International Journal of Neuroscience, 2014; 124(10): 771–776 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0020-7454 print / 1543-5245 online DOI: 10.3109/00207454.2013.879581
RESEARCH ARTICLE
Effects of ebselen on ischemia/reperfusion injury in rat brain M. Aras,1 M. Altas¸,1 S. Meydan,2 E. Nacar,3 M. Karcıo˘glu,4 K. T. Ulutas¸,5 and Y. Serarslan1 1
Department of Neurosurgery, Tayfur Ata S¨okmen Medical Faculty, Mustafa Kemal University, Hatay, Turkey; ˙ Department of Anatomy, Medical Faculty, Bezmialem Vakif University, Istanbul, Turkey; 3 School of Healthy Sciences, Department of Histology and Embryology, Mustafa Kemal University, Hatay, Turkey; 4 Department of Anesthesiology and Reanimation, Tayfur Ata S¨okmen Medical Faculty, Mustafa Kemal University, Hatay, Turkey; 5 Department of Clinical Biochemistry, Kadirli State Hospital, Osmaniye, Turkey
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Aim: Interruption of blood flow may result in considerable tissue damage via ischemia/reperfusion (I/R) injuryinduced oxidative stress in brain tissues. The aim of the present study was to investigate the effects of Ebselen treatment in short-term global brain I/R injury in rats. Material and Methods: The study was carried out on 27 Wistar-albino rats, divided into three groups including Sham group (n = 11), I/R group (n = 8) and I/R+Ebselen group (n = 8). Results: Malondialdehyde (MDA) levels were significantly increased in I/R group in comparison with the Sham group and I/R+Ebselen group (p < 0.001 and p < 0.01). Superoxide dismutase (SOD) activity was significantly lower in I/R group in comparison to both Sham (p < 0.001) and I/R+Ebselen (p < 0.01) groups. Similarly, SOD activity was decreased in I/R+Ebselen group when compared with Sham group (p < 0.001). Sham and I/R groups were similar in terms of nitric oxide (NO) levels. In contrast, the NO level was lower in I/R+Ebselen group when compared with Sham (p < 0.001) and I/R (p < 0.01) groups. There was no significant difference among the groups in terms of glutathione peroxidase and catalase activities. In histopathological examination, the brain tissues of rats that received Ebselen showed morphological improvement. Conclusion: Ebselen has neuron-protective effects due to its antioxidant properties as shown by the decrease in MDA overproduction, increase in SOD activity and the histological improvement after administration of Ebselen to I/R in brain tissue. KEYWORDS: Ebselen, brain injury, ischemia, reperfusion
Introduction A circulatory arrest causing any interference in blood flow may affect the brain that results in considerable tissue damage via ischemia/reperfusion injury [1]. Excitatory amino acid releasing, peri-infarct waves of depolarization, specific gene expression, and oxidative stress may all contribute to the development of ischemic damage [2]. Oxidative stress has been especially shown to play a critical role in ischemia/reperfusion-induced tissue damage through increased levels of reactive species leading to lipid peroxidation (LPO) and DNA damage [3]. Received 30 July 2013; revised 27 December 2013; accepted 27 December 2013. Correspondence: Mustafa Aras, M.D., Department of Neurosurgery, Tayfur ¨ Ata Sokmen Medical Faculty, Mustafa Kemal University, 31700 Hatay, Turkey. Tel: +90 32622 91000. Fax: +90 32624 55654; Mobile Phone: +90 50650 62322. E-mail: [email protected].
These reactive species are known as two subgroups including reactive oxygen species (ROS) and reactive nitrogen species. Perturbation in the production and/or metabolism of these two reactive species can have pathologic consequences for brain tissue [4]. Enhancement of antioxidant activities in brain tissues may be potentially beneficial for balancing emerged oxidant molecules and effecting neuronal recovery from I/R injury [5–7]. Determination of oxidative stress in cerebral ischemic damage focused on measuring the rate of consumption of endogenous protective molecules or enzymes and also formation of by-products of LPO [6]. Recent laboratory studies established that some antioxidants which scavenge free radicals may provide some protection from the cerebral infarction resulting from experimental cerebral ischemia [3]. Ebselen (2phenyl-1,2-benzisoselenazol-3(2H)-one) is a synthetic seleno-organic compound that has been developed as a free radical scavenger [8]. Clinical studies of the effects of Ebselen as a free radical scavenger indicated 771
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that the scavenging activity can be explained by the unique glutathione peroxidase (GSH-Px)-like activity [9,10]. However, no data are available on the actions of antioxidants in brain tissue during I/R injury, against which Ebselen supports neuroprotection from this injury. Therefore, the aim of the present study was to investigate the effect of Ebselen treatment against shortterm global brain I/R injury in rats with biochemical and histological analysis.
Materials and methods
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Animals and experimental procedures The animals were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by Mustafa Kemal University, Animal Ethical Committee. Male Wistar rats weighing 200–250 g were housed in polycarbonate cages and given the standard laboratory chow and water at 24◦ C with 42 ± 5% relative humidity in a 12-h–12-h light–dark cycle. Body temperature was maintained around 37 ± 5◦ C throughout the surgical procedure.
