Select Page

Sarahian et al (in press)

SHORT RESEARCH ARTICLE

PDF logo-blue 30

 

The Effects of the Peroxisome-Proliferator Activated Receptor-alpha Agonist, Fenofibrate, on the Antioxidant Capacity of the Brain in Pentylenetetrazol Kindling Seizures in Mice

 

Nahid Sarahian1, Mohammad Taghi Mohammadi1,2, Shamsi Darabi3, and Fatemeh Salem1

1Neuroscience Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran; 2Department of Physiology and Biophysics, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran; 3Department of Physiology, Faculty of Medicine, Qom University of Medical Sciences, Qom, Iran

Correspondence: mohammadimohammadt@bmsu.ac.ir (M.T.M.)

Sarahian N et al. Reactive Oxygen Species 7(19):xxx–xxx, 2019; ©2019 Cell Med Press

http://dx.doi.org/10.20455/ros.2019.807

(Received: June 24, 2018; Revised: September 1, 2018; Accepted: September 3, 2018)

ABSTRACT | It has been demonstrated that peroxisome-proliferator activated receptor-alpha (PPARα) has a potent neuroprotective role in various pathological events of the nervous tissue. Since oxidative damage is associated with development of seizure, we aimed to examine whether the PPARα agonist, fenofibrate, exerts protective effects against the repeated seizures in pentylenetetrazol (PTZ) kindling model in mice through improving the brain antioxidant capacity. The experiment was carried out in two groups of mice (each group, n = 12): PTZ-kindled mice and fenofibrate-treated kindled mice. Repetitive intraperitoneal injections of PTZ (65 mg/kg) once every 48 h were used to achieve the kindling seizures till day 21. The mice were administered orally fenofibrate (30 mg/kg/day) during the test. Latency and the brain activities of catalase and superoxide dismutase (SOD) as well as the brain content of reduced glutathione (GSH) were determined at termination of the experiment. The latency following the last injection of PTZ was considerably decreased in untreated kindled mice (49 ± 8 s), whereas fenofibrate treatment prevented this reduction in kindled mice (105 ± 16 s). Treatment with fenofibrate significantly increased the GSH content in kindled mice (20.22 ± 9.87 nmol/mg protein) compared to untreated kindled mice (5.37 ± 0.84 nmol/mg protein), (p < 0.05). Likewise, treatment with fenofibrate considerably increased the activities of catalase and SOD in kindled mice compared to untreated kindled mice by 78% and 55%, respectively. In view of the critical protective role of antioxidants in seizures, the findings of the present study suggested that the PPARα agonist, fenofibrate, might modulate the seizure behaviors in the PTZ kindling model in mice through improving the brain antioxidant capacity.

KEYWORDS | Antioxidant capacity; Fenofibrate; Pentylenetetrazol; Oxidative stress; PPARα agonist; Reactive oxygen species; Seizure

ABBREVIATIONS | GSH, reduced glutathione; NBT, nitroblue tetrazolium; PBS, phosphate-buffered saline; PPARα, peroxisome-proliferator activated receptor-alpha; PTZ, pentylenetetrazol; ROS, reactive oxygen species; SOD, superoxide dismutase


