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PHAREP-57; No. of Pages 5 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Review article
Vitamin C: A new auxiliary treatment of epilepsy? Edyta Sawicka-Glazer a, Stanisław J. Czuczwar a,b,* a b
Department of Pathophysiology, Medical University, Lublin, Poland Department of Physiopathology, Institute of Rural Health, Lublin, Poland
A R T I C L E I N F O
Article history: Received 18 February 2014 Accepted 20 February 2014 Available online xxx Keywords: Ascorbic acid Epilepsy Seizures Neurodegeneration
A B S T R A C T
Although many approaches to the therapy of epilepsy exist, most of antiepileptic drugs, beside certain and unquestioned benefits, have convinced disadvantages. That is the reason for looking for new methods of treatment. Ascorbic acid, as an antioxidant and electron donor accumulated in central nervous system, seems to take part in diminishing reactions of oxidative stress in brain and cooperate with other antioxidants like alpha-tocoferol. Vitamin C, easily transported through the blood–brain barrier, is proved to reduce injury in the hippocampus during seizures. Depending on type of seizures, it has mostly inhibitory activity and even decreases mortality. Moreover, vitamin C acts as a neuroprotective factor by consolidating cell membranes and decreasing lipid peroxidation. A possible adjunctive role of vitamin C in epileptic patients needs to be considered. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress – mechanism and consequences . . . . . . . . . . . . . . . Vitamin C – association with seizure activity . . . . . . . . . . . . . . . . . . Vitamin C – anticonvulsant and prooxidative activity . . . . . . . . . . . . Pentylenetetrazol (PTZ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pilocarpine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Postraumatic epilepsy model (intracortical administration of Other models of experimental epilepsy . . . . . . . . . . . . . . . . . . Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prooxidative action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motor and cognitive functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Epilepsy is a common neurological disorder, characterised by immediate and tilting seizures, affecting about 1–2% of population [4]. Especially susceptible to expand this disorder are immature brains [12]. In the group treated by modern antiepileptic drugs (AEDs) ascendancy of 70% is going into remission, but up to 30%
* Corresponding author. E-mail address: [email protected] (S.J. Czuczwar).
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will be still exposed to seizures [30,42]. That is the reason of interest of routine usage of vitamins as a prevention or probable new method of treatment. Vitamins have been considered to be important patterns in controlling certain types of seizures or even preventing adverse effects of AEDs [30]. Although, folic acid, thiamine, vitamin D or vitamin E have not been proved this kind of action [30], there is a strong persuasion of neuroprotective action of vitamin C on brain tissue, especially during seizures. Vitamin C is an exogenous water-soluble substance [30], nutritional supplement [10]. It is a cofactor in building blood
http://dx.doi.org/10.1016/j.pharep.2014.02.016 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Sawicka-Glazer E, Czuczwar SJ. Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.02.016
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vessels and takes role of an antioxidant [11]. Vitamin C is synthesised from glucose in livers of mammals, except humans by the reason of absence of specific enzyme which is gulonolactone oxidase [28,41]. Vitamin C is an electron donor and reducing agent, so it prevents lipid, protein and DNA oxidation [28]. The evidence of this action has been proved in extracellular fluids, e.g. plasma [6]. Ascorbic acid is also accumulated in adrenal glands and central nervous system [24]. It acts not only as a reactive oxygen species scavenger, but also helps to restore other substances with antioxidant properties, such as alpha-tocopherol or glutathione [6]. Oxidative stress – mechanism and consequences Oxidative stress is a state when there is a disproportion of proand antioxidants with the predominance of prooxidants which are considered to be reactive forms of oxygen (ROS) [16] and reactive nitrogen species [1]. Brain consumes 20% of inhaled oxygen but only 2–5% of it is reduced [1], so it causes a possibility of various chemical reactions. Moreover, central nervous system has its specific regions more vulnerable to oxidative reactions, like hippocampus, by the reason of lower concentration of antioxidants than in the other areas [31]. Hippocampus is very susceptible to concentration of vitamin C not only in adolescence, but its atrophy was noticed also in animals with prenatal deficiency of ascorbic acid [41]. Seizures are thought as high energy demanding reactions. It is noteworthy that seizure activity may cause meaningful changes especially in mitochondrial homeostasis [1]: diminishes the amount of energy by reduction of ATP, inhibits mitochondrial respiratory enzymes and lowers neuronal plasma membrane potential. Such a hyperactivity of neurons and excitotoxic process indicates also formation of ROS, causing prevalence of oxidative processes [29]. ROS disturb transport of electron chain enzymes and damage DNA [14,18]. Large contribution of mtDNA in cell is also thought to be one of the probable elements in increasing probability of seizures and interprets its role to epileptogenesis [17]. At normal conditions, such molecules as superoxide anion, hydroxyl radical or peroxinitrate participate in indispensable metabolic pathways [1], but in the excess, induce peroxidation of polyunsaturated fatty acids occurring in high concentration in brain tissue [15,32,43], peroxidation of proteins or DNA [1], which can result in neuronal cell damage and glial cell toxicity [31], especially in the immature brain [24], and neuronal deprivation in certain structures [39]. Brain tissue is ratable to lipid peroxidation during oxidative stress [14,15,43]. Membrane changes cause exempting of ‘‘arachidonic acid, diacylglycerols, eicosanoids, lipid peroxides and free radicals’’ [43]. Lipid peroxidation begins chain of reactions, which starts with emergence of elecrophilic aldehydes and than results in posttranslational modification of proteins [38]. There is, however, group of natural factors functioning as antiooxidants, like superoxide dismutase, catalase and glutathione peroxidase [43]. To such an important role of oxidative stress in epilepsy indicates the fact that a meaningful increase of RNA oxidation was observed during convulsions in rat brains [1]. Vitamin C – association with seizure activity Ascorbic acid is a basic micronutrient for all mammalians [8,37]; its highest concentration is encountered in spinal cord and brain [8]. Vitamin C co-operates in myelin building, plays here a role of an antioxidant and a neuromodulator for neuronal mediators [8]. It is transported through the blood-brain barrier in endothelial cells of brain vessels [13], its levels are proportional to N-methyl-Daspartate receptor activity and has high affinity for the SVCT2 transporter, which conveys neurotropic agents, e.g. certain drugs conjugated to ascorbic acid [7,18]. Ascorbic acid is transported to mitochondria in oxidised form of dehydroascorbic acid by facilitative glucose transporter GLUT1, GLUT3 and GLUT4 and is
