Different dose-dependent effects of ebselen in sciatic nerve ischemia-reperfusion injury in rats

Authors

  • Filiz Ozyigit Dumlupinar University, Faculty of Medicine, Department of Pharmacology, Kutahya, Turkey
  • Aysegul Kucuk Dumlupinar University, Faculty of Medicine, Department of Physiology, Kutahya, Turkey
  • Sezer Akcer Dumlupinar University, Faculty of Medicine, Department of Anatomy, Kutahya, Turkey
  • Murat Tosun Afyon Kocatepe University, Faculty of Medicine, Department of Histology, Afyon, Turkey
  • Emel Fatma Kocak Dumlupinar University, Faculty of Medicine, Department of Medical Biochemistry, Kutahya, Turkey
  • Cengiz Kocak Dumlupinar University, Faculty of Medicine, Department of Pathology, Kutahya, Turkey
  • Ahmet Kocak Dumlupinar University, Faculty of Medicine, Department of Histology, Kutahya, Turkey
  • Hasan Metineren Dumlupinar University, Faculty of Medicine, Department of Orthopedics and Traumatology, Kutahya, Turkey
  • Osman Genc Dumlupinar University, Faculty of Medicine, Department of Physiology, Kutahya, Turkey

DOI:

https://doi.org/10.17305/bjbms.2015.521

Keywords:

Ebselen, ischemia-reperfusion injury, oxidative stress, sciatic nerve.

Abstract

Ebselen is an organoselenium compound which has strong antioxidant and anti-inflammatory effects. We investigated the neuroprotective role of ebselen pretreatment in rats with experimental sciatic nerve ischemia-reperfusion (I/R) injury. Adult male Sprague Dawley rats were divided into four groups (N = 7 in each group). Before sciatic nerve I/R was induced, ebselen was injected intraperitoneally at doses of 15 and 30 mg/kg. After a 2 h ischemia and a 3 h reperfusion period, sciatic nerve tissues were excised. Tissue levels of malondialdehyde (MDA) and nitric oxide (NO), and activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were measured. Sciatic nerve tissues were also examined histopathologically. The 15 mg/kg dose of ebselen reduced sciatic nerve damage and apoptosis (P < 0.01), levels of MDA, NO, and inducible nitric oxide synthase (iNOS) positive cells (P < 0.01, P < 0.05, respectively), and increased SOD, GPx, and CAT activities (P < 0.001, P < 0.01, P < 0.05, respectively) compared with the I/R group that did not receive ebselen. Conversely, the 30 mg/kg dose of ebselen increased sciatic nerve damage, apoptosis, iNOS positive cells (P < 0.01, P < 0.05, P < 0.001) and MDA and NO levels (P < 0.05, P < 0.01) and decreased SOD, GPx, and CAT activities (P < 0.05) compared with the sham group. The results of this study suggest that ebselen may cause different effects depending on the dose employed. Ebselen may be protective against sciatic nerve I/R injury via antioxidant and antiapoptotic activities at a 15 mg/kg dose, conversely higher doses may cause detrimental effects.

Downloads

Download data is not yet available.

References

Gholami MR, Abolhassani F, Pasbakhsh P, Akbari M, Sobhani A, Eshraghian MR, et al. The effects of simvastatin on ischemia-reperfusion injury of sciatic nerve in adult rats. Eur J Pharmacol 2008;590:111-4.

http://dx.doi.org/10.1016/j.ejphar.2008.05.050

Bagdatoglu OT, Polat G, Bagdatoglu C, Atik U. Effects of peripheral nerve ischemia-reperfusion model on serum cytokine levels. Turk Neurosurg 2008;18:149-56.

Iida H, Schmeichel AM, Wang Y, Schmelzer JD, Low PA. Orchestration of the inflammatory response in ischemia-reperfusion injury. J Peripher Nerv Syst 2007;12:131-8.

http://dx.doi.org/10.1111/j.1529-8027.2007.00132.x

Nagamatsu M, Schmelzer JD, Zollman PJ, Smithson IL, Nickander KK, Low PA. Ischemic reperfusion causes lipid peroxidation and fiber degeneration. Muscle Nerve 1996;19:37-47.

http://dx.doi.org/10.1002/mus.880190103

Weisfeldt ML. Reperfusion and reperfusion injury. Clin Res 1987;35:13-20.

Schewe T. Molecular actions of ebselen--an antiinflammatory antioxidant. Gen Pharmacol 1995;26:1153-69.

http://dx.doi.org/10.1016/0306-3623(95)00003-J

Azad GK, Tomar RS. Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways. Mol Biol Rep 2014;41:4865-79.

http://dx.doi.org/10.1007/s11033-014-3417-x

Institute for Laboratory Animal Research, National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, D.C.: National Academy Press; 2010: 248.

Ledesma JC, Font L, Aragon CM. The H2O2 scavenger ebselen decreases ethanol-induced locomotor stimulation in mice. Drug Alcohol Depend 2012;124:42-9.

http://dx.doi.org/10.1016/j.drugalcdep.2011.12.003

Koizumi H, Fujisawa H, Suehiro E, Shirao S, Suzuki M. Neuroprotective effects of ebselen following forebrain ischemia: involvement of glutamate and nitric oxide. Neurol Med Chir 2011;51:337-43.

http://dx.doi.org/10.2176/nmc.51.337

Sayan H, Ozacmak VH, Ozen OA, Coskun O, Arslan SO, Sezen SC, et al. Beneficial effects of melatonin on reperfusion injury in rat sciatic nerve. J Pineal Res 2004;37:143-8.

http://dx.doi.org/10.1111/j.1600-079X.2004.00145.x

Yagi K. Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol Biol 1998;108:101-6.

http://dx.doi.org/10.1385/0-89603-472-0:101

Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 1982;126:131-8.

http://dx.doi.org/10.1016/0003-2697(82)90118-X

Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497–500.

Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158-69.

Johansson LH, Borg LA. A spectrophotometric method for determination of catalase activity in small tissue samples. Anal Biochem 1988;174:331-6.

http://dx.doi.org/10.1016/0003-2697(88)90554-4

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.

http://dx.doi.org/10.1016/0003-2697(76)90527-3

Coban YK, Ciralik H, Kurulas EB. Ischemic preconditioning reduces the severity of ischemia-reperfusion injury of peripheral nerve in rats. J Brachial Plex Peripher Nerve Inj 2006; 1:2.

Schmelzer JD, Zochodne DW, Low PA. Ischemic and reperfusion injury of rat peripheral nerve. Proc Natl Acad Sci 1989;86:1639-42.

http://dx.doi.org/10.1073/pnas.86.5.1639

Nagamatsu M, Schmelzer JD, Zollman PJ, Smithson IL, Nickander KK, Low PA. Ischemic reperfusion causes lipid peroxidation and fiber degeneration. Muscle Nerve 1996;19:37-47.

http://dx.doi.org/10.1002/mus.880190103

Coskun O, Yuncu M, Kanter M, Buyukbas S. Ebselen protects against oxidative and morphological effects of high concentration chronic toluene exposure on rat nerves. Eur J General Med 2006;3:64-72.

Gul S, Bahadir B, Hanci V, Acikgoz S, Bektas S, Ugurbas E, et al. Effects of ebselen versus nimodipine on cerebral vasospasm subsequent to experimental subarachnoid hemorrhage in rats. J Clin Neurosci 2010;17:608-11.

http://dx.doi.org/10.1016/j.jocn.2009.07.115

Aras M, Altas M, Meydan S, Nacar E, Karcıoglu M, Ulutas KT, et al. Effects of ebselen on ischemia/reperfusion injury in rat brain. Int J Neurosci 2014;124:771-6.

http://dx.doi.org/10.3109/00207454.2013.879581

Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 1994;63:175-95.

http://dx.doi.org/10.1146/annurev.bi.63.070194.001135

Endoh M, Maiese K, Wagner J. Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia. Brain Res 1994;651:92-100.

http://dx.doi.org/10.1016/0006-8993(94)90683-1

Poderoso JJ, Carreras MC, Lisdero C, Riobó NA, Schöpfer F, Boveris A. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys 1996; 328: 85–92.

http://dx.doi.org/10.1006/abbi.1996.0146

Porciúncula LO, Rocha JB, Cimarosti H, Vinadé L, Ghisleni G, Salbego CG, et al. Neuroprotective effect of ebselen on rat hippocampal slices submitted to oxygen-glucose deprivation: correlation with immunocontent of inducible nitric oxide synthase. Neurosci Lett 2003;346:101-4.

http://dx.doi.org/10.1016/S0304-3940(03)00580-9

Wei L, Zhang Y, Yang C, Wang Q, Zhuang Z, Sun Z. Neuroprotective effects of ebselen in traumatic brain injury model: involvement of nitric oxide and p38 mitogen-activated protein kinase signalling pathway. Clin Exp Pharmacol Physiol 2014;41:134-8.

http://dx.doi.org/10.1111/1440-1681.12186

Warner DS, Sheng H, Batinić-Haberle I. Oxidants, antioxidants and the ischemic brain. J Exp Biol 2004;207:3221-31.

http://dx.doi.org/10.1242/jeb.01022

Unsal C, Oran M, Albayrak Y, Aktas C, Erboga M, Topcu B, et al. Neuroprotective effect of ebselen against intracerebroventricular streptozotocin-induced neuronal apoptosis and oxidative stress in rats. Toxicol Ind Health 2013;14. [Epub ahead of print].

http://dx.doi.org/10.1177/0748233713509429

Kalayci M, Coskun O, Cagavi F, Kanter M, Armutcu F, Gul S, Acikgoz B. Neuroprotective effects of ebselen on experimental spinal cord injury in rats. Neurochem Res 2005;30:403-10.

http://dx.doi.org/10.1007/s11064-005-2615-2

Freude B, Masters TN, Robicsek F, Fokin A, Kostin S, Zimmermann R, et al. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J Mol Cell Cardiol 2000;32:197-208.

http://dx.doi.org/10.1006/jmcc.1999.1066

Yamagata K, Ichinose S, Miyashita A, Tagami M. Protective effects of ebselen, a seleno-organic antioxidant on neurodegeneration induced by hypoxia and reperfusion in stroke-prone spontaneously hypertensive rat. Neuroscience 2008;153:428-35.

http://dx.doi.org/10.1016/j.neuroscience.2008.02.028

Yoshizumi M, Kogame T, Suzaki Y, Fujita Y, Kyaw M, Kirima K, et al. Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells. Br J Pharmacol 2002;136:1023-32.

http://dx.doi.org/10.1038/sj.bjp.0704808

Yang CF, Shen HM, Ong CN. Ebselen induces apoptosis in HepG(2) cells through rapid depletion of intracellular thiols. Arch Biochem Biophys 2000;37:142-52.

http://dx.doi.org/10.1006/abbi.1999.1574

Xu JH, Hu HT, Liu Y, Qian YH, Liu ZH, Tan QR, et al. Neuroprotective effects of ebselen are associated with the regulation of Bcl-2 and Bax proteins in cultured mouse cortical neurons. Neurosci Lett 2006;399(3):210-4.