CONTENTS

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. Drugs and Chemicals

2.3. Experimental Design and Grouping

2.4. Determination of the Latency of Seizures

2.5. Brain Tissue Preparation

2.6. Measurement of GSH

2.7. Brain Activity of Catalase

2.8. Brain Activity of SOD

2.9. Statistical Analysis

3. Results

3.1. Effect of Fenofibrate on Latency of Kindled Mice

3.2. Effect of Fenofibrate on GSH Levels of the Brain

3.3. Effect of Fenofibrate on Catalase Activity of the Brain

3.4. Effect of Fenofibrate on SOD Activity of the Brain

4. Discussion


1. INTRODUCTION

Seizures generally result from an imbalance of excitation and inhibition due to a defect in inhibitory neurotransmission, particularly dysfunction of GABAergic inhibition [1, 2]. Additionally, excessive generation of reactive oxygen species (ROS) has been implicated in the development of seizures [3]. Under normal physiological conditions, ROS are generated to maintain a redox balance and are destructed by the brain antioxidant defense system [3, 4]. There is evidence indicating that the antioxidant capacity of the brain is weakened in several animal models of seizure, and antioxidant therapy may lessen the lesions induced by oxidative stress [4‒6]. It has been reported that lipid peroxidation and oxidative damage in accompany with mitochondrial dysfunction are observed following epileptic seizures [7, 8]. ROS accumulation caused by the seizures also induces oxidative damage to susceptible targets including proteins, lipids, and DNA [9, 10]. Consequently, the injuries induced by ROS may also aggravate the development of seizures [11]. In line with this notion, there is evidence showing that treatment with antioxidant substances may partially prevent the occurrence of seizures [7, 12]. Oxidative damage contributes to enhanced neuronal hyperexcitability by modifying the excitatory ion channels and inactivation of the energy-dependent glutamate transporters [3]. Therefore, there is a close relation between oxidative stress and seizure that has been frequently reported in experimental models and genetic studies [8, 13].

Previously, the robust neuroprotective functions of peroxisome-proliferator activated receptor-alpha (PPARα) have been demonstrated in various pathological events of the central nervous system [14‒17]. It has been reported that the agonists of this receptor such as fenofibrate have anti-inflammatory and antioxidant properties [15, 18]. PPARα activation also improves the endothelial function and vascular tight junction integrity in a variety of pathophysiological states of the brain [14]. Treatment with the PPARα agonist, fenofibrate, decreased the infarct volume in cerebral ischemia [17]. Fenofibrate also increased the activities of antioxidant enzymes catalase and superoxide dismutase (SOD) in the ischemic brain [18, 19]. The neuroprotective roles of PPARα and its agonist, fenofibrate, have been demonstrated in Alzheimer’s and Parkinson’s diseases through clinical and basic research [20, 21]. PPARα activation exerted a protective effect against beta-amyloid-induced neurodegeneration and inhibited beta-amyloid-induced pro-inflammatory responses [20, 21]. Treatment with fenofibrate also preserved cognitive function and locomotor activity in a toxin-induced model of Parkinson’s disease through preventing dopaminergic neurons loss and dysfunction [14, 20]. It has been demonstrated that these effects were mediated in part by decreased oxidative stress and pro-inflammatory mediators, suggesting that the PPARα agonist, fenofibrate, has anti-inflammatory and antioxidant properties [14, 16].

According to previous findings, the brain antioxidant capacity is weakened following the repetitive seizures [3, 6, 22]. Since the antioxidant properties of PPAR𝛼 agonists have been demonstrated in several pathological events of the nervous tissue, we aimed to evaluate whether the PPARα agonist, fenofibrate, exerts protective effects against the seizures in the pentylenetetrazol kindling model in mice through improvement of the brain antioxidant defense system.

2. MATERIALS AND METHODS

2.1. Animals 

All experimental protocols used in the current study were approved by the Institutional Animal Ethics Committee of the University of Baqiyatallah Medical Sciences (Tehran, Iran). Adult male NMRI mice, weighting about 23 ± 7 gram (aging 7‒8 weeks old), were employed in the current study. The mice acclimatized in the institutional animal house and were given standard chow and water ad libitum. The animals were housed under controlled conditions of light exposure (12 h light/dark cycle), temperature (22‒24˚C), and humidity (40‒60%).

2.2. Drugs and Chemicals

Pentylenetetrazol (PTZ) was purchased from Sigma-Aldrich (Steinheim, Germany). Fenofibrate was obtained from Hakim Pharmaceutical (Tehran, Iran). PTZ was dissolved in normal saline and the solution was injected to animals, intraperitoneally. Fenofibrate was suspended in 0.5% w/v of carboxy methyl cellulose (CMC) and the solution was administrated to the animals via oral gavage.

2.3. Experimental Design and Grouping

Twenty-four mice were randomly divided into untreated kindled mice and fenofibrate-treated kindled mice (each group, n = 12). In both groups, the repeated intraperitoneal injections of PTZ (65 mg/kg) once every 48 h were used for induction of seizures until day 21 [23, 24]. The mice were administered orally fenofibrate (30 mg/kg/day) five days before induction of seizures until day 21. Also, untreated kindled mice were administered the CMC solution without fenofibrate in the same volume.