immediately switched to ascorbic acid again [31,35]. Brain is one of few places where the substance is being clustered to various brain structures – hippocampus, amygdala and hypothalamus [35]. Vitamin C is transported primarily to mitochondria where ROS are in highest accumulation and there transfers an electron to ‘‘ascorbate-dependent peroxidase’’ [31]. The active form of ascorbic acid is an electron donor, so biochemically it acts as a reducing agent [12]. It controls in vivo biological actions of free radicals and behaves as a free radical scavenger [40], also playing a role of defence in cell’s structure. Interestingly, some AEDs, like for instance topiramate, may share ascorbic acid’s scavenging activity in epilepsy [5]. Vitamin C inhibits aggregating free fatty acids in rats’ brains during seizures [43]. It decreases peroxidation of lipids, most possibly by influence and escalating action of antioxidant enzymes like superoxide dismutase or catalase in the adult rat hippocampus [31,32] and works as a neuromodulator [40]. The accurate level of those enzymes prevents from seizures [31]. Concentration of ascorbic acid in plasma of patients with epilepsy is much lower than in non-epileptic patients [19,36]. As hippocampus and amygdala are considered as structures involved in transmitting seizures, their role is significant [12,32]. Vitamin C has been proved to reduce injury in the hippocampus during seizures [38]. It is also said to be a ‘‘positive modulator of GABA A receptors’’ [12]. Vitamin C, on the other hand, is involved in production of steroids and peptide hormones and gives progesterone-like effect [43]. As is proved, estrogen accompanies seizures, contrary progesterone and its metabolites (possibly allopregnanolone) prevent from seizure activity [12]. Ascorbic acid increases progesterone production, but also cooperates with it [12]. Ascorbic acid is one of many antioxidants in brain and strongly cooperates with others, like alpha-tocoferol, by regenerating its reduced form in cell membrane [40]. Vitamin C – anticonvulsant and prooxidative activity A few studies concerning usage of ascorbic acid in different seizure model have been conducted. Pentylenetetrazol (PTZ) Immature rats pretreated with ascorbic acid (500 mg/kg for 5 days) revealed a significant retardation of onset to myoclonic, clonic and tonic seizures or even the absence of convulsions was noted [12]. Vitamin C also decreased the number of seizures and mortality of rats. Moreover, a reduction in amino acid (GABA, glutamate, aspartate, glutamine) concentration, following vitamin C supplementation, has been observed in many regions of brain, especially in hippocampus and amygdala [12]. Ascorbic acid at 300 mg/kg was also shown to protect against seizures, to prolong seizure duration at 100 mg/kg, and to exert no effect on seizure activity at 30 mg/kg [33]. Further, inhibition of Na-K-ATPase activity induced by PTZ, which increases neuronal excitability and leads to convulsions, was reversed by ascorbic acid in the highest dose of 300 mg/kg [33]. Administration of ascorbic acid (200 mg/ kg) to guinea pigs led to a decreased intensity of seizures and prolongation of the latent period [27]. The scorbutic guinea-pigs showed shortened latent period to convulsions and their frequency was also higher [27]. Finally, vitamin C (250 mg/kg) significantly reduced the mortality rate in PTZ-treated rats although the evaluated seizure parameters, seizure duration, seizure severity, and latency were not affected [44]. Pilocarpine Pilocarpine model corresponds to temporal lobe epilepsy [34], which is the most frequent form of epilepsy in humans [40].
Please cite this article in press as: Sawicka-Glazer E, Czuczwar SJ. Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.02.016
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Neuropathologically, it affects mostly hippocampus causing its sclerosis and ‘‘atrophy of mesial temporal structures’’ [34], also including pyriform, entorhinal, frontal, parietal cortices, striatum and amygdaloid nucleus [23], like ‘‘neuronal loss, gliosis and vacuolar degeneration’’ [39]. The trials in adult rats, pretreated with ascorbic acid and then given pilocarpine, revealed the increase in latency to the onset of seizures and lower mortality rate [32,39,43,44], and even suppression of seizure episodes [40]. Furthermore, pilocarpine produced a high concentration of peroxide lipids in hippocampus, and ascorbic acid significantly reduced a probable cause of neuronal cell damage during pilocarpine induced seizures [40]. Vitamin C not only blocked accumulation of free fatty acids in neurons during seizures, but also sustained higher activity of catalase in hippocampus, as a defensive enzyme, and behaved as a neuromodulator by blocking the efflux of calcium from the neuronal cells [43]. Ascorbic acid (250 and 500 mg/kg) revealed a dose-dependent protection against hippocampal injury during pilocarpine seizures, even up to 60% [23,31,32,40]. Vitamin C pretreatment changed seizure activity in adult rats by reduction of free radicals [32]. An increase in antioxidant enzymes, in response to free radicals during pilocarpine-induced seizures, gives a strong persuasion on the meaningful role of vitamin C as an anticonvulsant agent [31]. Postraumatic epilepsy model (intracortical administration of iron ions) Yamamoto et al. [45] revealed that vitamin C connected to vitamin E by a phosphate and given in a dose of 200 mg/kg before intracortical injection of FeCl3, reduced and delayed the occurrence of epileptiform activities in electrocorticograms. Similar results were observed when the drug was co-administered with iron ions directly into the motor cortex. In the in vitro experiments, administration of the free radical scavenger was proved to inhibit the production of chemical compounds commonly known as peroxidants (thiobarbituric acid reactive substances and protein carbonyl). Other models of experimental epilepsy Ascorbic acid also proved to be efficient against penicillininduced seizures in rats [2]. When given 30 min after intracortical penicillin injection, vitamin C at 50 and 100 mg/kg reduced the frequency and amplitude of epileptiform activity observed in electrocoticograms. At doses of 200 and 400 mg/kg, it decreased the frequency but not amplitude, and at 25 or 800 mg/kg it exerted
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no protective effects. The greatest protective activity was noted at 100 mg/kg. Thus, this dose was selected for other experimental designs. When administered 30 min prior to penicillin, ascorbic acid significantly delayed the onset of epileptiform activity and when given for 7 days (the last dose of 100 mg/kg injected 24 h prior to penicillin), the epileptiform activity was not modified. Interestingly, vitamin C (100 mg/kg) also prevented the penicillininduced reduction in brain glutathione concentration and increase in lipid peroxidation level [2]. Moreover, vitamin C (250 mg/kg) did not prevent seizures in kainic acid model in rats, in which seizures more slowly develop [44]. On the other hand, ascorbic acid (30– 50 mg/kg) was proved to attenuate kainate-induced neuronal loss, caused mostly by lipid peroxidation in rat hippocampus [20]. Vitamin C also acts in a dose-dependent manner as a neuroprotectant by diminishing trimethyltin-induced seizures [35]. On the other hand, as the effect of audiogenic seizure, a significant decrease in the adrenal ascorbic acid was accomplished [9]. Most of the above data indicates a significant role of vitamin C in seizures. Effects of vitamin C on certain types of seizures are shown in Table 1.
