). Thus, administration of erdosteine reduces convulsion-induced oxygen stress and therefore may protect neurons.

*Mexidol,* novel original Russian synthetic anti-oxidant (2-ethylo-6-methyl-3-oxypiridine succinate) and effects of AEDs (PB, LTG, phenazepam) and alpha-tocopherol were studied in PTZ-induced seizure model in Wistar rats (Beshkatova et al., 2003). Fivefold elevation of NO production was found in the induced seizures. Also, the level of secondary products of lipid peroxydation (LPO) and thiobarbituric acid reactive substances was significantly increased in the cortex. The authors found that mexidol and PB were to be the most effective in preventing of PTZ-induced seizures among all the studied substances. The authors suppose that supres‐ sion of seizure-induced NO generation and LPO increase may be involved in the mechanism of AEDs action.

*Nigella sativa oil*. Nigella sativa oil, a powerful antioxidant which has been used in folk medicine and the kitchen for thousands of years, prevented PTZ-induced kindling in mice much more effectively than that of valproic acid (Ilhan et al., 2005a).

*Polyphenols (grape juice).* Epileptic seizures and AEDs may cause oxidative damage in hepato‐ cytes. Wistar rats received organic grap juice or conventional grape juice (rich in phenols) and saline for 17 days before PTZ-induced seizures (Rodrigues et al., 2012). The results showed that both juices conferred protection against lipid and protein oxidative damage of liver, and limited the increase in PTZ-induced NO metabolite content in liver and serum. Moreover, both juices inhibited the PTZ-induced reduction in enzymatic anti-oxidant defenses (SOD, CAT) and sulfhydryl protein in the liver and serum. These results indicate that grape juices can provide an insight into natural neuroprotective compounds and may lead to the development of new therapeutic strategies for epileptic patients (Rodrigues et al., 2012).

*Polyphenols (walnut kernels).* Walnuts have high concentration of phenols. Its supplementation was associated with increased seizure threshold and reduced mortality in PTZ seizure model in Wistar adult male rats (Asadi-Shekaari et al., 2012). Moreover, there was prevention of neurodegeneration. The authors suggest that this effect may be due to high concentration in walnuts of phenols, which have anticonvulsant properties. Recently, antiepileptic effects of phenols were updated by Lasoń (2013) with notion that phenols can have clinical relevance for novel approach to treatment of epilepsy.

*Physical activity.* It has been found that swimming training (6 weeks) protects against the increase of neural excitability and oxidative neuronal damage in PTZ-induced seizures in rats (Souza et al., 2009). EEG recordings showed that the spikes' amplitude in rats was decreased after PTZ administration in all doses following swimming.

*Topiramate (TPM) and selenium*. TPM and selenium had protective effects on PTZ-induced brain damage in rats by inhibiting free radical production, regulating calcium-dependent processes, and supporting the antioxidant redox system (Naziroglu et al., 2008). Recent studies indicate that selenium with/without topiramate administration in human and animals decreased seizure levels, although anti-oxidant values were increased (Naziroglu and Yürekli, 2013).

*Vitamin E and selenium*. Intraperitoneal administration of PTZ − an antagonist of the GABA Areceptor − induced seizures and ruptured the blood-brain barrier in rats. This has been demonstrated by means of Evans dye, used to mark the permeability of this barrier (Oztas et al., 2001). It has been suggested that free radicals are involved in the permeability of the bloodbrain barrier; this permeability leads to albumin extravasation to the thalamic nuclei, brain stem, frontal cortex and occipital cortex. Animals that have been given vitamin E or selenium (Se) prior to seizure induction, had less extravasation in these structures. It has also been demonstrated that in young rats and in normotermic conditions, barrier permeability was greater in males that in females (p<0.05) (Oztas et al., 2007).

#### *2.2.4. The kainic acid model (KA) and anti-oxidants*

model (Aldarmaa et al., 2010). Moreover, this effect was associated with an inhibition of PTZinduced increase of lipid and protein peroxidation and ROS. The authors suggest that anticonvulsant effect of AM may be mediated by its protective actions against oxidative

*Erdosteine*. Prior administration of erdosteine (mucoliticum), which acts as an antioxidant, attenuated OS and delayed onset of PTZ-induced seizures in mice (p<0.05) (Ilhan et al., 2005b). The erdosteine pretreated mice had lower levels of MDA and xanthine oxidase (oxidisers) and a higher level of SOD than control animals (p<0.001). Thus, administration of erdosteine reduces convulsion-induced oxygen stress and therefore may protect neurons.