Experimental design Rats were divided into three groups; Sham (n = 11), ischemia/reperfusion (I/R) (n = 8) (ischemia was induced by bilateral occlusion of the carotid arteries for 20 min and reperfusion was achieved by releasing the occlusion to restore the circulation for 20 min), and I/R+Ebselen (n = 8) (100 mg/kg/ for 2 h ago I/R).
Induction of cerebral ischemia Rats were anesthetized with ketamine hydrochloride (75 mg/kg) and Xylazin (8 mg/kg). Two common carotid arteries were exposed through lateral incisions and separated from the nervous vagus. Ischemia was induced by bilateral clamping of the common carotid arteries for 20 min. Reperfusion was achieved by declamping the arteries, then the circulation was restored for 20 min. Ebselen ampoules were obtained from Roche and administered intraperitoneally.
Tissue samples Brain tissues were rapidly excised, and right brain was used for microscopic examination. Left ones were used for biochemical analyses. The tissues were weighed and homogenized in 4 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) containing 0.50 mL/L Triton X-100 with a homogenizer (hKA Ultra-Turrax T 25 Basic)
for 2 min at 3000 rpm at +4◦ C. Tissue homogenates were centrifuged at 5000 g for 60 min to remove debris, and the clear supernatant fluids were separated and kept at −40◦ C until the enzyme activity measurements were performed (about a week later).
Biochemical determination The brain tissue samples were stored at −70◦ C until tissue analysis of MDA levels, NO, superoxide dismutase (SOD) and GSH-Px activities. Brain tissues were homogenized (for 2 min at 5000 rpm) in four volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) by using a glass Teflon homogenizer (Ultra Turrax hKA T10 Basic, Germany) for MDA, NO and protein measurement. The homogenates were then centrifuged at 5000 g for 60 min to remove debris and obtain supernatant. Supernatant fluids were collected and used for measuring GSH-Px activities and protein concentration. The supernatant solutions were mixed with an equal volume of an ethanol/chloroform mixture (5/3, volume per volume v/v). After centrifugation at 5000 g for 30 min, the clear upper layer (the ethanol phase) was collected and used in the analysis of SOD activity and protein assays. All preparation procedures were carried out at +4◦ C.
Determination of malondialdehyde level The tissue Malondialdehyde (MDA) level was determined using a method from Esterbauer and Cheeseman [11], based on its reaction with Thiobarbituric acid (TBA) at 90–100◦ C. In the TBA test reaction, MDA and TBA react to produce a pink pigment with an absorption maximum at 532 nm at pH 2–3 and at 90◦ C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was centrifuged and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling-water-bath for 10 min. After cooling, the absorbance was read at 532 nm. Results were expressed as nmol/g of wet tissue, by reference to a standard curve prepared from measurements made with a standard solution (1,1,3,3-tetramethoxypropane).
Determination of nitric oxide level in tissue samples The method for tissue nitric oxide levels was based on the Griess reaction. Samples were initially deproteinized with Somogy reagent. It was measured by spectrophotometry at 545 nm after conversion of nitrate to nitrite by coppered cadmium granules. A standard curve was established with a set of serial dilutions (10–8–10–3 mol/L) of sodium nitrite. Linear regression was done by using the peak area from the standards. The International Journal of Neuroscience
Effects of ebselen
resulting equation was used to calculate the unknown sample concentrations. The NO levels were expressed as μmol/g wet tissue.
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Determination of superoxide dismutase activity Total (Cu/Zn and Mn) SOD (EC 1.15.1.1) activity was determined based on the method of Sun et al. [12]. The principle of the method is based on the inhibition of Nitro Blue Tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant, after 1 mL of an ethanol–chloroform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the NBT reduction rate. The SOD activity was expressed as UI/g protein.
Determination of glutathione peroxidase activity Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured. The enzyme reaction in the tube containing NADPH, reduced glutathione (GSH) and sodium azide, and glutathione reductase was initiated by the addition of H2 O2 . The change in absorbance at 340 nm was evaluated by a spectrophotometer. Activity was expressed as UI/g protein.
Determination of catalase activity To analyze Catalase (CAT) activity, the brain tissue was sonicated in 50 mmol/L phosphate buffer (pH 7.0), and the resulting suspension was centrifuged at 3000 g for 10 min. The supernatant was used for enzyme assay. CAT activity was measured by the rate of decrease in hydrogen peroxide absorbance at 240 nm.
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Histopathological examination Brain hippocampus samples were fixed in 10% neutral buffered formalin. After dehydrating with graded alcohol series, tissues were then embedded in paraffin. Five micrometer thick transverse sections were cut and stained with hematoxylin and eosin for histological evaluation. Sections were examined and photographed with Olympus DP20 camera attached-Olympus CX41 photomicroscope for histopathological changes.