2.4. Determination of the Latency of Seizures

Immediately after PTZ injection, the treated and untreated kindled mice were transferred to a round open field, and the latency of seizures was determined. The time elapsed after injection of PTZ until appearance of the seizure behaviors, such as facial twitches/myoclonus and clonus of one fore-raw or bilateral myoclonus, was recorded as the latency.

2.5. Brain Tissue Preparation

Brains were quickly removed under deep anesthesia for evaluation of the antioxidant parameters. After homogenization of the brains in ice-cold phosphate-buffered saline (PBS), the homogenates were centrifuged at 14000 g at 4oC for 15 min. Then, the supernatants were used to determine the brain contents of reduced glutathione (GSH) as well as the activities of superoxide dismutase (SOD) and catalase. The method of Bradford was used to quantify the protein content of each hemisphere for data calculation [25].

2.6. Measurement of GSH

The GSH levels of the brain were assessed according to the method of Tietz [26]. First, the cellular protein was precipitated by adding sulfosalicylic acid (5%). After centrifugation at 2,000 g for 10 min, the supernatant was removed and GSH level was assayed as follows: 100 µl of the protein-free supernatant of the tissue lysate, 100 µl of 0.04% 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in 0.1% sodium citrate, and 800 µl of 0.3 mM Na2HPO4 were mixed. After 5 min, the absorbance was recorded at 412 nm. A standard curve was performed with GSH concentrations between 1 and 100 µM [27]. The GSH levels of the brain were calculated as nmol/mg protein.

2.7. Brain Activity of Catalase

The method of Aebi was used to determine the activity of catalase in the brain tissue homogenate [28]. First, the homogenate was incubated in the reaction mixture that contained 0.1 ml homogenate and 0.85 ml potassium phosphate buffer (50 mM and pH 7.0) at room temperature for 10 min. Then, the reaction was started by adding 0.05 ml H2O2 (30 mM prepared in 50 mM potassium phosphate buffer, pH 7.0). A decrease in the absorbance was recorded by a spectrophotometer at 240 nm for 3 min. Specific activity of catalase in the brain tissue was calculated as units/mg protein. One unit of catalase was defined as 1 µmol H2O2 decomposed per min.

2.8. Brain Activity of SOD

The SOD activity was measured using the method of Winterbourn et al. [29], based on the capability of SOD to inhibit the reduction of nitroblue tetrazolium (NBT). First, potassium phosphate buffer (0.067 M, pH 7.8) was added to 0.1 M EDTA containing 0.3 mM sodium cyanide, 1.5 mM NBT, and 0.1 ml of sample. Then, to initiate the reaction, riboflavin (0.12 mM) was added to each sample. After a 12 min-incubation, the absorbance of samples was recorded spectrophotometrically at 610 nm for five min. The amount of enzyme needed to induce 50% inhibition was taken as 1 unit [27]. The SOD activity in the brain tissue was calculated as units/mg protein.

2.9. Statistical Analysis

The SPSS (v.21, Chicago, IL, USA) was used for statistical analysis. The t-test was used to analyze the data between two groups (treated and untreated kindled mice). All data were expressed as mean ± SEM. A p < 0.05 was considered statistically significant.

3. RESULTS

3.1. Effect of Fenofibrate on Latency of Kindled Mice

Treatment with fenofibrate did not significantly affect the body weight changes during experimental period (Figure 1). As shown in Figure 2, the values of latency after first injection of PTZ for untreated and treated kindled mice were 116 ± 14 s and 111 ± 4 s, respectively; there was statistically no significant difference between them. The latency was considerably decreased in untreated kindled mice (49 ± 8 s) following the last injection of PTZ, whereas fenofibrate prevented the reduction of latency in kindled mice (105 ± 16 s) (p < 0.001).

FIGURE 1. The representative changes of body weight of untreated and treated kindled mice during the experiment. Body weight of kindled mice (both groups) gradually increased during the period of the test. All values are presented as mean ± SEM (n = 12).