Neuroprotection Recurrent seizures may lead to persistent changes in brain functioning and its histological structure like ‘‘enlargement of ventricles, deformation of the dentate gyres’’ and massive neuronal loss in several areas of brain [39]. Ascorbic acid may prevent from histopathological and functional damages caused by pilocarpineinduced seizures by succouring other antioxidants, like vitamin E [31], and enzymes, such as glutathione peroxidase or glutathione reductase in order to scavenge free radicals responsible for spreading seizure activity [32,39]. Usage of vitamin C significantly decreased number of animals with brain damage [39]. Vitamin C neuroprotection during PTZ-induced seizures was confirmed by Western blot analysis and Nissl staining. It proved lower expression of caspase-3 and, as a result, preventive role of ascorbic acid against apoptosis [25]. Vitamin C consolidates cell membrane, already unstable during seizures, by decreasing lipid peroxidation which is probably most important event in degenerative process. It also supports superoxide dismutase [31] and catalase activity, which play a role of the defence enzymes [32]. Considering a crucial role of oxidative stress during convulsions for the induction of neurodegeneration [31], inhibition of production of free radicals by ascorbic acid is essential in this regard [40].
Table 1 Effects of vitamin C on experimental seizures. Convulsant
Vitamin C (doses in mg/kg)
Effect on seizure activity Inhibition
Pentylenetetrazol
Pilocarpine
Penicilin
500 300 100 30
+ +
500 250
++ +
25 50–100 200–400 800
Kainic acid
250
Trimethyltin
50 100
Stimulation NI
+ ++
References
# NT NT NT
[12]
# # NI
+ +
Mortality
No influence (NI) or stimulation
[33]
[23,31]
NI NI
NT NT NT NT
NI
NT
[44]
NT NT
[35]
[2]
+, moderate inhibition; ++, potent inhibition; NT, not tested.
Please cite this article in press as: Sawicka-Glazer E, Czuczwar SJ. Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.02.016
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Still, vitamin C antioxidant and anticonvulsant functions do not correlate, and as neuroprotection has been proven for many times, it is probable that ascorbic acid has biphasic effect on convulsions. As a result, there may not exist a cause–effect relationship between those two functions and ascorbic acid may act as a neuromodulator [33]. An earlier study has suggested that vitamin C may participate in preservation against cognitive attenuation [37]. Because ascorbic acid is a modulator of cholinergic, serotonergic and dopaminergic systems and those are essential in memory functions, researchers wondered if there was a correlation between these particular actions [37]. It was established that treatment of phosphamidon intoxication with vitamin C reversed cognitive disturbances, oxidative stress in brain and probably intensified the reaction of neuronal cells to dopamine and glutamate [37]. The question is whether ascorbic acid can reverse [39] or just stop potential damages. However, reductive actions of vitamin C are commonly known, so there is a strong belief that it can be used in reducing brain injury [40] or even protecting against seizures and their detrimental effects.
brain weight’’ are the same as others without convulsions. On the other hand, it diminishes ‘‘working memory performance’’ [39]. It is also worth noticing that ascorbic acid conjugates of certain drugs are better absorbed and what comes with it – can act quicker than non-conjugated parent drug [22] and gives longer metabolic effect like combination of ascorbic acid with diclofenac [7]. Many studies are promising in that they recommend the usage of vitamin C for its neuroprotective role. However, more details are needed before findings on vitamin C are certain and can be used in clinical practice as a valuable part of treatment. There are also many interesting co-operations of vitamin C with other drugs, even life-threatening toxicity [3]. Still, ascorbic acid seems very promising as regards supplementary therapy of epilepsy and other neurodegenerative disorders. Last but not least, oxidative stress may be not only a consequence of seizure activity but it may be triggered by AEDs themselves [21]. In this context, vitamin C also seems beneficial in epileptic patients.