Statistical analysis Data were analyzed by using SPSSR 15 for Windows. Distributions of the groups were tested using the onesample Kolmogrov–Smirnov test. The measured parameters for all groups were normally distributed, and the parametric statistical method was used to analyze the data. One-way ANOVA test was performed, and post hoc multiple comparisons were done with LSD. Results were presented as mean ± standard error of means. p-Value lower than 0.05 was regarded as statistically significant.
Results Antioxidant enzyme activities Antioxidant enzyme activities of the groups are presented in Table 1. MDA levels were significantly increased in I/R group in comparison with the Sham group (p < 0.001) and I/R+Ebselen group (p < 0.01). SOD activity was highly decreased in I/R group in comparison to both Sham (p < 0.001) and I/R+Ebselen (p < 0.01) groups. Similarly, SOD activity was significantly lower in I/R+Ebselen group when compared with the Sham group (p < 0.001). However, the decrease in SOD activity in I/R+Ebselen group was not high as in I/R group. NO levels were similar in Sham and I/R groups. However, NO level in I/R+Ebselen group was significantly lower than that in Sham (p < 0.001)
Table 1. Analyze results of subgroups including control, IR and Ebselen.
CONTROL IR IR+E
MDA (nmol/g prt)
SOD (U/mg prt)
NO (μmol/g prt)
GSH-Px (U/g prt)
CAT (k/g prt)
23.4 ± 4.1 56.5 ± 8.5a 30.1 ± 5.6b,d
0.164 ± 0.038 0.047 ± 0.011a 0.105 ± 0.015c,d
0.026 ± 0.005 0.023 ± 0.003 0.016 ± 0.003c,d
4.84 ± 0.35 4.97 ± 0.53 4.87 ± 0.39
0.051 ± 0.009 0.045 ± 0.011 0.049 ± 0.012
All result were given as mean ± standard deviation. Abbreviations: IR, ischemia/reperfusion; E, Ebselen; Prt: protein. a p < 0.001 was found when compared with control. b p < 0.05 was found when compared with control. c p < 0.01 was found when compared with control. d p < 0.01 was found when compared with IR. C
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Figure 1. Normal neuronal structure in control group. Figure 3. Vacuolization (→) in I/R group.
and I/R (p < 0.01) groups. There was no significant difference between the glutathione peroxidase and catalase activities of the groups.
Histopathological results Microscopic examination revealed normal neuron and parenchyma structure in control group (Figure 1). In IR group, vascular and neuronal structures showed significant changes like shrunken neurons, eosinophilic cell bodies with damaged microvessels throughout sections (Figure 2). Vacuolization in neutrophil was also observed (Figure 3). In Ebselen group, microvessels and neurons were better preserved than IR group (Figure 4).
Discussion Recent studies have indicated that Ebselen has several pharmacological actions that can potentially account for its beneficial effects on the balance between oxidation and reduction reactions [7,11,13–15]. According to our literature search, this study is showing the neuroprotective effects of Ebselen in a rat model after a short-term global cerebral ischemia. Eosinophilic neurons (EN) are the hallmarks of ischemic encephalopathy [16]. Sun et al. [12] reported that intense EN areas transform into infarct areas when compared to areas of low-density EN. In our study, IR group revealed a large number of EN compared to Ebselen and control group. We saw shrunken neurons with pyknotic nuclei as the sign
Figure 2. Significant cell body shrinkage (→), increased
eosinophily (arrowhead) and damaged microvessel (∗ ) in I/R group.
Figure 4. Preserved neurons and improved neurophil in Ebselen group.