 

FIGURE 2. Latency in untreated kindled mice (PTZ) and treated kindled mice at the beginning and termination of the experiment. All values are presented as mean ± SEM (n = 12). ***, p < 0.001 at day 21.

3.2. Effect of Fenofibrate on GSH Levels of the Brain

The GSH levels of the brain in kindled mice at termination of the experiment are shown in Figure 3A. The mean value of brain GSH levels was 5.37 ± 0.84 nmol/mg protein for untreated kindled mice. Treatment with fenofibrate significantly increased the GSH levels of the brain in kindled mice (20.22 ± 9.87 nmol/mg protein) compared to untreated kindled mice (p < 0.05).

FIGURE 3. Effects of fenofibrate on the brain levels of GSH (A) and the activities of catalase (CAT) (B) and SOD (C) in untreated kindled mice (PTZ) and treated kindled mice at termination of the experiment. All values are presented as mean ± SEM (n = 4). *, p < 0.05; ***, p < 0.001 as compared with untreated kindled group.

3.3. Effect of Fenofibrate on Catalase Activity of the Brain

Figure 3B shows the catalase activity of the brain at termination of the experiment. The mean value of catalase activity in the brain of untreated kindled mice was 0.17 ± 0.01 units/mg protein. Fenofibrate significantly increased the catalase activity in the brain of kindled mice (0.78 ± 0.36 units/mg protein) compared to untreated kindled mice (p < 0.001).

3.4. Effect of Fenofibrate on SOD Activity of the Brain

As shown in Figure 3C, the SOD activity in the brain of untreated kindled mice was 1.08 ± 0.20 units/mg protein at termination of the experiment. Treatment with fenofibrate considerably increased the activity of SOD in the brain of kindled mice (2.40 ± 0.94 units/mg protein) compared to untreated kindled mice, but the difference was not statistically significant (p = 0.103).

4. DISCUSSION

Recently, the numerous reports have demonstrated the neuroprotective effects of the PPARα agonist, fenofibrate, in a variety of neurodegenerative diseases [16, 17]. In the present study, we showed that fenofibrate acts as an anticonvulsant in PTZ kindling model of seizure by preventing the reduction of seizures latency. Treatment with fenofibrate considerably enhanced the activities of antioxidant enzymes (SOD and catalase) in the brain of kindled mice compared to untreated kindled mice. Fenofibrate also significantly increased the GSH levels of the brain in kindled mice. Since oxidative damage plays a critical role in the pathophysiology of the seizure-induced brain damage [7, 11], it is suggested that the anticonvulsive effects of fenofibrate may correlate with the capability of this agonist to potentiate the antioxidant capacity of the brain in PTZ kindling model of seizure in mice.

A number of clinical and experimental studies have demonstrated that the increased free radical generation after occurrence of seizures results in membrane lipid peroxidation and oxidative damage [30, 31]. Also, low plasma levels of antioxidants have been reported in patients with status epilepticus, suggesting that seizures induce oxidative damage [30]. Additionally, long-term treatment with certain antiepileptic drugs such as valproic acid increases free radical formation in people [32]. Since the antioxidant imbalance is the main mechanism of free radical accumulation and oxidative damage after seizures [33], improving the antioxidant capacity of the brain would prevent the seizures-induced free radical accumulation and oxidative damage. Our results indicated that treatment with fenofibrate considerably enhanced the antioxidant capacity of the brain in kindled mice. The antioxidant functions of fenofibrate and PPARα activation have been reported in the various pathophysiological states of the nervous tissue [16, 17]. Therefore, it is suggested that fenofibrate may act as a neuroprotectant during seizure periods by enhancing the antioxidant capacity of the brain. Indeed, our findings revealed that fenofibrate prevented reduction of the latency of the kindling seizures.