*Mexidol,* novel original Russian synthetic anti-oxidant (2-ethylo-6-methyl-3-oxypiridine succinate) and effects of AEDs (PB, LTG, phenazepam) and alpha-tocopherol were studied in PTZ-induced seizure model in Wistar rats (Beshkatova et al., 2003). Fivefold elevation of NO production was found in the induced seizures. Also, the level of secondary products of lipid peroxydation (LPO) and thiobarbituric acid reactive substances was significantly increased in the cortex. The authors found that mexidol and PB were to be the most effective in preventing of PTZ-induced seizures among all the studied substances. The authors suppose that supres‐ sion of seizure-induced NO generation and LPO increase may be involved in the mechanism

*Nigella sativa oil*. Nigella sativa oil, a powerful antioxidant which has been used in folk medicine and the kitchen for thousands of years, prevented PTZ-induced kindling in mice much more

*Polyphenols (grape juice).* Epileptic seizures and AEDs may cause oxidative damage in hepato‐ cytes. Wistar rats received organic grap juice or conventional grape juice (rich in phenols) and saline for 17 days before PTZ-induced seizures (Rodrigues et al., 2012). The results showed that both juices conferred protection against lipid and protein oxidative damage of liver, and limited the increase in PTZ-induced NO metabolite content in liver and serum. Moreover, both juices inhibited the PTZ-induced reduction in enzymatic anti-oxidant defenses (SOD, CAT) and sulfhydryl protein in the liver and serum. These results indicate that grape juices can provide an insight into natural neuroprotective compounds and may lead to the development

*Polyphenols (walnut kernels).* Walnuts have high concentration of phenols. Its supplementation was associated with increased seizure threshold and reduced mortality in PTZ seizure model in Wistar adult male rats (Asadi-Shekaari et al., 2012). Moreover, there was prevention of neurodegeneration. The authors suggest that this effect may be due to high concentration in walnuts of phenols, which have anticonvulsant properties. Recently, antiepileptic effects of phenols were updated by Lasoń (2013) with notion that phenols can have clinical relevance

*Physical activity.* It has been found that swimming training (6 weeks) protects against the increase of neural excitability and oxidative neuronal damage in PTZ-induced seizures in rats (Souza et al., 2009). EEG recordings showed that the spikes' amplitude in rats was decreased

of new therapeutic strategies for epileptic patients (Rodrigues et al., 2012).

damage and amelioration of mitochondrial dysfunction.

10 Pharmacology and Nutritional Intervention in the Treatment of Disease

effectively than that of valproic acid (Ilhan et al., 2005a).

for novel approach to treatment of epilepsy.

after PTZ administration in all doses following swimming.

of AEDs action.

The KA model is used as a model substance in the assessment of neurotoxicity. It leads to excessive ROS production due to reduced antioxidant activity. When KA was administered to rats, lipid peroxidation of the neuronal membrane increased in proportion to seizure progres‐ sion (Ueda et al., 1997). In the same model, SOD and catalase activity increased significantly on day 5 following KA administration and returned to base level three weeks later; GSH-Px activity also increased significantly on day 5 but was still high three weeks later (Bruce and Baudry, 1995). Lipid and protein peroxidation, assessed by MDA concentration, increased significantly 8 and 16 hours later, then decreased on day 2 and day 5 following KA adminis‐ tration. The authors attribute the rapid increase in MDA and protein peroxidation to free radicals produced in this phase of the pathological KA effect; they conclude that the changes in enzymatic scavenger activity and the reduced MDA concentration may have been caused by glia proliferation due to neuronal death.

*Gastrodia elata B1* (GE). Hsieh et al. (2001) tested a traditional Chinese herb GE, used to treat epilepsy, in a controlled study using the rat KA seizure model. They found that prior admin‐ istration of GE significantly reduced in vitro lipid peroxidation in the brain, an effect analogous to the effect of phenytoin (PHT) – 20 mg/kg. In the authors' opiniont GE has an antiepileptic effect and is a free radical scavenger. This antiepileptic effect may be, at least, partly attributable to the GE's vanilla component (Hsieh et al., 2000).

*Melatonin*. In the mouse KA model, prior or simultaneous administration of melatonin (a powerful hydroxyl radical scavenger) (20 mg/kg i.p.) had an anti-oxidising effect and pre‐ vented lipid peroxidation, cerebral mitochondria DNA damage and seizures (Mohanan and Yamamoto, 2002).