http://dx.doi.org/10.1016/j.neulet.2006.02.024

Paunesku T, Mittal S, Protić M, Oryhon J, Korolev SV, Joachimiak A, et al. Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int J Radiat Biol 2001;77:1007-21.

http://dx.doi.org/10.1080/09553000110069335

Louis DN, Edgerton S, Thor AD, Hedley-Whyte ET. Proliferating cell nuclear antigen and Ki-67 immunohistochemistry in brain tumors: a comparative study. Acta Neuropathol 1991;81:675-9.

http://dx.doi.org/10.1007/BF00296379

Ino H, Chiba T. Expression of proliferating cell nuclear antigen (PCNA) in the adult and developing mouse nervous system. Brain Res Mol Brain Res 2000;78:163-74.

http://dx.doi.org/10.1016/S0169-328X(00)00092-9

Deng X, Wei H, Lou D, Sun B, Chen H, Zhang Y, et al. Changes in CLIP3 expression after sciatic nerve injury in adult rats. J Mol Histol 2012;43:669-79.

http://dx.doi.org/10.1007/s10735-012-9450-y

Shi H, Liu S, Miyake M, Liu KJ. Ebselen induced C6 glioma cell death in oxygen and glucose deprivation. Chem Res Toxicol 2006;19:655-60.

http://dx.doi.org/10.1021/tx0502544

Yang CF, Shen HM, Ong CN. Intracellular thiol depletion causes mitochondrial permeability transition in ebselen-induced apoptosis. Arch Biochem Biophys 2000;380:319-30.

http://dx.doi.org/10.1006/abbi.2000.1939

Gogvadze V, Klein SD, Shigenaga M, Ames BN, Richter C. Effect of ebselen on Ca2+ transport in mitochondria. Redox Rep 2000;5:359-63.

http://dx.doi.org/10.1179/135100000101535924

Different dose-dependent effects of ebselen in sciatic nerve ischemia-reperfusion injury in rats

Downloads

Additional Files

Published

2015-08-26

How to Cite

1.
Ozyigit F, Kucuk A, Akcer S, Tosun M, Kocak EF, Kocak C, Kocak A, Metineren H, Genc O. Different dose-dependent effects of ebselen in sciatic nerve ischemia-reperfusion injury in rats. Biomol Biomed [Internet]. 2015Aug.26 [cited 2023Sep.23];15(4):36-43. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/521

Issue

Section

Pharmacology

INTRODUCTION

Ischemia plays a major role in the development of pathological changes in many neuropathies. Diabetes mellitus, vascular occlusive diseases, and trauma are some pathologic conditions which cause neuropathy associated with ischemia of peripheral nerves [1]. A nerve must have a continuous and sufficient oxygen supply through intrinsic and extrinsic microvascular systems to carry out its normal functions [2]. Ischemia and reperfusion (I/R) causes ischemic fiber degeneration and oxidative injury in peripheral nerves. Schwann cells are the main target of oxidative injury due to I/R [3]. The neural tissue damage from I/R has ranged from demyelination to axonal degeneration and nerve infarction [4]. The degree of neural tissue damage is related to the duration of ischemia; the cellular and biochemical interactions which occur as a result of reperfusion may exacerbate tissue damage [5].

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one; PZ-51; DR-3305] is a synthetic anti-inflammatory, antioxidant, seleno-organic compound that mimics glutathione peroxidase (GPx) activity by catalyzing the reduction of reactive oxygen species (ROS) in a manner similar to GPx. It reacts with peroxynitrites generated during I/R and reduces hydroperoxides, including those arising in biomembranes and lipoproteins, to the corresponding hydroxy compounds. In addition, it reacts with a number of thiols such as glutathione, N-acetyl-L-cysteine, and dithiothreitol. At low concentrations, ebselen also inhibits a number of enzymes involved in inflammation, such as lipoxygenases, nitric oxide synthases, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, protein kinase C, and H+/K+-ATPase. This inhibitory effect may contribute to the anti-inflammatory potential of ebselen [6,7]. Ebselen is a lipid-soluble compound, therefore, it readily enters the cell. It exhibits strong electrophilic activity, which makes it capable of forming selenenyl-sulfide bonds with cysteine residues in proteins. The main mechanism of action of ebselen is through reactions with cysteine thiol groups in proteins. This compound also reacts with the thioredoxin (Trx) system and it is a highly suitable substrate for the mammalian thioredoxin reductase (TrxR) and acts as a highly efficient oxidant of the reduced Trx [7].

In this study, we investigated the effectiveness of ebselen in protecting the sciatic nerve against I/R injury, using biochemical (malondialdehyde, MDA; nitric oxide, NO; superoxide dismutase, SOD; glutathione peroxidase, GPx; and catalase, CAT), immunohistochemical (terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling, TUNEL; inducible nitric oxide synthase, iNOS; and proliferating cell nuclear antigen, PCNA), and histopathological analyses. Furthermore, we compared the protective effects of two different doses of ebselen, namely 15 mg/kg and 30 mg/kg, to determine an appropriate dosage for preventative treatment to minimize sciatic nerve I/R injury. To the best of our knowledge, the effect of ebselen has not been previously studied in rats with the sciatic nerve I/R injury using these biochemical and immunohistochemical parameters.

MATERIALS AND METHODS

This study was carried out in the Experimental Animals Research Center of Dumlupinar University. The study experiments were approved by the Local Ethics Committee for Experiments on Animals of Dumlupinar University (No: 2014.06.01). All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council [8].

Animals

Male Sprague-Dawley rats (300–350 g, 12–16 weeks, N = 28) were used. All rats were housed individually in transparent polycarbonate cages with a 12:12 h light-dark cycle at 22 ± 2 ºC and provided ad libitum access to fresh water and standard rat chow until the experiments commenced.