Prooxidative action Conflict of interests Ascorbic acid is probably one of most important antioxidants in human brain. But as its antioxidative and what comes with, anticonvulsive role is being discovered, vitamin C under certain circumstances, especially in the inherence of metals, becomes a prooxidative factor [40] and may induce neurotoxicity [24]. It is particularly evident in the presence of iron when it behaves as a prooxidant, while in its absence it attends an antioxidant function [2]. Other studies indicate that vitamin C acts as prooxidant in systems containing iron in vitro, but in vivo iron has no effect on ascorbic acid and it still reduces effects of oxidative stress [6]. The reason of such an action is the presence of proteins which sequester the iron [26]. In a different manner, it is possible that chronic supplementation of vitamin C at a dose of 60 mg/day in natural development period leads to its prooxidative results to brains in observed animals [24]. This is confirmed by higher ‘‘cortical spreading depression’’, which is defined as depletion of ‘‘neuronal excitability’’. On the other hand, there are antioxidants, e.g. astaxanthin, which attenuate ‘‘cortical spreading depression’’. It has been also found that vitamin C is able to react with lipid hydroperoxide and form metabolites influencing DNA, and as a result causing mutagenesis [12]. Homeostasis of neuronal cells is significant and balance of anti- and prooxidative actions is necessary. Considering that such a destructive process as seizures forms large amounts of free radicals, that can cause neurons more liable to cytotoxic effects, it may be inadvisable to support a patient with vitamin C, especially in high doses up to 10 g/day because of its possible interference with the antioxidant–prooxidant balance [12]. Motor and cognitive functions All trials with supplementation of vitamin C in seizures focus not only on biochemistry of neurons, but mostly on possible clinical appliance. Seizures themselves, starting with neuronal damage, cause neurodegeneration and behavioural problems like ‘‘cognitive dysfunction’’ [39]. Administration of ascorbic acid prior to pilocarpine-induced seizures comparing to ‘‘just pilocarpine group’’ does not affect motor functions like swimming or crossing squares with the four paws. It also does not generate behavioural changes and does not interfere with ‘‘spatial memory acquisition phase’’ in animals observed for 24 h. Histopathologically, less damage to the brain is observed. Moreover, vitamin C given before seizures is thought to improve metabolism of rats, so that their ‘‘physical growth and
S.J. Czuczwar has received an unrestricted grant from GlaxoSmithKline. He has also lectured for GlaxoSmithKline, UCB, SanofiAventis and Jannsen. E. Sawicka-Glazer reports no conflict of interests. Acknowledgement This paper has been supported by a grant from Medical University of Lublin (DS. 475/13).
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PHAREP-57; No. of Pages 5 E. Sawicka-Glazer, S.J. Czuczwar / Pharmacological Reports xxx (2014) xxx–xxx [17] Henshall DC, Meldrum BS. Cell death and survival mechanisms after single and repeated brief seizures. In: Jasper’s basic mechanisms of the epilepsies4th ed. Bethesda: National Center for Biotechnology Information; 2012. [18] Layton ME, Samson FE, Pazdernik TL. Kainic acid causes redox changes in cerebral cortex extracellular fluid: NMDA receptor activity increases ascorbic acid whereas seizure activity increases uric acid. Neuropharmacology 1998;37:149–57. [19] Liao KH, Mei QY, Zhou YC. Determination of antioxidants in plasma and erythrocyte in patients with epilepsy. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2004;29:72–4. [20] MacGregor DG, Higgins MJ, Jones PA, Maxwell WL, Watson MW, Graham DI, et al. Ascorbate attenuates the systemic kainate-induced neurotoxicity in the rat hippocampus. Brain Res 1996;727:133–44. [21] Majkowski J. Oxygen stress: epilepsy and antiepileptic drugs. Epileptologia 2011;19:143–57. [22] Manfredini S, Pavan B, Vertuani S, Scaglianti M, Compagnone D, Biondi C, et al. Design, synthesis and activity of ascorbic acid prodrugs of nipecotic, kynurenic and diclophenamic acids, liable to increase neurotropic activity. J Med Chem 2002;45:559–62. [23] Marinho MM, de Bruin VM, de Sousa FC, Aguiar LM, de Pinho RS, Viana GS. Inhibitory action of a calcium channel blocker (nimodipine) on seizures and brain damage induced by pilocarpine and lithium-pilocarpine in rats. Neurosci Lett 1997;235:13–6. [24] Monte-Guedes CK, Alves EV, Viana-da-Silva E, Guedes RC. Chronic treatment with ascorbic acid enhances cortical spreading depression in developing wellnourished and malnourished rats. Neurosci Lett 2011;496:191–4. [25] Naseer MI, Ullah I, Ullah N, Lee HY, Cheon EW, Chung J, et al. Neuroprotective effect of vitamin C against PTZ induced apoptotic neurodegeneration in adult rat brain. Pak J Pharm Sci 2011;24:263–8. [26] Niki E. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am J Clin Nutr 1991;54:1119S–24S. [27] Odumosu A, Wilson CW. The relationship of ascorbic acid to leptazol-induced convulsions in guinea-pigs. Br J Pharmacol 1974;50:471P–2P. [28] Padayatty SJ, Katz A, Wang Y, Eck P, Kwon O, Lee JH, et al. Vitamin C as an antioxidant: evaluation of its role in disease prevention. J Am Coll Nutr 2003;22:18–35. [29] Rambo LM, Ribeiro LR, Della-Pace ID, Stamm DN, da Rosa Gerbatin R, Prigol M, et al. Acute creatine administration improves mitochondrial membrane potential and protects against pentylenetetrazol-induced seizures. Amino Acids 2013;44:857–68. [30] Ranganathan LN, Ramaratnam S. Vitamins for epilepsy. The Cochrane Library 2009;4:1–31. [31] Santos IM, Tome´ Ada R, Saldanha GB, Ferreira PM, Milita˜o GC, Freitas RM. Oxidative stress in the hippocampus during experimental seizures can be ameliorated with the antioxidant ascorbic acid. Oxid Med Cell Longev 2009;2:214–21.