International Journal of Neuroscience
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Effects of ebselen
of neuron damage. The beneficial effects of Ebselen in brain I/R injury were shown previously. Yamagata et al. [7] reported decreased neuronal death in Ebselen administrated rats with brain I/R injury. Seo et al. [15] observed the neuroprotective effects of ebselen against ischemic brain damage with immunohistochemical methods. We also noticed that Ebselen improved neuron and neutrophil structure. Some recent studies regarding the therapeutic effects of Ebselen also suggest that Ebselen has beneficial effects on the neurological outcome of patients with cerebral infarct [9,10,14,15,17,18]. It was demonstrated that Ebselen has some protective effects on neurons during ischemic insults. The exact neuroprotective biochemical mechanism of ebselen is remaining unclear. ROS released following I/R leads to peroxidation of phospholipids, which results in the alteration in the cell membrane structure [19,20]. The consumption of endogenous protective molecules and formation of LPO products, such as MDA, determines tissue resistance against oxidative injury [21]. In several studies on global cerebral ischemia models, MDA was demonstrated to be increased in response to the ischemic condition. This increase was attributed to the reperfusion injury in rat brain [13,15]. In animals that were treated with Ebselen during ischemia reperfusion (I/R + Ebselen group), the balance between oxidant and antioxidant systems changed in favor of antioxidant systems. Similar to recent studies, MDA levels were reported to be decreased in Ebselen-treated animals in comparison to I/R group. A number of experimental studies on ischemia and reperfusion have recently documented the beneficial effect of SOD, an enzymatic scavenger of oxygen radicals, on cell viability. SOD and glutathione peroxidase are endogenous antioxidants, which function in the prevention of oxidative injury [22]. Toyoda and Lee [23] reported that the increase in SOD activity 6–24 h after transient focal ischemia was confined to the cortical penumbra with no increase in the ischemic core. Some studies reported a biphasic response to focal ischemia with initial reductions in SOD, GSH-Px, and catalase activities in the first hours after ischemic insult followed by increases around 72 h post insult [24–26]. Therefore, any upregulated enhancement of antioxidant activity in brain tissues may be potentially beneficial for neuronal recovery from I/R injury. In the current study, SOD activities in rats were dramatically decreased following ischemic insult. By Ebselen support, lowering effects of IR on SOD were relatively balanced and SOD indicated increase in comparison to I/R group (p < 0.01). These results are completely consistent with previously reported studies performed with other antioxidant agents. In the current study, GSH-Px activity was not altered neither by I/R nor Ebselen. It is thought that the preserved SOD activity in I/R+ebselen is not a direct effect of ebselen on C
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SOD. Ebselen inhibited oxidative stress as a result of glutathione peroxidase mimetic effect, then almost neurons are survived in I/R+ebselen. Therefore, the SOD activity of I/R+ebselen in the tissue homogenate showed similar to control. Similarly, catalase activity was unchanged after I/R and Ebselen. Although the results regarding GSH-Px was not compatible with the literature, the results were, however, compatible with our previous study investigated effects of ceftriaxone on ischemia/reperfusion injury in rat brain [27]. There are controversial results about the levels of GSH-Px activity in cerebral I/R injuries. We found no significant changes among groups. One of the reasons could be the GSH-Px-like activity of Ebselen in Ebselen + I/R group. In I/R group, the oxidative stress could be blocked by other antioxidant enzymes. Since brain has low GSH-Px activity, the changes in I/R group could be insignificant. Nitric oxide has been shown to have antioxidant properties not related to alterations in neutrophil migration or adhesion, which may account for much of the protective effects of NO during I/R injury [28,29]. Antioxidant properties of NO in the tissues may be related to the reduction of superoxide anion-mediated tissue toxicity by NO [30]. Thereby, after I/R injury NO reduces the generation of ROS, which can contribute to cellular injury, necrosis, and apoptosis. Kawachi et al. [31] observed eNOS-deficient mice subjected to a period of ischemia and found that eNOS-derived NO modulates nuclear neutrophils. This finding shows that NO may be involved enhanced reactive oxygen speciesdependent injury. In the current study, there was no significant alteration in NO levels during I/R injury. NO levels were, however, observed lower in I/R + Ebselen group in comparison to Sham and I/R groups. The decrease in I/R + Ebselen group may be related to the consumption of NO, which is triggered by Ebselen.
Conclusion The study showed that Ebselen has neuron-protective effects due to its anti-oxidant properties as shown by the decrease in MDA overproduction and the increased activity of SOD enzyme. However, additional studies including different doses of Ebselen should be conducted to clear consumption of NO levels. Thereby, exact mechanisms underlying this beneficial effect of Ebselen over I/R injury in brain tissue should be investigated by further studies.
Declaration of Interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.