Chemicals

Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] was purchased from Sigma-Aldrich Co. LLC (Ebselen®, St. Louis, MO, USA). It was dissolved in 10% Tween 80 (Sigma–Aldrich Co. LLC., St. Louis, MO, USA) [9].

Experimental study design

Experimental animals were randomly divided into four groups (N = 7). Group I, (sham) were subjected to abdominal median laparotomy and not to I/R conditions. Rats were treated with intraperitoneal (i.p.) injections of 1 mL of vehicle (10% Tween 80) 24 h before the laparotomy. Group II (I/R) was subjected to 2 h of ischemia followed by 3 h of reperfusion. The rats were treated with i.p. injections of 1 mL of vehicle 24 h before the laparotomy. Group III (I/R with 15 mg/kg ebselen) was treated similarly to the I/R group, but also received i.p. injections of 1 mL of 15 mg/kg ebselen, 24 h before the I/R. Group IV (I/R with 30 mg/kg ebselen) was treated similarly to Group III rats but received 1 mL of 30 mg/kg ebselen [10].

Surgical procedures

Rats were weighed and then anesthetized with intramuscular (i.m.) injections of 10 mg/kg xylazine hydrochloride (Rompun, Bayer, Istanbul, Turkey) and 70 mg/kg ketamine (Ketalar®, Pfizer, Istanbul, Turkey). After a suitable level of anesthesia was achieved, the rats were placed on a homeothermic table to maintain a stable body temperature of 37 ± 1 ºC, and then the anterior abdominal wall was shaved and sterilized with povidone-iodine solution. Ischemia was produced by ligation of the abdominal aorta at the level of iliolumbar arteries; these arteries supply the hindlimbs. The aorta and its branches were occluded with an atraumatic vascular clamp (Vascu®Stop Bulldog Clamp) for 2 h [11]. After 2 h of ischemia, the clamps were removed to allow reperfusion for 3 h. The abdomen was subsequently closed and the rats were observed during the 3 h of reperfusion. After reperfusion, the animals were euthanized under anesthesia, and the sciatic nerves were excised bilaterally. The sciatic nerve tissue samples were rinsed with a cold heparinized phosphate-buffered saline (PBS) to remove any red blood cells or clots. A portion of the sciatic nerve samples were fixed with 10% buffered formalin for histopathological and immunohistochemical analyses. Additional portions of sciatic nerve tissue samples were placed in Eppendorf tubes and immediately stored at -80ºC for biochemical analysis.

Biochemical analysis

For biochemical analysis, nerve tissue was mixed with cold working solution (50 mM phosphate buffer, pH 7.40), and homogenized with a mechanic homogenizer (Analytik Jena speedmill plus, Germany). The mixture was then centrifuged at 10,000 × g for 15 min at 4ºC, and the supernatant was preserved for biochemical analysis by storing on ice. MDA and NO levels, as well as SOD, GPx, and CAT activities were measured in sciatic nerve tissue homogenates using commercial enzyme-linked immunosorbent assay (ELISA) kits (Cayman Inc, Ann Arbor, MI, USA) on a microplate reader (BMG Labtech Spectrostar Nano, GmbH, Ortenberg, Germany). MDA concentration was measured based on the thiobarbituric acid reactive substances (TBARS) method [12]. NO concentration was measured based on the Griess method [13]. SOD activity was measured using a salt of tetrazolium to detect superoxide radicals generated by xanthine oxidase and hypoxanthine, in the presence of a range of concentrations of SOD [14]. Measurement of GPx activity was based on the method of Paglia and Valentine [15]. CAT activity was measured based on the reaction of the enzyme with methanol in the presence of an optimal concentration of hydrogen peroxide. The formaldehyde produced was measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (purpald) as a chromogen [16]. Tissue protein concentrations were measured based on the Bradford method on a Beckman Coulter AU680 analyzer (Beckman Coulter, Miami, FL, USA) [17].

Histopathological examinations

Sciatic nerve tissue samples were fixed in 10% formalin, embedded in paraffin, sectioned (4 µm), placed on slides, stained with hematoxylin and eosin (H&E), and examined under a light microscope (Olympus BX51, Tokyo, Japan) by a pathologist who was blinded to the treatment groups. The pathologic slides were graded histopathologically according to the edema and axonal vacuolization using a previously described scoring system by Coban et al. [18]. For each section, the vacuolization and edema were semi-quantitatively graded from 0 to 3 as follows: 0-normal, 1-mild, 2-moderate, and 3-severe.

In situ detection of sciatic nerve apoptosis

We used an in situ TUNEL assay to assess the degree of sciatic nerve apoptosis. Formalin-fixed sections were deparaffinized in xylene and rehydrated through graded concentrations of ethanol to water. DNA fragmentation in apoptosis was detected using a commercially available kit (ApopTag® peroxidase in situ apoptosis detection kit, Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. Processed samples were examined under a light microscope (Olympus BX51, Tokyo, Japan). The number of TUNEL-positive cells per 100 was calculated from randomly selected fields. The apoptotic index was calculated as the percentage of apoptotic (TUNEL-positive stained) cells.

Immunohistochemical examinations

iNOS and PCNA were examined immunohistochemically. Formaldehyde-fixed tissue was embedded in paraffin for further immunohistochemical examination. Immunohistochemical staining was performed using commercially available kits (Thermoscie.jpgic Lab Vision, Fremont, CA, USA) according to the manufacturer’s instructions and examined under a light microscope (Olympus BX51). The percentage of immunopositive cells was determined.