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[32] Santos LF, Freitas RL, Xavier SM, Saldanha GB, Freitas RM. Neuroprotective actions of vitamin C related to decreased lipid peroxidation and increased catalase activity in adult rats after pilocarpine-induced seizures. Pharmacol Biochem Behav 2008;89:1–5. [33] Schneider Oliveira M, Fla´via Furian A, Freire Royes LF, Rechia Fighera M, de Carvalho Myskiw J, Gindri Fiorenza N, et al. Ascorbate modulates pentylenetetrazol-induced convulsions biphasically. Neuroscience 2004;128:721–8. [34] Scorza FA, Arida RM, Naffah-Mazzacoratti Mda G, Scerni DA, Calderazzo L, Cavalheiro EA. The pilocarpine model of epilepsy: what have we learned? An Acad Bras Cienc 2009;81:345–65. [35] Shin EJ, Suh SK, Lim YK, Jhoo WK, Hjelle OP, Ottersen OP, et al. Ascorbate attenuates trimethyltin-induced oxidative burden and neuronal degeneration in the rat hippocampus by maintaining glutathione homeostasis. Neuroscience 2005;133:715–27. [36] Sudha K, Rao AV, Rao A. Oxidative stress and antioxidants in epilepsy. Clin Chim Acta 2001;303:19–24. [37] Suke SG, Shukla A, Mundhada D, Banerjee BD, Mediratta PK. Effect of phosphamidon on cognition and oxidative stress and its modulation by ascorbic acid and 40 -chlorodiazepam in rats. Pharmacol Biochem Behav 2013;103:637– 42. [38] Sultana R, Perluigi M, Allan Butterfield D. Lipid peroxidation triggers neurodegeneration: a redox proteomics view into the Alzheimer disease brain. Free Radic Biol Med 2013;65:1–14. [39] Tome´ Ada R, Feitosa CM, Freitas RM. Neuronal damage and memory deficits after seizures are reversed by ascorbic acid? Arq Neuropsiquiatr 2010;68:579–85. [40] Tome´ Ada R, Ferreira PM, Freitas RM. Inhibitory action of antioxidants (ascorbic acid or alpha-tocopherol) on seizures and brain damage induced by pilocarpine in rats. Arq Neuropsiquiatr 2010;68:355–61. [41] Tveden-Nyborg P, Vogt L, Schjoldager JG, Jeannet N, Hasselholt S, Paidi MD, et al. Maternal vitamin C deficiency during pregnancy persistently impairs hippocampal neurogenesis in offspring of guinea pigs. PLoS ONE 2012;7:e48488. http://dx.doi.org/10.1371/journal.pone.0048488. [42] Verma S, Sinha R, Kumar P, Amin F, Jain J, Tanwar S. Study of Convolvulus pluricaulis for antioxidant and anticonvulsant activity. Cent Nerv Syst Agents Med Chem 2012;12:55–9. [43] Xavier SM, Barbosa CO, Barros DO, Silva RF, Oliveira AA, Freitas RM. Vitamin C antioxidant effects in hippocampus of adult Wistar rats after seizures and status epilepticus induced by pilocarpine. Neurosci Lett 2007;420:76–9. [44] Xu K, Stringer JL. Antioxidants and free radical scavengers do not consistently delay seizure onset in animal models of acute seizures. Epilepsy Behav 2008;13:77–82. [45] Yamamoto N, Kabuto H, Matsumoto S, Ogawa N, Yokoi I. Alpha-tocopheryl-Lascorbate-2-O-phosphate diester, a hydroxyl radical scavenger, prevents the occurrence of epileptic foci in a rat model of post-traumatic epilepsy. Pathophysiology 2002;8:205–14.
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PHAREP-57; No. of Pages 5 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Review article
Vitamin C: A new auxiliary treatment of epilepsy? Edyta Sawicka-Glazer a, Stanisław J. Czuczwar a,b,* a b
Department of Pathophysiology, Medical University, Lublin, Poland Department of Physiopathology, Institute of Rural Health, Lublin, Poland
A R T I C L E I N F O
Article history: Received 18 February 2014 Accepted 20 February 2014 Available online xxx Keywords: Ascorbic acid Epilepsy Seizures Neurodegeneration
A B S T R A C T
Although many approaches to the therapy of epilepsy exist, most of antiepileptic drugs, beside certain and unquestioned benefits, have convinced disadvantages. That is the reason for looking for new methods of treatment. Ascorbic acid, as an antioxidant and electron donor accumulated in central nervous system, seems to take part in diminishing reactions of oxidative stress in brain and cooperate with other antioxidants like alpha-tocoferol. Vitamin C, easily transported through the blood–brain barrier, is proved to reduce injury in the hippocampus during seizures. Depending on type of seizures, it has mostly inhibitory activity and even decreases mortality. Moreover, vitamin C acts as a neuroprotective factor by consolidating cell membranes and decreasing lipid peroxidation. A possible adjunctive role of vitamin C in epileptic patients needs to be considered. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oxidative stress – mechanism and consequences . . . . . . . . . . . . . . . Vitamin C – association with seizure activity . . . . . . . . . . . . . . . . . . Vitamin C – anticonvulsant and prooxidative activity . . . . . . . . . . . . Pentylenetetrazol (PTZ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pilocarpine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Postraumatic epilepsy model (intracortical administration of Other models of experimental epilepsy . . . . . . . . . . . . . . . . . . Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prooxidative action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motor and cognitive functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Epilepsy is a common neurological disorder, characterised by immediate and tilting seizures, affecting about 1–2% of population [4]. Especially susceptible to expand this disorder are immature brains [12]. In the group treated by modern antiepileptic drugs (AEDs) ascendancy of 70% is going into remission, but up to 30%
* Corresponding author. E-mail address: [email protected] (S.J. Czuczwar).