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16. Crain BJ, Evenson DA, Polsky K, Nadler JV. Electron microscopic study of the gerbil dentate gyrus after transient forebrain ischemia. Acta Neuropathol 1990;79(4):409–17. 17. Wang YH, Wang WY, Chang CC, et al. Taxifolin ameliorates cerebral ischemia-reperfusion injury in rats through its antioxidative effect and modulation of NF-kappa B activation. J Biomed Sci 2006;13(1):127–41. 18. Zheng YQ, Liu JX, Wang JN, Xu L. Effects of crocin on reperfusion-induced oxidative/nitrative injury to cerebral microvessels after global cerebral ischemia. Brain Res 2007; 1138:86–94. 19. Aksoy Y, Yapanoglu T, Aksoy H, et al. Effects of endurance training on antioxidant defense mechanisms and lipid peroxidation in testis of rats. Arch Androl 2006;52(4):319–23. 20. Bukan N, Sancak B, Yavuz O, et al. Lipid peroxidation and scavenging enzyme levels in the liver of streptozotocin-induced diabetic rats. Indian J Biochem Biophys 2003;40(6):447–50. 21. Fujii J, Myint T, Okado A, et al. Oxidative stress caused by glycation of Cu,Zn-superoxide dismutase and its effects on intracellular components. Nephrol Dial Transplant 1996;11(Suppl 5):34–40. 22. Choi BH. Oxygen, antioxidants and brain dysfunction. Yonsei Med J 1993;34(1):1–10. 23. Toyoda T, Lee KS. Differential induction of superoxide dismutase in core and penumbra regions after transient focal ischemia in the rat neocortex. Neurosci Lett 1997;235(1–2):29–32. 24. Akhtar M, Pillai K, Vohora D. Effect of thioperamide on oxidative stress markers in middle cerebral artery occlusion model of focal cerebral ischemia in rats. Hum Exp Toxicol 2008;27(10):761–7. 25. Choi-Kwon S, Park KA, Lee HJ, et al. Temporal changes in cerebral antioxidant enzyme activities after ischemia and reperfusion in a rat focal brain ischemia model: effect of dietary fish oil. Brain Res Dev Brain Res 2004;152(1):11–8. 26. Saleem S, Ahmad M, Ahmad AS, et al. Behavioral and histologic neuroprotection of aqueous garlic extract after reversible focal cerebral ischemia. J Med Food 2006;9(4):537–44. 27. Altas M, Meydan S, Aras M, et al. Effects of ceftriaxone on ischemia/reperfusion injury in rat brain. J Clin Neurosci 2013;20(3):457–61. 28. Coeroli L, Renolleau S, Arnaud S, et al. Nitric oxide production and perivascular tyrosine nitration following focal ischemia in neonatal rat. J Neurochem 1998;70(6):2516–25. 29. Seif-el-Nasr M, Fahim AT. Antioxidant effect of N omeganitro-L-arginine methyl ester (L-NAME) on global cerebral ischemia in a rat model. Arzneimittel-Forschung 2001; 51(8):628–32. 30. Gaur V, Aggarwal A, Kumar A. Possible nitric oxide mechanism in the protective effect of hesperidin against ischemic reperfusion cerebral injury in rats. Indian J Exp Biol 2011;49(8):609–18. 31. Kawachi S, Cockrell A, Laroux FS, et al. Role of inducible nitric oxide synthase in the regulation of VCAM-1 expression in gut inflammation. Am J Physiol 1999;277(3 Pt 1):G572–6.
International Journal of Neuroscience
RESEARCH ARTICLE
Effects of ebselen on ischemia/reperfusion injury in rat brain M. Aras,1 M. Altas¸,1 S. Meydan,2 E. Nacar,3 M. Karcıo˘glu,4 K. T. Ulutas¸,5 and Y. Serarslan1 1
Department of Neurosurgery, Tayfur Ata S¨okmen Medical Faculty, Mustafa Kemal University, Hatay, Turkey; ˙ Department of Anatomy, Medical Faculty, Bezmialem Vakif University, Istanbul, Turkey; 3 School of Healthy Sciences, Department of Histology and Embryology, Mustafa Kemal University, Hatay, Turkey; 4 Department of Anesthesiology and Reanimation, Tayfur Ata S¨okmen Medical Faculty, Mustafa Kemal University, Hatay, Turkey; 5 Department of Clinical Biochemistry, Kadirli State Hospital, Osmaniye, Turkey
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Aim: Interruption of blood flow may result in considerable tissue damage via ischemia/reperfusion (I/R) injuryinduced oxidative stress in brain tissues. The aim of the present study was to investigate the effects of Ebselen treatment in short-term global brain I/R injury in rats. Material and Methods: The study was carried out on 27 Wistar-albino rats, divided into three groups including Sham group (n = 11), I/R group (n = 8) and I/R+Ebselen group (n = 8). Results: Malondialdehyde (MDA) levels were significantly increased in I/R group in comparison with the Sham group and I/R+Ebselen group (p < 0.001 and p < 0.01). Superoxide dismutase (SOD) activity was significantly lower in I/R group in comparison to both Sham (p < 0.001) and I/R+Ebselen (p < 0.01) groups. Similarly, SOD activity was decreased in I/R+Ebselen group when compared with Sham group (p < 0.001). Sham and I/R groups were similar in terms of nitric oxide (NO) levels. In contrast, the NO level was lower in I/R+Ebselen group when compared with Sham (p < 0.001) and I/R (p < 0.01) groups. There was no significant difference among the groups in terms of glutathione peroxidase and catalase activities. In histopathological examination, the brain tissues of rats that received Ebselen showed morphological improvement. Conclusion: Ebselen has neuron-protective effects due to its antioxidant properties as shown by the decrease in MDA overproduction, increase in SOD activity and the histological improvement after administration of Ebselen to I/R in brain tissue. KEYWORDS: Ebselen, brain injury, ischemia, reperfusion
Introduction A circulatory arrest causing any interference in blood flow may affect the brain that results in considerable tissue damage via ischemia/reperfusion injury [1]. Excitatory amino acid releasing, peri-infarct waves of depolarization, specific gene expression, and oxidative stress may all contribute to the development of ischemic damage [2]. Oxidative stress has been especially shown to play a critical role in ischemia/reperfusion-induced tissue damage through increased levels of reactive species leading to lipid peroxidation (LPO) and DNA damage [3]. Received 30 July 2013; revised 27 December 2013; accepted 27 December 2013. Correspondence: Mustafa Aras, M.D., Department of Neurosurgery, Tayfur ¨ Ata Sokmen Medical Faculty, Mustafa Kemal University, 31700 Hatay, Turkey. Tel: +90 32622 91000. Fax: +90 32624 55654; Mobile Phone: +90 50650 62322. E-mail: [email protected].
