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Efficiacy of Resveratrol and Quercetin After Experimental Spinal Cord Injury

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Efficiacy of Resveratrol and Quercetin After Experimental Spinal Cord Injury EXPERIMENTAL STUDY Efficiacy of resveratrol and quercetin after experimental spinal cord injury Ulvi Çiftçi, M.D.,1 Emre Delen, M.D.,2 Murat Vural, M.D.,3 Onur Uysal, M.D.,4 Didem Turgut Coşan, M.D.,5 Canan Baydemir, M.D.,6 Fulya Doğaner, M.D.5 1Department of Neurosurgery, Bakırköy Dr. Sadi Konuk Training and Research Hospital, İstanbul-Turkey 2Department of Neurosurgery, Trakya University Faculty of Medicine, Edirne-Turkey 3Department of Neurosurgery, Eskişehir Osmangazi University Faculty of Medicine, Eskişehir-Turkey 4Department of Histology, Eskişehir Osmangazi University Faculty of Medicine, Eskişehir-Turkey 5Department of Medical Biology, Eskişehir Osmangazi University Faculty of Medicine, Eskişehir-Turkey 6Department of Biostatistics, Kocaeli University Faculty of Medicine, Kocaeli-Turkey ABSTRACT BACKGROUND: The aim of this study was to investigate the effect of natural antioxidants resveratrol and quercetin on oxidative stress and secondary cell damage in rats with acute spinal cord injury. METHODS: In this experimental study, 42 male Sprague-Dawley rats were used. Spinal cord injury was performed with clip compres- sion method at level of T4-5. The study was conducted using 6 groups: control, trauma, trauma and solvent, trauma and resveratrol, trauma and quercetin, and trauma with combined resveratrol and quercetin. All rats were euthanized 48 hours after the procedure. Effects of resveratrol and quercetin on serum and tissue total antioxidant capacity and paraoxanase activity level were examined. RESULTS: Compared to trauma group, there was a significant increase in total antioxidant capacity and paraoxanase activity level in resveratrol, quercetin, and combined treatment groups. There was no significant difference between resveratrol and quercetin groups with regard to total antioxidant capacity and paraoxanase activity level. Total antioxidant capacity and paraoxanase activity level were significantly higher in solvent group than trauma group. In histopathological evaluation, there was a decrease in polymorphonuclear leukocyte infiltration in solvent, resveratrol, quercetin, and combined treatment groups. CONCLUSION: Biochemical and histological staining results of present study showed that resveratrol and quercetin may be effec- tive in preventing secondary damage in spinal cord injury. Keywords: Antioxidant; experimental spinal cord injury; quercetin; resveratrol. INTRODUCTION nosis. Poor prognosis and devastating outcomes of SCI jus- tify scientific interest in the pathophysiology of SCI as well Spinal cord injury (SCI) is a clinical condition that often as in protective strategies and development of treatment causes devastating outcomes such as permanent incapacity, options.[1,2] Resveratrol and quercetin are naturally occurring especially in the younger population. It is well known that antioxidative compounds that are referred to as polyphe- SCI is a type of trauma with no known effective therapeutic nols. Resveratrol reduces oxidation of low-density lipopro- methods, and accordingly it is still difficult to estimate prog- tein (LDL) by chelating copper (II) and phagocytizing oxygen free radicals. It also inhibits platelet aggregation and prevents Address for correspondence: Ulvi Çiftçi, M.D. thrombus formation and atherosclerosis, acting as a thera- Bakırköy Dr. Sadi Konuk Eğitim ve Araştırma Hastanesi, peutic agent to prevent potential cardiovascular disease.[2–5] Nöroşirürji Kliniği, İstanbul, Turkey Quercetin has a broad range of pharmacological characteris- Tel: +90 212 - 414 7171 E-mail: [email protected] tics such as anticancer, antiviral, antihistaminic, antitumoral, immunomodulatory, antioxidant, and anti-inflammatory Qucik Response Code Ulus Travma Acil Cerrahi Derg [5–10] 2016;22(5):423–431 properties. Recently, there have been studies conducted doi: 10.5505/tjtes.2016.44575 on antioxidant effects of these agents. The current study explored therapeutic effects and antioxidant properties of Copyright 2016 TJTES post-SCI administered resveratrol and quercetin on second- ary cell damage. Ulus Travma Acil Cerrahi Derg, September 2016, Vol. 22, No. 5 423 Çiftçi et al. Efficiacy of resveratrol and quercetin after experimental spinal cord injury MATERIALS AND METHODS immediate removal with clip holder. Post-SCI paraplegia was observed in all rats. Resveratrol and quercetin were dissolved Male Sprague-Dawley rats (weight: 250±50 g; n=42) were in dimethyl sulfoxide (DMSO) and single dose 1 hour follow- supplied for the study by the experimental research center of ing SCI was administered. All rats were euthanized 48 hours Eskisehir Osmangazi University. Animals had 2-week period after