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will be still exposed to seizures [30,42]. That is the reason of interest of routine usage of vitamins as a prevention or probable new method of treatment. Vitamins have been considered to be important patterns in controlling certain types of seizures or even preventing adverse effects of AEDs [30]. Although, folic acid, thiamine, vitamin D or vitamin E have not been proved this kind of action [30], there is a strong persuasion of neuroprotective action of vitamin C on brain tissue, especially during seizures. Vitamin C is an exogenous water-soluble substance [30], nutritional supplement [10]. It is a cofactor in building blood
http://dx.doi.org/10.1016/j.pharep.2014.02.016 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Sawicka-Glazer E, Czuczwar SJ. Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.02.016
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vessels and takes role of an antioxidant [11]. Vitamin C is synthesised from glucose in livers of mammals, except humans by the reason of absence of specific enzyme which is gulonolactone oxidase [28,41]. Vitamin C is an electron donor and reducing agent, so it prevents lipid, protein and DNA oxidation [28]. The evidence of this action has been proved in extracellular fluids, e.g. plasma [6]. Ascorbic acid is also accumulated in adrenal glands and central nervous system [24]. It acts not only as a reactive oxygen species scavenger, but also helps to restore other substances with antioxidant properties, such as alpha-tocopherol or glutathione [6]. Oxidative stress – mechanism and consequences Oxidative stress is a state when there is a disproportion of proand antioxidants with the predominance of prooxidants which are considered to be reactive forms of oxygen (ROS) [16] and reactive nitrogen species [1]. Brain consumes 20% of inhaled oxygen but only 2–5% of it is reduced [1], so it causes a possibility of various chemical reactions. Moreover, central nervous system has its specific regions more vulnerable to oxidative reactions, like hippocampus, by the reason of lower concentration of antioxidants than in the other areas [31]. Hippocampus is very susceptible to concentration of vitamin C not only in adolescence, but its atrophy was noticed also in animals with prenatal deficiency of ascorbic acid [41]. Seizures are thought as high energy demanding reactions. It is noteworthy that seizure activity may cause meaningful changes especially in mitochondrial homeostasis [1]: diminishes the amount of energy by reduction of ATP, inhibits mitochondrial respiratory enzymes and lowers neuronal plasma membrane potential. Such a hyperactivity of neurons and excitotoxic process indicates also formation of ROS, causing prevalence of oxidative processes [29]. ROS disturb transport of electron chain enzymes and damage DNA [14,18]. Large contribution of mtDNA in cell is also thought to be one of the probable elements in increasing probability of seizures and interprets its role to epileptogenesis [17]. At normal conditions, such molecules as superoxide anion, hydroxyl radical or peroxinitrate participate in indispensable metabolic pathways [1], but in the excess, induce peroxidation of polyunsaturated fatty acids occurring in high concentration in brain tissue [15,32,43], peroxidation of proteins or DNA [1], which can result in neuronal cell damage and glial cell toxicity [31], especially in the immature brain [24], and neuronal deprivation in certain structures [39]. Brain tissue is ratable to lipid peroxidation during oxidative stress [14,15,43]. Membrane changes cause exempting of ‘‘arachidonic acid, diacylglycerols, eicosanoids, lipid peroxides and free radicals’’ [43]. Lipid peroxidation begins chain of reactions, which starts with emergence of elecrophilic aldehydes and than results in posttranslational modification of proteins [38]. There is, however, group of natural factors functioning as antiooxidants, like superoxide dismutase, catalase and glutathione peroxidase [43]. To such an important role of oxidative stress in epilepsy indicates the fact that a meaningful increase of RNA oxidation was observed during convulsions in rat brains [1]. Vitamin C – association with seizure activity Ascorbic acid is a basic micronutrient for all mammalians [8,37]; its highest concentration is encountered in spinal cord and brain [8]. Vitamin C co-operates in myelin building, plays here a role of an antioxidant and a neuromodulator for neuronal mediators [8]. It is transported through the blood-brain barrier in endothelial cells of brain vessels [13], its levels are proportional to N-methyl-Daspartate receptor activity and has high affinity for the SVCT2 transporter, which conveys neurotropic agents, e.g. certain drugs conjugated to ascorbic acid [7,18]. Ascorbic acid is transported to mitochondria in oxidised form of dehydroascorbic acid by facilitative glucose transporter GLUT1, GLUT3 and GLUT4 and is
immediately switched to ascorbic acid again [31,35]. Brain is one of few places where the substance is being clustered to various brain structures – hippocampus, amygdala and hypothalamus [35]. Vitamin C is transported primarily to mitochondria where ROS are in highest accumulation and there transfers an electron to ‘‘ascorbate-dependent peroxidase’’ [31]. The active form of ascorbic acid is an electron donor, so biochemically it acts as a reducing agent [12]. It controls in vivo biological actions of free radicals and behaves as a free radical scavenger [40], also playing a role of defence in cell’s structure. Interestingly, some AEDs, like for instance topiramate, may share ascorbic acid’s scavenging activity in epilepsy [5]. Vitamin C inhibits aggregating free fatty acids in rats’ brains during seizures [43]. It decreases peroxidation of lipids, most possibly by influence and escalating action of antioxidant enzymes like superoxide dismutase or catalase in the adult rat hippocampus [31,32] and works as a neuromodulator [40]. The accurate level of those enzymes prevents from seizures [31]. Concentration of ascorbic acid in plasma of patients with epilepsy is much lower than in non-epileptic patients [19,36]. As hippocampus and amygdala are considered as structures involved in transmitting seizures, their role is significant [12,32]. Vitamin C has been proved to reduce injury in the hippocampus during seizures [38]. It is also said to be a ‘‘positive modulator of GABA A receptors’’ [12]. Vitamin C, on the other hand, is involved in production of steroids and peptide hormones and gives progesterone-like effect [43]. As is proved, estrogen accompanies seizures, contrary progesterone and its metabolites (possibly allopregnanolone) prevent from seizure activity [12]. Ascorbic acid increases progesterone production, but also cooperates with it [12]. Ascorbic acid is one of many antioxidants in brain and strongly cooperates with others, like alpha-tocoferol, by regenerating its reduced form in cell membrane [40]. Vitamin C – anticonvulsant and prooxidative activity A few studies concerning usage of ascorbic acid in different seizure model have been conducted. Pentylenetetrazol (PTZ) Immature rats pretreated with ascorbic acid (500 mg/kg for 5 days) revealed a significant retardation of onset to myoclonic, clonic and tonic seizures or even the absence of convulsions was noted [12]. Vitamin C also decreased the number of seizures and mortality of rats. Moreover, a reduction in amino acid (GABA, glutamate, aspartate, glutamine) concentration, following vitamin C supplementation, has been observed in many regions of brain, especially in hippocampus and amygdala [12]. Ascorbic acid at 300 mg/kg was also shown to protect against seizures, to prolong seizure duration at 100 mg/kg, and to exert no effect on seizure activity at 30 mg/kg [33]. Further, inhibition of Na-K-ATPase activity induced by PTZ, which increases neuronal excitability and leads to convulsions, was reversed by ascorbic acid in the highest dose of 300 mg/kg [33]. Administration of ascorbic acid (200 mg/ kg) to guinea pigs led to a decreased intensity of seizures and prolongation of the latent period [27]. The scorbutic guinea-pigs showed shortened latent period to convulsions and their frequency was also higher [27]. Finally, vitamin C (250 mg/kg) significantly reduced the mortality rate in PTZ-treated rats although the evaluated seizure parameters, seizure duration, seizure severity, and latency were not affected [44]. Pilocarpine Pilocarpine model corresponds to temporal lobe epilepsy [34], which is the most frequent form of epilepsy in humans [40].