Since oxidative stress is thought to play a role in pathogenesis of hypertention and epilepto‐ genesis, it could be used as a tool for studying co-morbidity of both conditions (Atanasova et al., 2013). The authors studied efficacy of chronic pretreatment with *melatonin*, infused via subcutaneous osmotic mini pump for 14 days, on KA-induced status epilepticus, oxidative stress and expression of heat shock protein (HSP) 72 in spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. SHRs showed increase in the level of LP in frontal cortex and hippocampus and decreased cytosolic superoxide dismutase (SOD/CuZn) production in the frontal cortex compared to normotensive Wistar rats. Status epilepticus induced by KA was associated with increased LP and expression of HSP 72 in the hippocampus in the two strains, and increased SOD/CuZn production in frontal cortex of SHRs. Melatonin failed to suppress seizure incidence and intensity. However, latency was significantly increased in SHRs. Increased activity in SOD/CuZn and mitochondrial SOD Mn as well and reduced expression of HSP 72 in hippocampus was observed in Wistar rats pretreated with melatonin. **The observed strain differences in the efficacy of chronic melatonin expression before status epilepticus suggests a lack of direct link between the seizure activity and markers of oxygen stress and neurotoxicity.**

mechanisms leading to seizures or status epilepticus are unknown. It is thought that oxygen stress plays an important role but it is still unknown which brain structures are more sensitive.

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*The pilocarpine-induced status epilepticus* had differential effects on catalase level in discrete brain structures (Freitas et al., 2004; Freitas et al., 2005). The highest elevation of the enzyme level was found in the hippocampus (36%), striatum (31%) and frontal cortex (15%); no changes were found in the cerebellum. According to the authors, the endogenous increase in catalase activity, responsible for removal of free oxygen radicals which are produced during convul‐ sions, may be an auto-regulatory compensating defence mechanism which counteracts the negative effects of oxygen stress in the status epilepticus. In this model of epilepsy, results show that oxidative stress, lipid peroxidation and nitric oxide could be responsible for neuronal damage in the hippocampus (Freitas, 2009).Other researchers have come to similar conclusions (Kawakami et al., 2006; Tejada et al., 2007). The later authors investigated pilo‐ carpine-evoked status epilepticus and found that MDA levels increased significantly in the brain cortex (64%), suggesting oxygen injury. They found a simultaneous increase in the antioxidising activity of catalase enzymes (28%), GSH-Px (28%) and SOD (21%). On the other hand, vitamin E concentration in the cerebral cortex was reduced (15%) due to increased lipid peroxydation following pilocarpine administration. The amount of lipid peroxydation product in cortical neurons of the cerebral hemispheres decreased by 30% at the peak of convulsions observed 10-15 min. after i.p. picrotoxin injection. In neuroglial cells of control animals the

intensity of lipid peroxydation was 1.7-2.0 times lower (Flerov et al., 2004).

effect could be achieved using lipoic acid.

*Apocynin*. In pilocarpine-induced status epilepticus in rats, ROS generation was increased in CA1, CA3 and dentate gyrus of dorsal hippocampus (Pestana et al., 2010). The authors found that administration of apocynin (NADPH oxidase inhibitor) for 7 days prior to pilocarpineinduced status epilepticus had protective activity: ROS production and neurodegeneration in

*Lipoic acid*. In pilocarpine-induced seizures in rats there was significant increase in lipid peroxidation, nitrite level and GPx, however, no alterations were found in SOD and catalase activities (Militão et al., 2010). Administration of lipoic acid significantly reduced the lipid peroxidation level and nitrite content, and increased the SOD, catalase and GPx activities in striatum after seizures. The study supports hypothesis that brain damage induced by the oxidative process plays a crucial role in seizures pathogenic consequences, and a protective

*Vitamin C*. In the same model in rats, prior administration of vitamin C (250 mg/kg i.p.) reduced the negative effects of oxygen stress and neuronal lesion (Santos et al., 2008). The latency time to convulsion onset following pilocarpine administration was longer and mortality in the status epilepticus was reduced compared with the group which did not receive vitamin C or received physiological saline. This study also found that in the group which only received vitamin C the level of lipid peroxidation was lower than in the group which a) received pilocarpine and b) received pilocarpine and vitamin C. In all the experimental groups, catalase activity in the hippocampus increased compared with the control group which only received physiological saline. In the authors' opinion, the neuroprotective function of vitamin C in adult

the studied structures were decreased by an average of 20% and 61%, respectively.