procedure. All procedures and group data are summa- of adaptation to surroundings before initiation of study. They rized in Table 1. were housed in polycarbonate cages in a temperature- and humidity-controlled room (21±10°C and 45-55%, respec- Biochemical Analyses tively) with 12/12 reversed light cycle. They were fed with Thoracic cavity of rats was opened under general anesthe- standard rat chow and allowed to drink ad libitum. Proce- sia in order to determine total antioxidant status (TAS) level dures regarding animal care, surgery, and sample preparation and paraoxonase-1 (PON-1) activity. A blood sample of 3 were approved by the institutional animal care and experi- mL was drawn from heart, centrifuged at 1000 rpm for 10 ments committee of Eskisehir Osmangazi University Faculty minutes, and plasma was collected. Tissue samples and spinal of Medicine. Regulations and guidelines for the care and use cord samples of approximately 15 mm in length were taken of laboratory animals of institutional animal care and experi- from all groups and frozen in liquid nitrogen. Samples were ments committee of Eskisehir Osmangazi University Faculty transferred to tubes containing 1 mL 1x phosphate buffer of Medicine (19.08.2009/130) were observed, as well as US saline and processed in ultrasonic homogenizer. The resultant National Institutes of Health guidelines regarding the care homogenate was centrifuged at 3500 rpm for 15 minutes and and use of animals for experimental procedures. supernatant was used for measurements. In plasma and tissue homogenates, TAS level was read using Shimadzu UV-1601 Surgical Procedure spectrophotometer (Shimadzu Corp., Kyoto, Japan) at absor- The experimental SCI model was applied to 42 rats divided bance level of 660 nm according to TAS assay kit (Rel Assay into 6 groups. Sedation was ensured with intramuscularly Diagnostics, Gaziantep, Turkey) procedures, while PON-1 ac- (i.m.) administered xylazine (Rompun; Bayer AG, Leverkusen, tivity was measured using same device at absorbance level of Germany) at dose of 10 mg/kg, followed by ketamine hydro- 412 nm according to the PON-1 kit (Rel Assay Diagnostics, chloride (Ketalar; Pfizer, New York, NY, USA) i.m. at dose Gaziantep, Turkey) procedures. of 50mg/kg for general anesthesia. Rivlin-Tator compression model (1978) was used: Surgical mark point was spinous pro- Histological Examinations cess of T2 vertebrae, the most remarkable point of the rat Approximately 15 mm-long samples of spinal cord taken spine. The area from T3 to T6 was shaved, and local surgery from control and experiment groups for histological evalua- site antisepsis and environmental isolation were ensured. tion included white matter, grey matter, and trauma site. All Midline skin incision was made through cutaneous and subcu- specimens were carefully excised and fixed in neutral buffered taneous layers. Fascia was opened and paravertebral muscles formalin for histological analyses. After fixation, tissue was em- were subperiostally lateralized. T4-T5 laminectomy was per- bedded in paraffin and serial sections (4 μm) were prepared formed. Spinal cord was exposed and an aneurysm clip with for each paraffin block; on average, 50 sections were collected extradural closing pressure of 50 g (Yaşargil clip FE 619K, per rat. Sections were stained with hematoxylin and eosin for Aesculap AG, Tuttlingen, Germany) was used to induce SCI. assessment of SCI. Digital images were obtained using Olym- In SCI groups, the clip was in place for 1 minute followed by pus BX-61 microscope (Olympus Corp., Tokyo, Japan) with a Table 1. Procedures performed and substances used according to experiment group Group n Procedure Substance T4 and T5 Spinal cord DMSO Resveratrol Quercetin laminectomy injury (dissolved in DMSO) (dissolved in DMSO) C 7 √ – – – – SCI 7 √ √ – – – SCI+DMSO 7 √ √ 0.3 mL i.p. – – SCI+R 7 √ √ – 100 mg/kg i.p. – SCI+Q 7 √ √ – – 200 mg/kg i.p. SCI+R+Q 7 √ √ – 100 mg/kg i.p. 200 mg/kg i.p. C: Control group; DMSO: Dimethyl sulfoxide; i.p: Intraperitoneal; Q: Quercetin; R: Resveratrol; SCI: Spinal cord injury group; SCI+DMSO: Post-SCI DMSO group; SCI+Q: Post-SCI quercetin group; SCI+R: Post-SCI resveratrol group; SCI+R+Q: Post-SCI combined treatment group. 424 Ulus Travma Acil Cerrahi Derg, September 2016, Vol. 22, No. 5 Çiftçi et al. Efficiacy of resveratrol and quercetin after experimental spinal cord injury DP70 digital camera. Histological scoring of SCI was as follows: ter of groups. When multiple comparison tests were exam- no damage (0), very mild (1), mild (2), moderate (3), and se- ined in order to determine inter-group differences, significant vere (4). Tissue samples were also examined for hemorrhage, increase was observed in hemorrhage in SCI and post-SCI edema, necrosis, axonal swelling, chromatolysis, polymorpho- DMSO groups compared with control group (p<0.05) (Figs. nuclear leukocyte
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