Please cite this article in press as: Sawicka-Glazer E, Czuczwar SJ. Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.02.016
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Neuropathologically, it affects mostly hippocampus causing its sclerosis and ‘‘atrophy of mesial temporal structures’’ [34], also including pyriform, entorhinal, frontal, parietal cortices, striatum and amygdaloid nucleus [23], like ‘‘neuronal loss, gliosis and vacuolar degeneration’’ [39]. The trials in adult rats, pretreated with ascorbic acid and then given pilocarpine, revealed the increase in latency to the onset of seizures and lower mortality rate [32,39,43,44], and even suppression of seizure episodes [40]. Furthermore, pilocarpine produced a high concentration of peroxide lipids in hippocampus, and ascorbic acid significantly reduced a probable cause of neuronal cell damage during pilocarpine induced seizures [40]. Vitamin C not only blocked accumulation of free fatty acids in neurons during seizures, but also sustained higher activity of catalase in hippocampus, as a defensive enzyme, and behaved as a neuromodulator by blocking the efflux of calcium from the neuronal cells [43]. Ascorbic acid (250 and 500 mg/kg) revealed a dose-dependent protection against hippocampal injury during pilocarpine seizures, even up to 60% [23,31,32,40]. Vitamin C pretreatment changed seizure activity in adult rats by reduction of free radicals [32]. An increase in antioxidant enzymes, in response to free radicals during pilocarpine-induced seizures, gives a strong persuasion on the meaningful role of vitamin C as an anticonvulsant agent [31]. Postraumatic epilepsy model (intracortical administration of iron ions) Yamamoto et al. [45] revealed that vitamin C connected to vitamin E by a phosphate and given in a dose of 200 mg/kg before intracortical injection of FeCl3, reduced and delayed the occurrence of epileptiform activities in electrocorticograms. Similar results were observed when the drug was co-administered with iron ions directly into the motor cortex. In the in vitro experiments, administration of the free radical scavenger was proved to inhibit the production of chemical compounds commonly known as peroxidants (thiobarbituric acid reactive substances and protein carbonyl). Other models of experimental epilepsy Ascorbic acid also proved to be efficient against penicillininduced seizures in rats [2]. When given 30 min after intracortical penicillin injection, vitamin C at 50 and 100 mg/kg reduced the frequency and amplitude of epileptiform activity observed in electrocoticograms. At doses of 200 and 400 mg/kg, it decreased the frequency but not amplitude, and at 25 or 800 mg/kg it exerted
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no protective effects. The greatest protective activity was noted at 100 mg/kg. Thus, this dose was selected for other experimental designs. When administered 30 min prior to penicillin, ascorbic acid significantly delayed the onset of epileptiform activity and when given for 7 days (the last dose of 100 mg/kg injected 24 h prior to penicillin), the epileptiform activity was not modified. Interestingly, vitamin C (100 mg/kg) also prevented the penicillininduced reduction in brain glutathione concentration and increase in lipid peroxidation level [2]. Moreover, vitamin C (250 mg/kg) did not prevent seizures in kainic acid model in rats, in which seizures more slowly develop [44]. On the other hand, ascorbic acid (30– 50 mg/kg) was proved to attenuate kainate-induced neuronal loss, caused mostly by lipid peroxidation in rat hippocampus [20]. Vitamin C also acts in a dose-dependent manner as a neuroprotectant by diminishing trimethyltin-induced seizures [35]. On the other hand, as the effect of audiogenic seizure, a significant decrease in the adrenal ascorbic acid was accomplished [9]. Most of the above data indicates a significant role of vitamin C in seizures. Effects of vitamin C on certain types of seizures are shown in Table 1.