*Petasites japonicum* (BMP). Sok et al. (2006), studied the anticonvulsive effects of the plant grown in East Asia and used for both culinary purposes and in folk medicine. Its root extracts are still used for treating headaches and asthma. Prolonged administration of BMP, prior to KA administration, reduced mortality in mice by one half. Administration of the BMP-I subfraction reduced convulsive seizures and also significantly reduced neuronal loss in parts CA1 and CA3 of the hippocampus. The authors suggest that BMP-I is the factor responsible for prevention of oxidisation lesion in mouse brain.

*SCH 58261 – a selective adenosine A(2A) receptor (A(2A)R) antagonist.* KA induced seizures in young rats (21-day-old) were pretreated with SCH 58261 before (i.p.) KA administration (Bortolatto et al., 2012). It resulted in prolonged latency for the onset of the first clonic seizure and at the highest dose decreased the appearance of clonic seizures and mortality rate. The adenosine antagonist was also effective in protecting against alteration in oxidative stress parameters (ROS, CAT, GPx, and GST (glutathione S-tansferaze)) activities. Thus, SCH 58261 was protective against the induced neurotoxicity, and might represent a novel approach for the treatment of seizures.

*Sesame seeds*. Sesamin is a well-known antioxidant from sesame seeds and it scavenges free radicals. In KA–induced status epilepticus in mice and rats sesamin significantly decreased ROS, MDA and the mortality was decreased from 22% to 0% in rats (Hsieh et al., 2011).

*Vineatrol.* In the rat KA model, prior administration of vineatrol significantly reduced brain MDA levels but had no effect on the GSH levels (Gupta and Briyal, 2006). Doses exceeding 20 and 40 mg/kg lengthened the latency time to the first seizures. Additional administration of vineatrol 30 and 60 minutes after KA administration significantly reduced seizure incidence. The authors suggest that vineatrol could potentially be useful in status epilepticus.

#### *2.2.5. The pilocarpine model of seizures, status epilepticus, oxidants and anti-oxidants*

*Pilocarpine*, an imidasole alkaloid extracted from the leaves of the Pilocarpus jaborandi shrub, is a parasympathomimetic, a cholinergic agonist which acts similarly to acetylcholine. It is often used to evoke epileptic convulsions and status epilepticus in animal models. The mechanisms leading to seizures or status epilepticus are unknown. It is thought that oxygen stress plays an important role but it is still unknown which brain structures are more sensitive.

subcutaneous osmotic mini pump for 14 days, on KA-induced status epilepticus, oxidative stress and expression of heat shock protein (HSP) 72 in spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. SHRs showed increase in the level of LP in frontal cortex and hippocampus and decreased cytosolic superoxide dismutase (SOD/CuZn) production in the frontal cortex compared to normotensive Wistar rats. Status epilepticus induced by KA was associated with increased LP and expression of HSP 72 in the hippocampus in the two strains, and increased SOD/CuZn production in frontal cortex of SHRs. Melatonin failed to suppress seizure incidence and intensity. However, latency was significantly increased in SHRs. Increased activity in SOD/CuZn and mitochondrial SOD Mn as well and reduced expression of HSP 72 in hippocampus was observed in Wistar rats pretreated with melatonin. **The observed strain differences in the efficacy of chronic melatonin expression before status epilepticus suggests a lack of direct link between the seizure activity and markers of oxygen**

*Petasites japonicum* (BMP). Sok et al. (2006), studied the anticonvulsive effects of the plant grown in East Asia and used for both culinary purposes and in folk medicine. Its root extracts are still used for treating headaches and asthma. Prolonged administration of BMP, prior to KA administration, reduced mortality in mice by one half. Administration of the BMP-I subfraction reduced convulsive seizures and also significantly reduced neuronal loss in parts CA1 and CA3 of the hippocampus. The authors suggest that BMP-I is the factor responsible for

*SCH 58261 – a selective adenosine A(2A) receptor (A(2A)R) antagonist.* KA induced seizures in young rats (21-day-old) were pretreated with SCH 58261 before (i.p.) KA administration (Bortolatto et al., 2012). It resulted in prolonged latency for the onset of the first clonic seizure and at the highest dose decreased the appearance of clonic seizures and mortality rate. The adenosine antagonist was also effective in protecting against alteration in oxidative stress parameters (ROS, CAT, GPx, and GST (glutathione S-tansferaze)) activities. Thus, SCH 58261 was protective against the induced neurotoxicity, and might represent a novel approach for

*Sesame seeds*. Sesamin is a well-known antioxidant from sesame seeds and it scavenges free radicals. In KA–induced status epilepticus in mice and rats sesamin significantly decreased ROS, MDA and the mortality was decreased from 22% to 0% in rats (Hsieh et al., 2011).