Statistical analyses

Statistical analyses were performed using GraphPad Prism version 6.05 (GraphPad Software, Inc., CA, USA). All data were expressed as mean ± standard error of the mean (SEM). Because of the small experimental groups, we used non-parametric statistics. Quantitative data were tested using the Kruskal-Wallis analysis of variance on ranks and Dunn’s method was used for post hoc testing. A P value < 0.05 was considered to be statistically significant.

RESULTS

Significant differences were observed between MDA and NO levels in the assay groups (P < 0.001). Our results revealed that tissue MDA and NO levels were significantly higher in the I/R group compared to the sham group (P < 0.001, Table 1). Tissue MDA and NO levels were also significantly higher in the I/R with 30 mg/kg ebselen group compared with the sham group (P < 0.05 and P < 0.01, respectively, Table 1). When the I/R with 15 mg/kg ebselen and the I/R groups were compared, MDA and NO levels showed significant differences (P < 0.01 and P < 0.05, respectively, Table 1).

TABLE 1: Comparisons of measured biochemical parameters in sciatic nerve tissue samples between assay groups

When SOD, GPx, and CAT activities were evaluated, significant differences were observed between assay groups (P < 0.001, Table 1). SOD, GPx, and CAT activities were significantly lower in the I/R group compared to the sham group (P < 0.001, Table 1). Significant differences were observed between the I/R with 15 mg/kg ebselen and the I/R groups for SOD, GPx, and CAT (P < 0.01, P < 0.01, P < 0.05, respectively, Table 1). SOD, GPx, and CAT activities were significantly lower in the I/R with 30 mg/kg ebselen group compared to sham group (P < 0.05, Table 1).

In the histopathological examinations, our results showed that I/R injury caused a marked increase in total histopathological sciatic nerve damage score compared with the sham group (P < 0.001, Table 2). The dose of 15 mg/kg of ebselen significantly reduced the histological damage scores compared with the I/R group (P < 0.01, Table 2). Although the 15 mg/kg dose of ebselen significantly attenuated morphologic changes, which occurred as a result of I/R injury in the sciatic nerve tissue, the 30 mg/kg dose of ebselen did not reduce the histological damage scores compared to the I/R group (P > 0.05, Table 2). Furthermore, the 30 mg/kg dose of ebselen caused a significant increase in the total histopathological sciatic nerve damage score compared with the sham group (P < 0.01, Table 2). Compared with the sham group, a significant increase in axonal cytoplasmic vacuolization and degeneration in axons of the sciatic nerve was observed in the I/R group and I/R with 30 mg/kg ebselen group. In addition, intramyelinic edema within nerve fibers was observed, not only in the perivascular region, but also in endoneurial vessels in the I/R group and I/R with 30 mg/kg ebselen group. However, the histological appearance was near normal in the I/R with 15 mg/kg ebselen group. Considerably preserved neuronal structures were seen in the sciatic nerve pretreated with a 15 mg/kg dose of ebselen (Figure 1A, 1B, 1C, 1D).

TABLE 2: Comparisons of scored histopathological and immunohistochemical values in sciatic nerve tissue samples between assay groups
FIGURE 1: Histopathological examination of the sciatic nerve tissues of assay groups, H&E × 100; H&E × 200. Axonal cytoplasmic vacuolization, degeneration in the axons of the sciatic nerve, intramyelinic edema within nerve fibers in the perivascular region and in endoneurial vessels were seen in the I/R group (B: H&E × 100, B*: H&E × 200) and I/R with 30 mg/kg ebselen group (D). Considerably preserved neuronal structures were seen in the sciatic nerve treated with a 15 mg/kg dose of ebselen (C). IE: Intramyelinic edema, VC: Vascular congestion, CV: Cytoplasmic vacuolization, AD: Axonal degeneration. A. Sham group, B. I/R group, C. I/R with 15 mg/kg Ebselen group, D. I/R with 30 mg/kg Ebselen group.

When apoptosis in the sciatic nerve tissue was examined using the TUNEL method, we observed that I/R injury caused a significant increase in the apoptotic index compared with the sham group (P < 0.01). A dose of 15 mg/kg of ebselen significantly reduced the apoptotic index compared with the I/R group (P < 0.01). However, the 30 mg/kg dose of ebselen increased the apoptotic index compared with the sham group and the I/R with 15 mg/kg ebselen group (P > 0.05 and P < 0.05, respectively, Table 2, Figure 2A, 2B, 2C, 2D).

FIGURE 2: Examination of apoptosis in sciatic nerve tissues of assay groups, TUNEL × 400. Apoptosis in the sciatic nerve tissue was examined using the TUNEL method. The number of TUNEL-positive cells per 100 was calculated from randomly selected fields. The apoptotic index was calculated as the percentage of apoptotic (TUNEL-positive stained) cells. I/R injury caused a significant increase in the apoptotic index (B). A dose of 15 mg/kg of ebselen significantly reduced the apoptotic index (C). The 30 mg/kg dose of ebselen increased the apoptotic index compared with the sham group (D). A. Sham group, B. I/R group, C. I/R with 15 mg/kg ebselen group, D. I/R with 30 mg/kg ebselen group.

iNOS and PCNA expressions were examined using immunohistochemical staining. Our results demonstrated that I/R injury caused a significant increase in the number of iNOS and PCNA positive cells compared with the sham group (P < 0.001 and P < 0.01, respectively). The 15 mg/kg dose of ebselen partially reduced the number of iNOS and PCNA positive cells, but these reductions were not statistically significant (Table 2, Figure 3A, 3B, 3C, 3D and Figure 4A, 4B, 4C, 4D). Moreover, the 30 mg/kg dose of ebselen also significantly increased the number of iNOS and PCNA positive cells compared with sham group (P < 0.001, Table 2, Figure 3A, 3B, 3C, 3D and Figure 4A, 4B, 4C, 4D).