Neuroprotection Recurrent seizures may lead to persistent changes in brain functioning and its histological structure like ‘‘enlargement of ventricles, deformation of the dentate gyres’’ and massive neuronal loss in several areas of brain [39]. Ascorbic acid may prevent from histopathological and functional damages caused by pilocarpineinduced seizures by succouring other antioxidants, like vitamin E [31], and enzymes, such as glutathione peroxidase or glutathione reductase in order to scavenge free radicals responsible for spreading seizure activity [32,39]. Usage of vitamin C significantly decreased number of animals with brain damage [39]. Vitamin C neuroprotection during PTZ-induced seizures was confirmed by Western blot analysis and Nissl staining. It proved lower expression of caspase-3 and, as a result, preventive role of ascorbic acid against apoptosis [25]. Vitamin C consolidates cell membrane, already unstable during seizures, by decreasing lipid peroxidation which is probably most important event in degenerative process. It also supports superoxide dismutase [31] and catalase activity, which play a role of the defence enzymes [32]. Considering a crucial role of oxidative stress during convulsions for the induction of neurodegeneration [31], inhibition of production of free radicals by ascorbic acid is essential in this regard [40].
Table 1 Effects of vitamin C on experimental seizures. Convulsant
Vitamin C (doses in mg/kg)
Effect on seizure activity Inhibition
Pentylenetetrazol
Pilocarpine
Penicilin
500 300 100 30
+ +
500 250
++ +
25 50–100 200–400 800
Kainic acid
250
Trimethyltin
50 100
Stimulation NI
+ ++
References
# NT NT NT
[12]
# # NI
+ +
Mortality
No influence (NI) or stimulation
[33]
[23,31]
NI NI
NT NT NT NT
NI
NT
[44]
NT NT
[35]
[2]
+, moderate inhibition; ++, potent inhibition; NT, not tested.
Please cite this article in press as: Sawicka-Glazer E, Czuczwar SJ. Vitamin C: A new auxiliary treatment of epilepsy? Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2014.02.016
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Still, vitamin C antioxidant and anticonvulsant functions do not correlate, and as neuroprotection has been proven for many times, it is probable that ascorbic acid has biphasic effect on convulsions. As a result, there may not exist a cause–effect relationship between those two functions and ascorbic acid may act as a neuromodulator [33]. An earlier study has suggested that vitamin C may participate in preservation against cognitive attenuation [37]. Because ascorbic acid is a modulator of cholinergic, serotonergic and dopaminergic systems and those are essential in memory functions, researchers wondered if there was a correlation between these particular actions [37]. It was established that treatment of phosphamidon intoxication with vitamin C reversed cognitive disturbances, oxidative stress in brain and probably intensified the reaction of neuronal cells to dopamine and glutamate [37]. The question is whether ascorbic acid can reverse [39] or just stop potential damages. However, reductive actions of vitamin C are commonly known, so there is a strong belief that it can be used in reducing brain injury [40] or even protecting against seizures and their detrimental effects.
brain weight’’ are the same as others without convulsions. On the other hand, it diminishes ‘‘working memory performance’’ [39]. It is also worth noticing that ascorbic acid conjugates of certain drugs are better absorbed and what comes with it – can act quicker than non-conjugated parent drug [22] and gives longer metabolic effect like combination of ascorbic acid with diclofenac [7]. Many studies are promising in that they recommend the usage of vitamin C for its neuroprotective role. However, more details are needed before findings on vitamin C are certain and can be used in clinical practice as a valuable part of treatment. There are also many interesting co-operations of vitamin C with other drugs, even life-threatening toxicity [3]. Still, ascorbic acid seems very promising as regards supplementary therapy of epilepsy and other neurodegenerative disorders. Last but not least, oxidative stress may be not only a consequence of seizure activity but it may be triggered by AEDs themselves [21]. In this context, vitamin C also seems beneficial in epileptic patients.
Prooxidative action Conflict of interests Ascorbic acid is probably one of most important antioxidants in human brain. But as its antioxidative and what comes with, anticonvulsive role is being discovered, vitamin C under certain circumstances, especially in the inherence of metals, becomes a prooxidative factor [40] and may induce neurotoxicity [24]. It is particularly evident in the presence of iron when it behaves as a prooxidant, while in its absence it attends an antioxidant function [2]. Other studies indicate that vitamin C acts as prooxidant in systems containing iron in vitro, but in vivo iron has no effect on ascorbic acid and it still reduces effects of oxidative stress [6]. The reason of such an action is the presence of proteins which sequester the iron [26]. In a different manner, it is possible that chronic supplementation of vitamin C at a dose of 60 mg/day in natural development period leads to its prooxidative results to brains in observed animals [24]. This is confirmed by higher ‘‘cortical spreading depression’’, which is defined as depletion of ‘‘neuronal excitability’’. On the other hand, there are antioxidants, e.g. astaxanthin, which attenuate ‘‘cortical spreading depression’’. It has been also found that vitamin C is able to react with lipid hydroperoxide and form metabolites influencing DNA, and as a result causing mutagenesis [12]. Homeostasis of neuronal cells is significant and balance of anti- and prooxidative actions is necessary. Considering that such a destructive process as seizures forms large amounts of free radicals, that can cause neurons more liable to cytotoxic effects, it may be inadvisable to support a patient with vitamin C, especially in high doses up to 10 g/day because of its possible interference with the antioxidant–prooxidant balance [12]. Motor and cognitive functions All trials with supplementation of vitamin C in seizures focus not only on biochemistry of neurons, but mostly on possible clinical appliance. Seizures themselves, starting with neuronal damage, cause neurodegeneration and behavioural problems like ‘‘cognitive dysfunction’’ [39]. Administration of ascorbic acid prior to pilocarpine-induced seizures comparing to ‘‘just pilocarpine group’’ does not affect motor functions like swimming or crossing squares with the four paws. It also does not generate behavioural changes and does not interfere with ‘‘spatial memory acquisition phase’’ in animals observed for 24 h. Histopathologically, less damage to the brain is observed. Moreover, vitamin C given before seizures is thought to improve metabolism of rats, so that their ‘‘physical growth and
S.J. Czuczwar has received an unrestricted grant from GlaxoSmithKline. He has also lectured for GlaxoSmithKline, UCB, SanofiAventis and Jannsen. E. Sawicka-Glazer reports no conflict of interests. Acknowledgement This paper has been supported by a grant from Medical University of Lublin (DS. 475/13).
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