These reactive species are known as two subgroups including reactive oxygen species (ROS) and reactive nitrogen species. Perturbation in the production and/or metabolism of these two reactive species can have pathologic consequences for brain tissue [4]. Enhancement of antioxidant activities in brain tissues may be potentially beneficial for balancing emerged oxidant molecules and effecting neuronal recovery from I/R injury [5–7]. Determination of oxidative stress in cerebral ischemic damage focused on measuring the rate of consumption of endogenous protective molecules or enzymes and also formation of by-products of LPO [6]. Recent laboratory studies established that some antioxidants which scavenge free radicals may provide some protection from the cerebral infarction resulting from experimental cerebral ischemia [3]. Ebselen (2phenyl-1,2-benzisoselenazol-3(2H)-one) is a synthetic seleno-organic compound that has been developed as a free radical scavenger [8]. Clinical studies of the effects of Ebselen as a free radical scavenger indicated 771
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M. Aras et al.
that the scavenging activity can be explained by the unique glutathione peroxidase (GSH-Px)-like activity [9,10]. However, no data are available on the actions of antioxidants in brain tissue during I/R injury, against which Ebselen supports neuroprotection from this injury. Therefore, the aim of the present study was to investigate the effect of Ebselen treatment against shortterm global brain I/R injury in rats with biochemical and histological analysis.
Materials and methods
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Animals and experimental procedures The animals were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals by Mustafa Kemal University, Animal Ethical Committee. Male Wistar rats weighing 200–250 g were housed in polycarbonate cages and given the standard laboratory chow and water at 24◦ C with 42 ± 5% relative humidity in a 12-h–12-h light–dark cycle. Body temperature was maintained around 37 ± 5◦ C throughout the surgical procedure.
Experimental design Rats were divided into three groups; Sham (n = 11), ischemia/reperfusion (I/R) (n = 8) (ischemia was induced by bilateral occlusion of the carotid arteries for 20 min and reperfusion was achieved by releasing the occlusion to restore the circulation for 20 min), and I/R+Ebselen (n = 8) (100 mg/kg/ for 2 h ago I/R).
Induction of cerebral ischemia Rats were anesthetized with ketamine hydrochloride (75 mg/kg) and Xylazin (8 mg/kg). Two common carotid arteries were exposed through lateral incisions and separated from the nervous vagus. Ischemia was induced by bilateral clamping of the common carotid arteries for 20 min. Reperfusion was achieved by declamping the arteries, then the circulation was restored for 20 min. Ebselen ampoules were obtained from Roche and administered intraperitoneally.
Tissue samples Brain tissues were rapidly excised, and right brain was used for microscopic examination. Left ones were used for biochemical analyses. The tissues were weighed and homogenized in 4 volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) containing 0.50 mL/L Triton X-100 with a homogenizer (hKA Ultra-Turrax T 25 Basic)
for 2 min at 3000 rpm at +4◦ C. Tissue homogenates were centrifuged at 5000 g for 60 min to remove debris, and the clear supernatant fluids were separated and kept at −40◦ C until the enzyme activity measurements were performed (about a week later).
Biochemical determination The brain tissue samples were stored at −70◦ C until tissue analysis of MDA levels, NO, superoxide dismutase (SOD) and GSH-Px activities. Brain tissues were homogenized (for 2 min at 5000 rpm) in four volumes of ice-cold Tris-HCl buffer (50 mM, pH 7.4) by using a glass Teflon homogenizer (Ultra Turrax hKA T10 Basic, Germany) for MDA, NO and protein measurement. The homogenates were then centrifuged at 5000 g for 60 min to remove debris and obtain supernatant. Supernatant fluids were collected and used for measuring GSH-Px activities and protein concentration. The supernatant solutions were mixed with an equal volume of an ethanol/chloroform mixture (5/3, volume per volume v/v). After centrifugation at 5000 g for 30 min, the clear upper layer (the ethanol phase) was collected and used in the analysis of SOD activity and protein assays. All preparation procedures were carried out at +4◦ C.
Determination of malondialdehyde level The tissue Malondialdehyde (MDA) level was determined using a method from Esterbauer and Cheeseman [11], based on its reaction with Thiobarbituric acid (TBA) at 90–100◦ C. In the TBA test reaction, MDA and TBA react to produce a pink pigment with an absorption maximum at 532 nm at pH 2–3 and at 90◦ C for 15 min. The sample was mixed with 2 volumes of cold 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was centrifuged and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling-water-bath for 10 min. After cooling, the absorbance was read at 532 nm. Results were expressed as nmol/g of wet tissue, by reference to a standard curve prepared from measurements made with a standard solution (1,1,3,3-tetramethoxypropane).
