Inflammation ( # 2015) DOI: 10.1007/s10753-015-0179-0

The Neuroprotective Effect of Coumaric Acid on Spinal Cord Ischemia/Reperfusion Injury in Rats Mustafa Guven,1,8 Muserref Hilal Sehitoglu,2 Yasemin Yuksel,3 Mehmet Tokmak,4 Adem Bozkurt Aras,1 Tarik Akman,1 Umut Hatay Golge,5 Ergun Karavelioglu,6 Ercan Bal,7 and Murat Cosar1

Abstract—The main causes of spinal cord ischemia are a variety of vascular pathologies causing acute arterial occlusions. We investigated neuroprotective effects of coumaric acid on spinal cord ischemia injury in rats. Rats were divided randomly into four groups of eight animals as follows: control, ischemia, ischemia + coumaric acid, and ischemia + methylprednisolone. In the control group, only a laparotomy was performed. In all other groups, the spinal cord ischemia was performed by the infrarenal aorta cross-clamping model. Levels of malondialdehyde and nuclear respiratory factor 1 were analyzed, as were the activity of superoxide dismutase. Histopathological and immunohistochemical evaluations were performed. Neurological evaluation was performed with the Tarlov scoring system. The ischemia + coumaric acid group was compared with the ischemia group, and a significant decrease in malondialdehyde and levels was observed. Nuclear respiratory factor 1 level and superoxide dismutase activity of the ischemia + coumaric acid group were significantly higher than in the ischemia group. In histopathological samples, the ischemia + coumaric acid group is compared with the ischemia group, and there was a significant increase in numbers of normal neurons. In immunohistochemical staining, hypoxia-inducible factor-1α and NF-kappa B immunopositive neurons were significantly decreased in the ischemia + coumaric acid group compared with that in the ischemia group. The neurological deficit scores of the ischemia + coumaric acid group were significantly higher than the ischemia group at 24 h. Our results revealed for the first time that coumaric acid exhibits meaningful neuroprotective activity following ischemia-reperfusion injury of the spinal cord. KEY WORDS: coumaric acids; spinal cord ischemia; hypoxia-inducible factor 1; NF-kappa B.

INTRODUCTION 1

Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 2 Department of Medical Biochemistry, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 3 Department of Histology & Embryology, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey 4 Department of Neurosurgery, Faculty of Medicine, Medipol University, Istanbul, Turkey 5 Department of Orthopaedics, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey 6 Department of Neurosurgery, Faculty of Medicine, Afyon Kocatepe University, Afyon, Turkey 7 Department of Neurosurgery, Ataturk Education and Research Hospital, Ankara, Turkey 8 To whom correspondence should be addressed at Department of Neurosurgery, Faculty of Medicine, Canakkale Onsekiz Mart University, Canakkale, Turkey. E-mail: [email protected]

Spinal cord ischemia (SCI) and reperfusion (SCIR) may develop in a variety of situations. The main causes of situations with reduced perfusion are a variety of vascular pathologies causing acute arterial occlusion, surgical interventions requiring clamping, trauma-causing ischemia, transplantation, and shock. A wide range of complications, including everything from paraparesis to death, may develop after SCIR damage. Reversible or irreversible cell damage related to ischemia is linked to insufficient blood flow perfusing organs or tissues. Ischemia disrupts oxidative phosphorylation in the cell, causing reduced intercellular adenosine triphosphate and phosphocreatine synthesis. This situation disrupts the ionic pump function related to ATP in the cell membrane and results in increased calcium, sodium, and

0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York

Guven, Sehitoglu, Yuksel, Tokmak, Aras, Akman, Golge, Karavelioglu, Bal, and Cosar water entering the cell. During ischemia, degradation of adenine nucleotides increases. This causes increased accumulation of hypoxanthine, the precursor of reactive oxygen species (ROS), within the cell. After ischemia, the reperfusion of the region and the renewed presence of molecular oxygen in the cell create ROS [1]. It is thought that the ROS linked to high levels of malondialdehyde (MDA) causes lipid peroxidation. Lipid peroxidation results in damage to cell membranes. ROS is cleared from the cell by superoxide dismutase (SOD) enzyme [2]. SCI, on the other hand, is known to damage mitochondria. Therefore, in the present study, we analyzed the protein marker of mitochondrial biogenesis (nuclear respiratory factor-1, NRF1) [3]. Coumaric acid (CA), assumed in the polyphenolic compounds, is synthesized from cinnamic acid by means of the P450-dependent 4-cinnamic acid hydroxylase enzyme [4]. The main sources of CA are chocolate; apples and pears; beans, soybeans, potato, and tomato; and tea, coffee, wine, and beer [5]. In our study, the antioxidant and protective effects of CA on SCIR damage in rats were investigated. With this aim, both the effects of CA on SOD activity and MDA and NRF1 levels after SCIR were analyzed. Additionally, the dead neurons and the immunopositive cells were counted in spinal cord histological samples. Toluidine-blue staining was used to evaluate general histological examination, and hypoxia-inducible factor-1α (HIF-1α) and nuclear factor kappa B (NF-kappa B) primary antibodies were used to label and evaluate these proteins immunohistochemically.