FIGURE 3: Examination of iNOS expression in sciatic nerve tissues of assay groups, iNOS × 400. iNOS expression was examined using immunohistochemical staining. The percentage of iNOS-positive cells was determined. I/R injury caused a significant increase in the number of iNOS positive cells (B). The 15 mg/kg dose of ebselen partially reduced the number of iNOS positive cells (C). The 30 mg/kg dose of ebselen increased the number of iNOS positive cells compared with the sham group (D). A. Sham group, B. I/R group, C. I/R with 15 mg/kg Ebselen group, D. I/R with 30 mg/kg Ebselen group.
FIGURE 4: Examination of PCNA expression in sciatic nerve tissues of assay groups, PCNA x 400. PCNA expression was examined using immunohistochemical staining. The percentage of PCNA positive cells was determined. I/R injury caused a significant increase in the number of PCNA positive cells (B). The 15 mg/kg dose of ebselen partially reduced the number of PCNA positive cells (C). The 30 mg/kg dose of ebselen increased the number of PCNA positive cells compared with the sham group (D). A. Sham group, B. I/R group, C. I/R with 15 mg/kg Ebselen group, D. I/R with 30 mg/kg Ebselen group.

DISCUSSION

In this study, we investigated the neuroprotective effects of ebselen pre-treatment with two different doses in rats subjected to experimental sciatic nerve I/R injury. Our results revealed that the 15 mg/kg dose of ebselen had remarkable protective effects against sciatic nerve I/R injury. This protective effect was evident as a decrease in MDA and NO levels and an increase in antioxidant enzyme activities in sciatic nerve tissue. In addition, the histopathological examination of sciatic nerve injury, apoptosis, and immunohistochemical markers corroborated this result. Although the 30 mg/kg dose of ebselen pretreatment did not worsen the I/R injury, it revealed comparable level of results with those of the no-drug I/R injury group. The 30 mg/kg dose of ebselen pretreatment led to increases in sciatic nerve damage, apoptosis, and oxidative stress compared with sham group. Therefore, we suggest that ebselen may be protective against sciatic nerve I/R injury at 15 mg/kg dose. Conversely, higher doses may be harmful.

Reperfusion of the ischemic tissue increases the production of ROS, which causes damage to a variety of molecules and impairs cellular functions [19]. Production and accumulation of ROS causes lipid peroxidation (LPO) in cellular membranes and subsequent generation of MDA. Therefore, MDA is a useful quantitative indicator of LPO [20]. In this study, sciatic nerve I/R caused significant LPO accumulation. Although the 15 mg/kg dose of ebselen significantly reduced LPO, the 30 mg/kg dose of ebselen did not reduce LPO compared with the I/R group. Moreover, the 30 mg/kg dose of ebselen significantly increased LPO accumulation compared with the sham group. The results of this study were consistent with previous studies [21-23]. Consequently, while the 15 mg/kg dose of ebselen pretreatment may be protective against sciatic nerve I/R injury by reducing LPO, the 30 mg/kg dose of ebselen appears to have little or no effect on LPO.

Nitric oxide is a signaling molecule produced in various cell types in the nerve tissue, and is synthesized by two isoforms of NOS: neuronal and endothelial. A third isoform of NOS, namely iNOS, is expressed in numerous cell types as a result of the inflammatory processes. Higher levels of NO, which are produced by iNOS, contribute to the inflammatory process and induce I/R injury [24-26]. Our results showed that the 15 mg/kg dose of ebselen reduced the tissue NO levels and the number of iNOS positive cells compared with the I/R group. However, the 30 mg/kg dose of ebselen increased tissue NO levels and the number of iNOS positive cells compared with the sham group. Our results were consistent with a previous study [27]. Unlike our study, Wei et al. demonstrated that both a 10 mg/kg and a 30 mg/kg dose of ebselen administered by an intragastric route significantly reduced the NO and iNOS mRNA levels in rats with a traumatic brain injury model in a dose-dependent manner [28]. In our study, a 30 mg/kg dose of ebselen did not reduce NO levels or the number of iNOS positive cells. This discrepancy may be related to differences in bioavailability and the LD50 dose of ebselen between an oral and a parenteral route. In addition, the dose-dependent effect of ebselen may be different in brain and sciatic nerve tissues. The results of this study led us to propose that the 15 mg/kg dose of ebselen may be neuroprotective against I/R injury in sciatic nerve by decreasing NO levels and inhibiting iNOS activity. However, the 30 mg/kg dose of ebselen appears to have little or no effect on iNOS activity.

Endogenous antioxidant enzymes are an important defense system that scavenges ROS including SOD, GPx, and CAT, thereby reducing their detrimental effects [29]. In this study, the 15 mg/kg dose of ebselen significantly increased antioxidant enzyme activities. However, the 30 mg/kg dose of ebselen did not significantly increase antioxidant enzyme activities. Our results were consistent with previous studies [21-23,30,31]. Therefore, we suggest that the protective effects of the 15 mg/kg dose of ebselen may be exerted via improvement of the antioxidant defense system in sciatic nerve I/R injury.