Determination of nitric oxide level in tissue samples The method for tissue nitric oxide levels was based on the Griess reaction. Samples were initially deproteinized with Somogy reagent. It was measured by spectrophotometry at 545 nm after conversion of nitrate to nitrite by coppered cadmium granules. A standard curve was established with a set of serial dilutions (10–8–10–3 mol/L) of sodium nitrite. Linear regression was done by using the peak area from the standards. The International Journal of Neuroscience
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resulting equation was used to calculate the unknown sample concentrations. The NO levels were expressed as μmol/g wet tissue.
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Determination of superoxide dismutase activity Total (Cu/Zn and Mn) SOD (EC 1.15.1.1) activity was determined based on the method of Sun et al. [12]. The principle of the method is based on the inhibition of Nitro Blue Tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the supernatant, after 1 mL of an ethanol–chloroform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the amount of enzyme causing 50% inhibition in the NBT reduction rate. The SOD activity was expressed as UI/g protein.
Determination of glutathione peroxidase activity Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured. The enzyme reaction in the tube containing NADPH, reduced glutathione (GSH) and sodium azide, and glutathione reductase was initiated by the addition of H2 O2 . The change in absorbance at 340 nm was evaluated by a spectrophotometer. Activity was expressed as UI/g protein.
Determination of catalase activity To analyze Catalase (CAT) activity, the brain tissue was sonicated in 50 mmol/L phosphate buffer (pH 7.0), and the resulting suspension was centrifuged at 3000 g for 10 min. The supernatant was used for enzyme assay. CAT activity was measured by the rate of decrease in hydrogen peroxide absorbance at 240 nm.
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Histopathological examination Brain hippocampus samples were fixed in 10% neutral buffered formalin. After dehydrating with graded alcohol series, tissues were then embedded in paraffin. Five micrometer thick transverse sections were cut and stained with hematoxylin and eosin for histological evaluation. Sections were examined and photographed with Olympus DP20 camera attached-Olympus CX41 photomicroscope for histopathological changes.
Statistical analysis Data were analyzed by using SPSSR 15 for Windows. Distributions of the groups were tested using the onesample Kolmogrov–Smirnov test. The measured parameters for all groups were normally distributed, and the parametric statistical method was used to analyze the data. One-way ANOVA test was performed, and post hoc multiple comparisons were done with LSD. Results were presented as mean ± standard error of means. p-Value lower than 0.05 was regarded as statistically significant.
Results Antioxidant enzyme activities Antioxidant enzyme activities of the groups are presented in Table 1. MDA levels were significantly increased in I/R group in comparison with the Sham group (p < 0.001) and I/R+Ebselen group (p < 0.01). SOD activity was highly decreased in I/R group in comparison to both Sham (p < 0.001) and I/R+Ebselen (p < 0.01) groups. Similarly, SOD activity was significantly lower in I/R+Ebselen group when compared with the Sham group (p < 0.001). However, the decrease in SOD activity in I/R+Ebselen group was not high as in I/R group. NO levels were similar in Sham and I/R groups. However, NO level in I/R+Ebselen group was significantly lower than that in Sham (p < 0.001)
Table 1. Analyze results of subgroups including control, IR and Ebselen.
CONTROL IR IR+E
MDA (nmol/g prt)
SOD (U/mg prt)
NO (μmol/g prt)
GSH-Px (U/g prt)
CAT (k/g prt)
23.4 ± 4.1 56.5 ± 8.5a 30.1 ± 5.6b,d
0.164 ± 0.038 0.047 ± 0.011a 0.105 ± 0.015c,d
0.026 ± 0.005 0.023 ± 0.003 0.016 ± 0.003c,d
4.84 ± 0.35 4.97 ± 0.53 4.87 ± 0.39
0.051 ± 0.009 0.045 ± 0.011 0.049 ± 0.012
All result were given as mean ± standard deviation. Abbreviations: IR, ischemia/reperfusion; E, Ebselen; Prt: protein. a p < 0.001 was found when compared with control. b p < 0.05 was found when compared with control. c p < 0.01 was found when compared with control. d p < 0.01 was found when compared with IR. C
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Figure 1. Normal neuronal structure in control group. Figure 3. Vacuolization (→) in I/R group.
and I/R (p < 0.01) groups. There was no significant difference between the glutathione peroxidase and catalase activities of the groups.
Histopathological results Microscopic examination revealed normal neuron and parenchyma structure in control group (Figure 1). In IR group, vascular and neuronal structures showed significant changes like shrunken neurons, eosinophilic cell bodies with damaged microvessels throughout sections (Figure 2). Vacuolization in neutrophil was also observed (Figure 3). In Ebselen group, microvessels and neurons were better preserved than IR group (Figure 4).
Discussion Recent studies have indicated that Ebselen has several pharmacological actions that can potentially account for its beneficial effects on the balance between oxidation and reduction reactions [7,11,13–15]. According to our literature search, this study is showing the neuroprotective effects of Ebselen in a rat model after a short-term global cerebral ischemia. Eosinophilic neurons (EN) are the hallmarks of ischemic encephalopathy [16]. Sun et al. [12] reported that intense EN areas transform into infarct areas when compared to areas of low-density EN. In our study, IR group revealed a large number of EN compared to Ebselen and control group. We saw shrunken neurons with pyknotic nuclei as the sign
Figure 2. Significant cell body shrinkage (→), increased
eosinophily (arrowhead) and damaged microvessel (∗ ) in I/R group.