Based on our results, the brain GSH content of fenofibrate-treated kindled mice significantly increased compared with untreated mice. GSH is a key antioxidant with several cellular functions. It directly reacts with different oxygen and nitrogen free radicals [34]. Also, GSH acts as an essential cofactor for critical antioxidant enzymes, such as glutathione peroxidase that converts hydrogen peroxide to H2O [35]. Furthermore, GSH protected the neurons against calcium-induced cell death and glutamate-mediated neurotoxicity [36, 37]. The GSH levels of the brain were shown to be decreased during the seizures [38]. Our results suggested that fenofibrate might potentiate the brain antioxidant system by increasing the GSH levels during the kindling seizures. Previously, Prigol et al. reported that GSH protected against seizure incidents by decreasing oxidative damage and antagonizing the glycine and glutamate modulatory locations in the NMDA receptor [37].

Pronounced alterations of antioxidant enzymes such as SOD and catalase have been reported following status epilepticus [39]. According to previous findings, the catalase activity of cerebral cortex was noticeably decreased following experimentally induced repeated seizures [4]. Likewise, the total activity of SOD was considerably decreased following acute seizures in kindled rats [40]. Accordingly, improving the antioxidant capacity of the brain might provide a beneficial effect against seizures-induced neuronal and oxidative damages. Based on our results, fenofibrate administration improved the antioxidant capacity of the brain in kindled mice through increasing the antioxidant enzymes, including SOD and catalase activities. SOD is a key antioxidant enzyme that neutralizes superoxide [22]. Similarly, catalase is another key enzyme of the brain antioxidant system that is responsible for the neutralization of H2O2 derived from SOD-mediated dismutation of superoxide [27, 41]. According to our results, fenofibrate was able to potentiate the brain enzymatic antioxidant capacity by enhancing the activities of catalase and SOD in the kindling seizures. Therefore, fenofibrate administration might improve the seizures-induced oxidative and neuronal damages by enhancing both the enzymatic and non-enzymatic antioxidant capacity of the brain.

ACKNOWLEDGMENTS

This study was performed in the Department of Physiology and Biophysics, Faculty of Medicine, Baqiyatallah University of Medical Sciences, Tehran, Iran. The authors are cordially appreciating Student Research Committee of Baqiyatallah University of Medical Sciences. The authors declare no conflicts of interest.