Apoptosis triggered by oxidative stress is an important pathogenic mechanism in I/R-induced cell death [32]. In this study, results of the TUNEL assay demonstrated that the 15 mg/kg dose of ebselen reduced the apoptotic index. Conversely, the 30 mg/kg dose of ebselen significantly increased the apoptotic index. Our results were consistent with previous studies [30,33-36]. Yang et al. demonstrated that ebselen led to a dose-dependent increase of TUNEL-positive cells, which depleted intracellular glutathione (GSH) and protein thiols [35]. The authors have suggested that the rapid depletion of intracellular thiols and GSH may lead to a severe imbalance of the cellular redox status, and that this depletion may play a role in ebselen-induced apoptosis [35]. Another study reported that, although a low dose of ebselen revealed a protective effect against glutamate toxicity by decreasing Bax and increasing Bcl-2, higher doses caused a significant decrease both in cell viability and protection against glutamate toxicity. This suggests that the ebselen lacks neurotrophic effects and, at higher concentrations, is toxic to cells [36]. Consequently, our results indicate that the 15 mg/kg dose of ebselen may ameliorate sciatic nerve I/R injury by inhibiting the apoptotic pathways. Conversely, the 30 mg/kg dose of ebselen may induce apoptosis by activating the apoptotic pathways.

Paunesku et al. used PCNA immunostaining to detect proliferating cells. PCNA is a nuclear auxilliary protein of DNA polymerase δ which accumulates in the nucleus during the S phase of the cell cycle; it is required for DNA replication and repair. Because its expression correlates to cell proliferation and cell cycle, it is commonly used as a physiological or pathological marker of proliferating cells [37]. Schwann cells of normal peripheral nerves were reported to be immunoreactive for PCNA [38]. In addition, it has been reported that PCNA mRNA is expressed not only in proliferating cells but also in nonproliferating cells such as neurons [39]. Deng et al. demonstrated that the proliferation of Schwann cells and PCNA increased after sciatic nerve injury. The researchers suggested that the proliferation of Schwann cells is important for peripheral nerve regeneration and that they proliferate during myelination after sciatic nerve injury [40]. In our study, the no-drug I/R group and the group given 30 mg/kg ebselen both significantly increased the number of PCNA positive cells compared with the sham group. Although the PCNA positivity was increased in the I/R with 15 mg/kg ebselen group, this increase was not significant compared with the sham group. Consequently, we suggest that because the sciatic nerve tissue was well protected by the dose of 15 mg/kg ebselen against I/R injury, the increase in PCNA and the corresponding proliferation of Schwann cells may be less in the I/R with 15 mg/kg ebselen group than in the I/R and the I/R with 30 mg/kg ebselen groups because of a reduced requirement for regenerating tissue.

The most interesting result of this study was the dose-dependent manner of the effect of ebselen in relation to sciatic nerve I/R injury. In spite of the positive effects of ebselen, certain signs were observed in response to selenium-containing compounds that could be the basis of potentially damaging actions. Several in vitro studies reported that high concentrations of ebselen may be toxic to cells [35,41-43]. Although the mechanism underlying the toxicity of ebselen is not fully understood, it has been shown that ebselen treatment causes a dose-dependent depletion of cellular GSH levels [35,41]; a dose-dependent loss of mitochondrial membrane potential and release of cytochrome c [42]; and deterioration of mitochondrial functioning and inhibition of the Trx system [43]. Consequently, in our study, while the 15 mg/kg dose of ebselen demonstrated a protective effect against sciatic nerve I/R injury, the 30 mg/kg dose of ebselen did not demonstrate any protective effect. Ebselen may be neuroprotective at low concentrations, however, it may cause detrimental effects at high concentrations.

In conclusion, the results of this study revealed that the low and high doses of ebselen have different effects in sciatic nerve I/R injury. Ebselen may be an effective neuroprotective agent at low doses against sciatic nerve I/R injury depending on antioxidant and antiapoptotic effects. On the other hand, high doses of ebselen may cause detrimental effects, therefore, these observations are of particular importance because ebselen is currently used in clinical trials for possible use as a therapeutic agent for stroke. Further studies should be performed to ide.jpgy a reliable therapeutic window for this promising, clinically used agent.

DECLARATION OF INTERESTS

The authors declare that they have no conflicts of interest to disclose.

Acknowledgements

ACKNOWLEDGMENTS

This study was supported by a grant from the Scie.jpgic Research Project of Dumlupinar University (Project no: 2013-28).