Figure 4. Preserved neurons and improved neurophil in Ebselen group.
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of neuron damage. The beneficial effects of Ebselen in brain I/R injury were shown previously. Yamagata et al. [7] reported decreased neuronal death in Ebselen administrated rats with brain I/R injury. Seo et al. [15] observed the neuroprotective effects of ebselen against ischemic brain damage with immunohistochemical methods. We also noticed that Ebselen improved neuron and neutrophil structure. Some recent studies regarding the therapeutic effects of Ebselen also suggest that Ebselen has beneficial effects on the neurological outcome of patients with cerebral infarct [9,10,14,15,17,18]. It was demonstrated that Ebselen has some protective effects on neurons during ischemic insults. The exact neuroprotective biochemical mechanism of ebselen is remaining unclear. ROS released following I/R leads to peroxidation of phospholipids, which results in the alteration in the cell membrane structure [19,20]. The consumption of endogenous protective molecules and formation of LPO products, such as MDA, determines tissue resistance against oxidative injury [21]. In several studies on global cerebral ischemia models, MDA was demonstrated to be increased in response to the ischemic condition. This increase was attributed to the reperfusion injury in rat brain [13,15]. In animals that were treated with Ebselen during ischemia reperfusion (I/R + Ebselen group), the balance between oxidant and antioxidant systems changed in favor of antioxidant systems. Similar to recent studies, MDA levels were reported to be decreased in Ebselen-treated animals in comparison to I/R group. A number of experimental studies on ischemia and reperfusion have recently documented the beneficial effect of SOD, an enzymatic scavenger of oxygen radicals, on cell viability. SOD and glutathione peroxidase are endogenous antioxidants, which function in the prevention of oxidative injury [22]. Toyoda and Lee [23] reported that the increase in SOD activity 6–24 h after transient focal ischemia was confined to the cortical penumbra with no increase in the ischemic core. Some studies reported a biphasic response to focal ischemia with initial reductions in SOD, GSH-Px, and catalase activities in the first hours after ischemic insult followed by increases around 72 h post insult [24–26]. Therefore, any upregulated enhancement of antioxidant activity in brain tissues may be potentially beneficial for neuronal recovery from I/R injury. In the current study, SOD activities in rats were dramatically decreased following ischemic insult. By Ebselen support, lowering effects of IR on SOD were relatively balanced and SOD indicated increase in comparison to I/R group (p < 0.01). These results are completely consistent with previously reported studies performed with other antioxidant agents. In the current study, GSH-Px activity was not altered neither by I/R nor Ebselen. It is thought that the preserved SOD activity in I/R+ebselen is not a direct effect of ebselen on C
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SOD. Ebselen inhibited oxidative stress as a result of glutathione peroxidase mimetic effect, then almost neurons are survived in I/R+ebselen. Therefore, the SOD activity of I/R+ebselen in the tissue homogenate showed similar to control. Similarly, catalase activity was unchanged after I/R and Ebselen. Although the results regarding GSH-Px was not compatible with the literature, the results were, however, compatible with our previous study investigated effects of ceftriaxone on ischemia/reperfusion injury in rat brain [27]. There are controversial results about the levels of GSH-Px activity in cerebral I/R injuries. We found no significant changes among groups. One of the reasons could be the GSH-Px-like activity of Ebselen in Ebselen + I/R group. In I/R group, the oxidative stress could be blocked by other antioxidant enzymes. Since brain has low GSH-Px activity, the changes in I/R group could be insignificant. Nitric oxide has been shown to have antioxidant properties not related to alterations in neutrophil migration or adhesion, which may account for much of the protective effects of NO during I/R injury [28,29]. Antioxidant properties of NO in the tissues may be related to the reduction of superoxide anion-mediated tissue toxicity by NO [30]. Thereby, after I/R injury NO reduces the generation of ROS, which can contribute to cellular injury, necrosis, and apoptosis. Kawachi et al. [31] observed eNOS-deficient mice subjected to a period of ischemia and found that eNOS-derived NO modulates nuclear neutrophils. This finding shows that NO may be involved enhanced reactive oxygen speciesdependent injury. In the current study, there was no significant alteration in NO levels during I/R injury. NO levels were, however, observed lower in I/R + Ebselen group in comparison to Sham and I/R groups. The decrease in I/R + Ebselen group may be related to the consumption of NO, which is triggered by Ebselen.
Conclusion The study showed that Ebselen has neuron-protective effects due to its anti-oxidant properties as shown by the decrease in MDA overproduction and the increased activity of SOD enzyme. However, additional studies including different doses of Ebselen should be conducted to clear consumption of NO levels. Thereby, exact mechanisms underlying this beneficial effect of Ebselen over I/R injury in brain tissue should be investigated by further studies.
Declaration of Interest The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.
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