REFERENCES

  1. Goldberg EM, Coulter DA. Mechanisms of epileptogenesis: a convergence on neural circuit dysfunction. Nat Rev Neurosci 2013; 14(5):337‒49. doi: 10.1038/nrn3482.
  2. Badawy RA, Harvey AS, Macdonell RA. Cortical hyperexcitability and epileptogenesis: understanding the mechanisms of epilepsy: part 1. J Clin Neurosci 2009; 16(3):355‒65. doi: 10.1016/j.jocn.2008.08.026.
  3. Puttachary S, Sharma S, Stark S, Thippeswamy T. Seizure-induced oxidative stress in temporal lobe epilepsy. Biomed Res Int 2015; 2015:745613. doi: 10.1155/2015/745613.
  4. Folbergrova J, Jesina P, Nuskova H, Houstek J. Antioxidant enzymes in cerebral cortex of immature rats following experimentally-induced seizures: upregulation of mitochondrial MnSOD (SOD2). Int J Dev Neurosci 2013; 31(2):123‒30. doi: 10.1016/j.ijdevneu.2012.11.011.
  5. Cardenas-Rodriguez N, Gonzalez-Trujano ME, Aguirre-Hernandez E, Ruiz-Garcia M, Sampieri A, 3rd, Coballase-Urrutia E, et al. Anticonvulsant and antioxidant effects of Tilia americana var. mexicana and flavonoids constituents in the pentylenetetrazole-induced seizures. Oxid Med Cell Longev 2014; 2014:329172. doi: 10.1155/2014/329172.
  6. Folbergrova J. Oxidative stress in immature brain following experimentally-induced seizures. Physiol Res 2013; 62 Suppl 1:S39‒48.
  7. Aguiar CC, Almeida AB, Araujo PV, de Abreu RN, Chaves EM, do Vale OC, et al. Oxidative stress and epilepsy: literature review. Oxid Med Cell Longev 2012; 2012:795259. doi: 10.1155/2012/795259.
  8. Shin EJ, Jeong JH, Chung YH, Kim WK, Ko KH, Bach JH, et al. Role of oxidative stress in epileptic seizures. Neurochem Int 2011; 59(2):122‒37. doi: 10.1016/j.neuint.2011.03.025.
  9. Liang LP, Jarrett SG, Patel M. Chelation of mitochondrial iron prevents seizure-induced mitochondrial dysfunction and neuronal injury. J Neurosci 2008; 28(45):11550‒6. doi: 10.1523/JNEUROSCI.3016-08.2008.
  10. Patel M. Mitochondrial dysfunction and oxidative stress: cause and consequence of epileptic seizures. Free Radic Biol Med 2004; 37(12):1951‒62. doi: 10.1016/j.freeradbiomed.2004.08.021.
  11. Waldbaum S, Patel M. Mitochondrial dysfunction and oxidative stress: a contributing link to acquired epilepsy? J Bioenerg Biomembr 2010; 42(6):449‒55. doi: 10.1007/s10863-010-9320-9.
  12. Kong Q, Lin CL. Oxidative damage to RNA: mechanisms, consequences, and diseases. Cell Mol Life Sci 2010; 67(11):1817‒29. doi: 10.1007/s00018-010-0277-y.
  13. Menon B, Ramalingam K, Kumar RV. Oxidative stress in patients with epilepsy is independent of antiepileptic drugs. Seizure 2012; 21(10):780‒4. doi: 10.1016/j.seizure.2012.09.003.
  14. Moran EP, Ma JX. Therapeutic effects of PPAR alpha on neuronal death and microvascular impairment. PPAR Res 2015; 2015:595426. doi: 10.1155/2015/595426.
  15. Moran E, Ding L, Wang Z, Cheng R, Chen Q, Moore R, et al. Protective and antioxidant effects of PPARalpha in the ischemic retina. Invest Ophthalmol Vis Sci 2014; 55(7):4568‒76. doi: 10.1167/iovs.13-13127.
  16. Fidaleo M, Fanelli F, Ceru MP, Moreno S. Neuroprotective properties of peroxisome proliferator-activated receptor alpha (PPARalpha) and its lipid ligands. Curr Med Chem 2014; 21(24):2803‒21.
  17. Bordet R, Ouk T, Petrault O, Gele P, Gautier S, Laprais M, et al. PPAR: a new pharmacological target for neuroprotection in stroke and neurodegenerative diseases. Biochem Soc Trans 2006; 34(Pt 6):1341‒6. doi: 10.1042/BST0341341.
  18. Collino M, Aragno M, Mastrocola R, Benetti E, Gallicchio M, Dianzani C, et al. Oxidative stress and inflammatory response evoked by transient cerebral ischemia/reperfusion: effects of the PPAR-alpha agonist WY14643. Free Radic Biol Med 2006; 41(4):579‒89. doi: 10.1016/j.freeradbiomed.2006.04.030.
  19. Chen XR, Besson VC, Palmier B, Garcia Y, Plotkine M, Marchand-Leroux C. Neurological recovery-promoting, anti-inflammatory, and anti-oxidative effects afforded by fenofibrate, a PPAR alpha agonist, in traumatic brain injury. J Neurotrauma 2007; 24(7):1119‒31. doi: 10.1089/neu.2006.0216.