*Vineatrol.* In the rat KA model, prior administration of vineatrol significantly reduced brain MDA levels but had no effect on the GSH levels (Gupta and Briyal, 2006). Doses exceeding 20 and 40 mg/kg lengthened the latency time to the first seizures. Additional administration of vineatrol 30 and 60 minutes after KA administration significantly reduced seizure incidence.

*Pilocarpine*, an imidasole alkaloid extracted from the leaves of the Pilocarpus jaborandi shrub, is a parasympathomimetic, a cholinergic agonist which acts similarly to acetylcholine. It is often used to evoke epileptic convulsions and status epilepticus in animal models. The

The authors suggest that vineatrol could potentially be useful in status epilepticus.

*2.2.5. The pilocarpine model of seizures, status epilepticus, oxidants and anti-oxidants*

**stress and neurotoxicity.**

the treatment of seizures.

prevention of oxidisation lesion in mouse brain.

12 Pharmacology and Nutritional Intervention in the Treatment of Disease

*The pilocarpine-induced status epilepticus* had differential effects on catalase level in discrete brain structures (Freitas et al., 2004; Freitas et al., 2005). The highest elevation of the enzyme level was found in the hippocampus (36%), striatum (31%) and frontal cortex (15%); no changes were found in the cerebellum. According to the authors, the endogenous increase in catalase activity, responsible for removal of free oxygen radicals which are produced during convul‐ sions, may be an auto-regulatory compensating defence mechanism which counteracts the negative effects of oxygen stress in the status epilepticus. In this model of epilepsy, results show that oxidative stress, lipid peroxidation and nitric oxide could be responsible for neuronal damage in the hippocampus (Freitas, 2009).Other researchers have come to similar conclusions (Kawakami et al., 2006; Tejada et al., 2007). The later authors investigated pilo‐ carpine-evoked status epilepticus and found that MDA levels increased significantly in the brain cortex (64%), suggesting oxygen injury. They found a simultaneous increase in the antioxidising activity of catalase enzymes (28%), GSH-Px (28%) and SOD (21%). On the other hand, vitamin E concentration in the cerebral cortex was reduced (15%) due to increased lipid peroxydation following pilocarpine administration. The amount of lipid peroxydation product in cortical neurons of the cerebral hemispheres decreased by 30% at the peak of convulsions observed 10-15 min. after i.p. picrotoxin injection. In neuroglial cells of control animals the intensity of lipid peroxydation was 1.7-2.0 times lower (Flerov et al., 2004).

*Apocynin*. In pilocarpine-induced status epilepticus in rats, ROS generation was increased in CA1, CA3 and dentate gyrus of dorsal hippocampus (Pestana et al., 2010). The authors found that administration of apocynin (NADPH oxidase inhibitor) for 7 days prior to pilocarpineinduced status epilepticus had protective activity: ROS production and neurodegeneration in the studied structures were decreased by an average of 20% and 61%, respectively.

*Lipoic acid*. In pilocarpine-induced seizures in rats there was significant increase in lipid peroxidation, nitrite level and GPx, however, no alterations were found in SOD and catalase activities (Militão et al., 2010). Administration of lipoic acid significantly reduced the lipid peroxidation level and nitrite content, and increased the SOD, catalase and GPx activities in striatum after seizures. The study supports hypothesis that brain damage induced by the oxidative process plays a crucial role in seizures pathogenic consequences, and a protective effect could be achieved using lipoic acid.

*Vitamin C*. In the same model in rats, prior administration of vitamin C (250 mg/kg i.p.) reduced the negative effects of oxygen stress and neuronal lesion (Santos et al., 2008). The latency time to convulsion onset following pilocarpine administration was longer and mortality in the status epilepticus was reduced compared with the group which did not receive vitamin C or received physiological saline. This study also found that in the group which only received vitamin C the level of lipid peroxidation was lower than in the group which a) received pilocarpine and b) received pilocarpine and vitamin C. In all the experimental groups, catalase activity in the hippocampus increased compared with the control group which only received physiological saline. In the authors' opinion, the neuroprotective function of vitamin C in adult rats may be due to reduced lipid peroxidation and increased catalase activity following convulsions and status epilepticus.