the temperature and humidity were held at 21±2 °C and 55–60 %. Experimental Design Rats were randomly divided into four equal groups consisting of eight rats each: Group I Sham-operated control group: Laparotomy and infrarenal abdominal aorta dissection was completed, but occlusion was not performed. Group II A single-dose 1-ml intraperitoneal vehicle (isotonic NaCl 0.9 %) was administered to rats following 20-min spinal cord ischemia and 24-h reperfusion (ischemia group). Group III Intraperitoneal CA (single-dose 100 mg/kg body weight) was administered to rats following 20-min spinal cord ischemia, reperfusion was performed 24 h after ischemia, and rats were sacrificed 24 h after ischemia (ischemia + CA group). Group IV Intraperitoneal methylprednisolone (single-dose 30 mg/kg body weight) was administered to rats following 20-min spinal cord ischemia, reperfusion was performed 24 h after ischemia, and rats were sacrificed 24 h after ischemia (ischemia + methylprednisolone (MP) group). Dosage

MATERIALS AND METHODS Animals The methods used for animal experiments were in accordance with the international guiding principles for biomedical research involving animals recommended by the World Health Organization. Permission was granted by Canakkale Onsekiz Mart University Animal Experiments Local Ethics Committee (protocol number: 2012/08-14, date of approval: December 27, 2012). This study was conducted at the Canakkale Onsekiz Mart University Experimental Research Center. In this study, we used 32 male Sprague-Dawley rats weighing 250–350 g. All animals were fed ad libitum with 7–8 mm pellet rat food (Bil-Yem Ltd, Ankara, Turkey) and tap water. To establish a 12-h darkness/12-h light environment, photoperiodic white fluorescent light was used, and

The CA dosage was set at 100 mg/kg body weight based on preliminary studies with various doses (50, 75, 100 mg) to reveal the biological effects of CA [4, 6–11]. The ischemia + MP group received a single intraperitoneal 30 mg/kg dose of MP immediately after the occlusion clips were removed. This MP dosage was selected based on earlier studies [12–14]. Reagents Coumaric acid (≥98 % HPLC) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Methylprednisolone was obtained from Mustafa Nevzat Drug Industry Inc. (Istanbul, Turkey). The drugs were dispersed with isotonic NaCl 0.9 %. The protein concentrations were determined by the Bradford method using Bradford reagent (Cat. No. B6916-1KT; Sigma-Aldrich, St. Louis, MO, USA). An SOD assay kit (Cat. No. 19160) and a lipid peroxidation (MDA) assay kit (Cat. No. MAK085) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A rat nuclear

Neuroprotective Effect of Coumaric Acid on Spinal Cord Ischemia respiratory factor 1 ELISA kit (Cat. No. CK-E90555) was obtained from Hangzhou Eastbiopharm Co. Ltd. (Hangzhou, China). HIF-1α primary antibody (Cat. No. PA116601) was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). NF-kappa B primary antibody (Cat. No. sc-109) was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Surgical Procedure SCIR was induced as reported by Zivin et al. [15]. A Biopac MP36 (BIOPAC Systems Inc., Goleta, CA, USA) device was used as a monitor. Mean arterial pulse was 375 beats/min during surgery. Body temperature was monitored with a rectal probe and was adjusted to 37.1–37.4 °C with a heating pad during surgery. Rats were given premedication with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg). Anesthesia was continued with ketamine injections at intervals without intubation or mechanical ventilation. Heparin (100 U/kg) was administered intravenously 5 min before aortic clipping. The surgical approach was via the supine position. After the operating field was prepared in a sterile fashion, laparotomy was performed with a standard midline incision. After retracting the intestines laterally, the retroperitoneum was opened and was approached to the abdominal aorta. Spinal cord ischemia was induced by crossclamping the aorta with mini aneurysm clips of 70-g closing force (Yasargil, FE721, Aesculap, Germany) between just below the left renal artery and just proximal to the aortic bifurcation. Loss of aortic pulse was confirmed visually and by palpation. The duration of ischemia was set at 20 min, and later, the clips were removed and distal reperfusion was observed visually. At the end of the procedure, the abdominal wall was closed with 5/0 Prolene sutures. Animals in the control group underwent a surgical procedure similar to that performed on the other groups, but the aorta was not clamped. This group of animals was used to elicit the effects of anesthesia and operation on the results and to determine the biochemical parameters studied in the normal spinal cord tissue. After surgery, animals were fed on a standard diet and water ad libitum in their cages. At the 24th hour, all animals were anesthetized with pentobarbital (20 mg/kg) and sacrificed. The lumbar spinal cord was harvested immediately via the posterior approach. Each spinal cord was longitudinally divided into two equal parts with a fine scalpel. Half of the specimen was kept for histopathological investigation, and it was fixed in