  20. Uppalapati D, Das NR, Gangwal RP, Damre MV, Sangamwar AT, Sharma SS. Neuroprotective potential of peroxisome proliferator activated receptor- alpha agonist in cognitive impairment in Parkinson’s disease: behavioral, biochemical, and PBPK profile. PPAR Res 2014; 2014:753587. doi: 10.1155/2014/753587.
  21. Santos MJ, Quintanilla RA, Toro A, Grandy R, Dinamarca MC, Godoy JA, et al. Peroxisomal proliferation protects from beta-amyloid neurodegeneration. J Biol Chem 2005; 280(49):41057‒68. doi: 10.1074/jbc.M505160200.
  22. Rowley S, Patel M. Mitochondrial involvement and oxidative stress in temporal lobe epilepsy. Free Radic Biol Med 2013; 62:121‒31. doi: 10.1016/j.freeradbiomed.2013.02.002.
  23. Li B, Wang L, Sun Z, Zhou Y, Shao D, Zhao J, et al. The anticonvulsant effects of SR 57227 on pentylenetetrazole-induced seizure in mice. PLoS One 2014; 9(4):e93158. doi: 10.1371/journal.pone.0093158.
  24. Zhu X, Shen K, Bai Y, Zhang A, Xia Z, Chao J, et al. NADPH oxidase activation is required for pentylenetetrazole kindling-induced hippocampal autophagy. Free Radic Biol Med 2016; 94:230‒42. doi: 10.1016/j.freeradbiomed.2016.03.004.
  25. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248‒54.
  26. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969; 27(3):502‒22.
  27. Mohammadi MT, Amini R, Jahanbakhsh Z, Shekarforoush S. Effects of atorvastatin on the hypertension-induced oxidative stress in the rat brain. Iran Biomed J 2013; 17(3):152‒7.
  28. Aebi H. Catalase in vitro. Methods Enzymol 1984; 105:121‒6.
  29. Winterbourn CC, Hawkins RE, Brian M, Carrell RW. The estimation of red cell superoxide dismutase activity. J Lab Clin Med 1975; 85(2):337‒41.
  30. Menon B, Ramalingam K, Kumar RV. Low plasma antioxidant status in patients with epilepsy and the role of antiepileptic drugs on oxidative stress. Ann Indian Acad Neurol 2014; 17(4):398‒404. doi: 10.4103/0972-2327.144008.
  31. Rauca C, Zerbe R, Jantze H. Formation of free hydroxyl radicals after pentylenetetrazol-induced seizure and kindling. Brain Res 1999; 847(2):347‒51.
  32. Gualtieri CT, Johnson LG. Comparative neurocognitive effects of 5 psychotropic anticonvulsants and lithium. MedGenMed 2006; 8(3):46.
  33. Lin Y, Han Y, Xu J, Cao L, Gao J, Xie N, et al. Mitochondrial DNA damage and the involvement of antioxidant defense and repair system in hippocampi of rats with chronic seizures. Cell Mol Neurobiol 2010; 30(6):947‒54. doi: 10.1007/s10571-010-9524-x.
  34. Mari M, Morales A, Colell A, Garcia-Ruiz C, Fernandez-Checa JC. Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal 2009; 11(11):2685‒700. doi: 10.1089/ARS.2009.2695.
  35. Morris G, Anderson G, Dean O, Berk M, Galecki P, Martin-Subero M, et al. The glutathione system: a new drug target in neuroimmune disorders. Mol Neurobiol 2014; 50(3):1059‒84. doi: 10.1007/s12035-014-8705-x.
  36. Levy DI, Sucher NJ, Lipton SA. Glutathione prevents N-methyl-D-aspartate receptor-mediated neurotoxicity. Neuroreport 1991; 2(6):345‒7.
  37. Prigol M, Bruning CA, Nogueira CW, Zeni G. The role of the glutathione system in seizures induced by diphenyl diselenide in rat pups. Chem Biol Interact 2011; 193(1):65‒70. doi: 10.1016/j.cbi.2011.05.004.
  38. Reid M, Jahoor F. Glutathione in disease. Curr Opin Clin Nutr Metab Care 2001; 4(1):65‒71.
  39. Ahmad M, Abu-Taweel GM, Aboshaiqah AE, Ajarem JS. The effects of quinacrine, proglumide, and pentoxifylline on seizure activity, cognitive deficit, and oxidative stress in rat lithium-pilocarpine model of status epilepticus. Oxid Med Cell Longev 2014; 2014:630509. doi: 10.1155/2014/630509.
  40. Rauca C, Wiswedel I, Zerbe R, Keilhoff G, Krug M. The role of superoxide dismutase and alpha-tocopherol in the development of seizures and kindling induced by pentylenetetrazol – influence of the radical scavenger alpha-phenyl-N-tert-butyl nitrone. Brain Res 2004; 1009(1‒2):203‒12. doi: 10.1016/j.brainres.2004.01.082.
  41. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39(1):44‒84. doi: 10.1016/j.biocel.2006.07.001.