REFERENCES

  1. , , , , , (). The effects of simvastatin on ischemia-reperfusion injury of sciatic nerve in adult rats. Eur J Pharmacol. http://dx.doi.org/10.1016/j.ejphar.2008.05.050
  2. , , , (). Effects of peripheral nerve ischemia-reperfusion model on serum cytokine levels. Turk Neurosurg.
  3. , , , , (). Orchestration of the inflammatory response in ischemia-reperfusion injury. J Peripher Nerv Syst. http://dx.doi.org/10.1111/j.1529-8027.2007.00132.x
  4. , , , , , (). Ischemic reperfusion causes lipid peroxidation and fiber degeneration. Muscle Nerve. http://dx.doi.org/10.1002/mus.880190103
  5. (). Reperfusion and reperfusion injury. Clin Res.
  6. (). Molecular actions of ebselen--an antiinflammatory antioxidant. Gen Pharmacol. http://dx.doi.org/10.1016/0306-3623(95)00003-J
  7. , (). Ebselen, a promising antioxidant drug: mechanisms of action and targets of biological pathways. Mol Biol Rep. http://dx.doi.org/10.1007/s11033-014-3417-x
  8. (). Guide for the Care and Use of Laboratory Animals.
  9. , , (). The H2O2 scavenger ebselen decreases ethanol-induced locomotor stimulation in mice. Drug Alcohol Depend. http://dx.doi.org/10.1016/j.drugalcdep.2011.12.003
  10. , , , , (). Neuroprotective effects of ebselen following forebrain ischemia: involvement of glutamate and nitric oxide. Neurol Med Chir. http://dx.doi.org/10.2176/nmc.51.337
  11. , , , , , (). Beneficial effects of melatonin on reperfusion injury in rat sciatic nerve. J Pineal Res. http://dx.doi.org/10.1111/j.1600-079X.2004.00145.x
  12. (). Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol Biol. http://dx.doi.org/10.1385/0-89603-472-0:101
  13. , , , , , (). Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. http://dx.doi.org/10.1016/0003-2697(82)90118-X
  14. , , (). A simple method for clinical assay of superoxide dismutase. Clin Chem.
  15. , (). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med.
  16. , (). A spectrophotometric method for determination of catalase activity in small tissue samples. Anal Biochem. http://dx.doi.org/10.1016/0003-2697(88)90554-4
  17. (). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. http://dx.doi.org/10.1016/0003-2697(76)90527-3
  18. , , (). Ischemic preconditioning reduces the severity of ischemia-reperfusion injury of peripheral nerve in rats. J Brachial Plex Peripher Nerve Inj.
  19. , , (). Ischemic and reperfusion injury of rat peripheral nerve. Proc Natl Acad Sci. http://dx.doi.org/10.1073/pnas.86.5.1639
  20. , , , , , (). Ischemic reperfusion causes lipid peroxidation and fiber degeneration. Muscle Nerve. http://dx.doi.org/10.1002/mus.880190103
  21. , , , (). Ebselen protects against oxidative and morphological effects of high concentration chronic toluene exposure on rat nerves. Eur J General Med.
  22. , , , , , (). Effects of ebselen versus nimodipine on cerebral vasospasm subsequent to experimental subarachnoid hemorrhage in rats. J Clin Neurosci. http://dx.doi.org/10.1016/j.jocn.2009.07.115
  23. , , , , , (). Effects of ebselen on ischemia/reperfusion injury in rat brain. Int J Neurosci. http://dx.doi.org/10.3109/00207454.2013.879581
  24. , (). Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem. http://dx.doi.org/10.1146/annurev.bi.63.070194.001135
  25. , , (). Expression of the inducible form of nitric oxide synthase by reactive astrocytes after transient global ischemia. Brain Res. http://dx.doi.org/10.1016/0006-8993(94)90683-1
  26. , , , , , (). Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys. http://dx.doi.org/10.1006/abbi.1996.0146
  27. , , , , , (). Neuroprotective effect of ebselen on rat hippocampal slices submitted to oxygen-glucose deprivation: correlation with immunocontent of inducible nitric oxide synthase. Neurosci Lett. http://dx.doi.org/10.1016/S0304-3940(03)00580-9
  28. , , , , , (). Neuroprotective effects of ebselen in traumatic brain injury model: involvement of nitric oxide and p38 mitogen-activated protein kinase signalling pathway. Clin Exp Pharmacol Physiol. http://dx.doi.org/10.1111/1440-1681.12186
  29. , , (). Oxidants, antioxidants and the ischemic brain. J Exp Biol. http://dx.doi.org/10.1242/jeb.01022
  30. , , , , , (). Neuroprotective effect of ebselen against intracerebroventricular streptozotocin-induced neuronal apoptosis and oxidative stress in rats. Toxicol Ind Health. [Epub ahead of print] http://dx.doi.org/10.1177/0748233713509429
  31. , , , , , , (). Neuroprotective effects of ebselen on experimental spinal cord injury in rats. Neurochem Res. http://dx.doi.org/10.1007/s11064-005-2615-2
  32. , , , , , (). Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J Mol Cell Cardiol. http://dx.doi.org/10.1006/jmcc.1999.1066
  33. , , , (). Protective effects of ebselen, a seleno-organic antioxidant on neurodegeneration induced by hypoxia and reperfusion in stroke-prone spontaneously hypertensive rat. Neuroscience. http://dx.doi.org/10.1016/j.neuroscience.2008.02.028
  34. , , , , , (). Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells. Br J Pharmacol. http://dx.doi.org/10.1038/sj.bjp.0704808
  35. , , (). Ebselen induces apoptosis in HepG(2) cells through rapid depletion of intracellular thiols. Arch Biochem Biophys. http://dx.doi.org/10.1006/abbi.1999.1574
  36. , , , , , (). Neuroprotective effects of ebselen are associated with the regulation of Bcl-2 and Bax proteins in cultured mouse cortical neurons. Neurosci Lett. http://dx.doi.org/10.1016/j.neulet.2006.02.024
  37. , , , , , (). Proliferating cell nuclear antigen (PCNA): ringmaster of the genome. Int J Radiat Biol. http://dx.doi.org/10.1080/09553000110069335
  38. , , , (). Proliferating cell nuclear antigen and Ki-67 immunohistochemistry in brain tumors: a comparative study. Acta Neuropathol. http://dx.doi.org/10.1007/BF00296379
  39. , (). Expression of proliferating cell nuclear antigen (PCNA) in the adult and developing mouse nervous system. Brain Res Mol Brain Res. http://dx.doi.org/10.1016/S0169-328X(00)00092-9
  40. , , , , , (). Changes in CLIP3 expression after sciatic nerve injury in adult rats. J Mol Histol. http://dx.doi.org/10.1007/s10735-012-9450-y
  41. , , , (). Ebselen induced C6 glioma cell death in oxygen and glucose deprivation. Chem Res Toxicol. http://dx.doi.org/10.1021/tx0502544
  42. , , (). Intracellular thiol depletion causes mitochondrial permeability transition in ebselen-induced apoptosis. Arch Biochem Biophys. http://dx.doi.org/10.1006/abbi.2000.1939
  43. , , , , (). Effect of ebselen on Ca2+transport in mitochondria. Redox Rep. http://dx.doi.org/10.1179/135100000101535924