formalin for 7 days. The other half was stored in a freezer at −80 °C for biochemical estimations. Evaluation of Neurological Status The neurological status of the animals was assessed blindly by a neurologist at 1, 12, and 24 h after SCIR. To assess the motor function of rats after SCIR, rats were scored by a modified Tarlov’s scale [16] as follows: 0, no lower extremity movement; 1, lower extremity motion without gravity; 2, lower extremity motion against gravity; 3, able to stand with assistance; 4, able to walk with assistance; and 5, normal. Biochemical Investigations of Spinal Cord Tissues For biochemical investigation, MDA and NRF1 levels and SOD activity from each supernatant were measured in duplicate with highly sensitive ELISA spectrophotometry, respectively. All the data were defined as mean±standard deviation (SD) results based on per milligram of protein. The protein concentrations were determined by the Lowry method [17] using commercial protein standards (Sigma-Aldrich, St. Louis, MO, USA, total protein kit-TP0300-1KT). NRF1 Assay Principles A rat NRF1 ELISA kit was used to assay NRF1 on the basis of the biotin double antibody sandwich technology [18]. Absorbances of each well were measured under 450-nm wavelength. The results were expressed as nanogram per milliliter NRF1 per milligram protein. SOD Activity Assay Principles Tissue SOD activity was measured with a modified spectrophotometric method at 450 nm as described by Sun et al. [19]. It was also measured with an SOD assay kit using highly sensitive ELISA spectrophotometry. The results were expressed as unit per milliliter SOD per milligram protein. MDA Assay Principles Tissue MDA levels were determined according to Buege’s method [20]. Lipid peroxidation was determined by the reaction of MDA with thiobarbituric acid (TBA) to form a colorimetric (532 nm) product, proportional to the MDA present. A lipid peroxidation (MDA) assay kit was used to determine MDA levels. The results were expressed as nanomole per milliliter MDA per milligram protein.

Guven, Sehitoglu, Yuksel, Tokmak, Aras, Akman, Golge, Karavelioglu, Bal, and Cosar Histopathological Examination The spinal cord samples were fixed in 10 % neutral formalin. After dehydration in graded ethanol, tissue samples were embedded in paraffin and cut into sections of 7 μm in thickness. Tissue samples were stained with hematoxylin-eosin (H-E) and toluidine blue for general histopathological examination. HIF-1α and NF-kappa B primary antibodies were used to label these proteins in the spinal cord following ischemic injury. Nissl Staining After being dewaxed and rehydrated, the spinal cord sections were stained with 0.1 % toluidine blue for 3 min, rinsed in distilled water, dehydrated in ethanol solutions with increasing concentrations, and cleared in xylene. The neurons containing Nissl bodies, loose chromatin, and prominent nucleoli were considered to be normal neurons, and the damaged neurons were identified by the loss of Nissl bodies, cavitation around nucleus, and the presence of pyknotic nuclei. Immunohistochemistry Samples mounted on polylysine-coated slides were deparaffinized and hydrated in graded alcohols and immunohistochemically stained with HIF-1α and NFkappa B primary antibodies. Citrate buffer (pH=6.0) was used for antigen retrieval by microwaving for 20 min. It was then washed in TBS, and endogenous peroxidase activity was blocked by using 3 % hydrogen peroxidase in methanol for 12 min. Ultraviolet-blocking solution was used for 5 min, then sections were incubated with HIF-1α (1:100 dilution) and NF-kappa B (1:100 dilution) primary antibodies at 4 °C overnight. The next day, sections were washed in TBS. Biotinylated goat anti-polyvalent antibody (UltraVision Large Volume Detection System, HRP (ready-to-use), TP-125-HL, Thermo Scientific Inc., Waltham, MA, USA) was used without dilution as secondary antibodies. Sections were incubated for 20 min at room temperature (20–22 °C). Then, sections were visualized with an AEC kit (Labvision Corp., Fremont, CA, USA) for chromogen. Finally, all the slides were counterstained with Mayer’s hematoxylin (Thermo Scientific Inc., Waltham, MA, USA) and mounted with a water-based mounting medium. Image analysis All the stained sections were observed under a light microscope (Eclipse E-600 Nikon, Tokyo, Japan). Image

analysis of sections after immunohistochemistry was done by Image Analysis Software (NIS Elements Nikon, Tokyo, Japan). Intact motor neuron cells and immunopositive cells with HIF-1α and NF-kappa B were counted by Image Analysis Software (NIS Elements Nikon, Tokyo, Japan). Gray matter damage was assessed on the basis of the number of normal neurons per microscopic field in the right and left ventral horn with ×200 magnification. Neurons with properly shaped and granular cytoplasm containing Nissl bodies, loose chromatin, and prominent nucleoli were considered to be normal neurons. Neurons with intensely darkening-shrunken or vacuolated cytoplasm, pyknotic nuclei, and disappearance of Nissl bodies were considered to be necrotic and degenerated neurons. For the evaluation of protein expression of NF-kappa B and HIF-1α, the immunopositive cells in the gray matter of spinal cord sections were counted and the data was statistically analyzed.

Statistical Analysis Biochemical results were subjected to one-way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS 19.0, SPSS Inc., Chicago, IL, USA) software. Differences among the groups were obtained using Tukey’s test option. For histopathological results, the Kruskal-Wallis test was used to analyze the differences between the groups, and the Mann-Whitney U test was used for pairwise comparisons. Statistical significance was set at p