**3.2. Carbamazepine**

**3.3. Lamotrigine**

Ferraro, 2007).

**3.4. Levetiracetam**

case of other AEDs.

**3.5. Phenobarbital (PB)**

**3.6. Phenytoin (PHT)**

A1 against PHT toxicity and teratogenesis.

1997).

Short term CBZ administration to roinbout trout and a low level of oxidative stress could induce adaptive responses of antioxidant enzymes, however, long-term exposure to CBZ could

Epilepsy Treatment and Nutritional Intervention

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15

LTG does not lead to detectable increases in lipid peroxidation in rats in vivo (Lu and Uetrecht, 2007). The anti-epileptic effectiveness of LTG in the *partial complex epilepsy model* (stimulation of the dentate gyrus) in rats was in reverse proportion to the level of nitric oxide (Sardo and

LEV (2000 mg/kg i.p.) administered prior to *pilocarpine administration* (400 mg/kg s.c.) in mice prevented lipid peroxidation increase in the hippocampus (but did not increase nitrate level or reduce catalase activity in the hippocampus or cortical glutathione) (Oliveira et al., 2007). Perhaps the anti-oxidising, neuroprotective effect of LEV and the consequent reduction of oxygen stress can be attributed to a different mechanism than the one which is active in the

Male Sprague – Dawley rats were pretreated with PB – a well known cytochrome P450 inducer (Dostalek et al., 2007). The markers of in vivo oxygen stress were influenced by PB resulting in significantly increased malondialdehyde, H2O2 generation and NADPH oxidation in vitro

PHT is known to produce ROS, which are involved in mechanism of the PHT-evoked terato‐ genesis and developmental toxicity. PHT initiates the oxidation damage to proteins and fats in the *maternal and embryonic liver tissue* organelle in murine rodents (Mahle and Dasgupta,

Gallagher and Sheehy (2010) used *cultured human prenatal liver slices* to study the effects of the human teratogen PHT on cell toxicity. Their findings in a relevant human model system are supportive of a protective role of GSH and alpha class glutathione S – transferases izoenzymes

Using mutant catalase deficient mice and transgenic mice expressing human catalase, Abramov and Wells (2011) investigated protective importance of *embryonic catalase* against endogenous ROS and the ROS-imitating teratogen PHT in embryo culture. They provided evidence that the low level of embryonic catalase protects from developmental and xenobioticenhanced oxygen stress and that embryonic variations of this enzyme affect development.

and significantly enhanced formation in vivo in liver and plasma.

lead to serious oxidative damage of *fish brain* (Li et al., 2010).

*Vitamin E and status epilepticus*. Barros et al. (2007) applied the same model and found that administration of vitamin E (200 mg/kg i.p.) 30 minutes prior to the administration of pilocar‐ pine (400 mg/kg s.c.) leads to increased (214%) catalase activity in the hippocampus compared with rats which were only given policarpine (67%) or physiological saline. The authors conclude that increased catalase activity may be responsible for the regulation of free radicals evoked by the status epilepticus.

In pilocarpine-induced status epilepticus in rats autophagy – a process of bulk degeneration of cellular constituents through autophagosome-lysosomal pathways was studied (Cao et al., 2009). Status epilepticus induces an excess production of ROS resulting in an increase of autophagy which was a partially inhibited by pretreatment with vitamin E. The strong protective effect of vitamin E could be achieved in the same pilocarpine seizure model (Tome et al., 2010) The authors confirmed that oxidative stress occurs in rat hippocampus resulting in the brain damage and this plays a crucial role in seizure pathogenic consequences.

#### *2.2.6. The audiogenic seizure model*

*Melatonin and valproic acid*. Prolonged melatonin administration in rats congenitally predis‐ posed to audiogenic convulsions (the Krushinsky-Molodkina model) had no effect on seizures evoked by a 20 times more powerful auditory stimulus (Savina et al., 2006). VPA administra‐ tion significantly reduced convulsions but VPA and melatonin combination had a significantly larger anti-seizure effect: it lengthened latency time and reduced seizure severity. However, combined treatment led to much more rapid onset of myoclonia than in groups receiving either VPA or melatonin.
