Original Article
Effects of Intrathecal Caffeic Acid Phenethyl Ester and Methylprednisolone on Oxidant/ Antioxidant Status in Traumatic Spinal Cord Injuries Cuneyt Gocmez1 Feyzi Celik2 Osman Evliyaoglu5
Kagan Kamasak1
1 Department of Neurosurgery, University of Dicle, Diyarbakir, Turkey 2 Department of Anesthesiology, University of Dicle, Diyarbakir, Turkey 3 Department of Neurosurgery, University of Fırat, Elazığ, Turkey 4 Department of Neurology, University of Dicle, Diyarbakir, Turkey 5 Department of Biochemistry, University of Dicle, Diyarbakir, Turkey
Metin Kaplan3
Ertugrul Uzar4
Adalet Arıkanoglu4
Address for correspondence Cuneyt Gocmez, MD, Dicle Üniversitesi, Beyin Cerrahisi AD, Diyarbakir 21280, Turkey (e-mail: [email protected]).
J Neurol Surg A 2015;76:20–24.
Abstract
Keywords
► spinal cord injury ► caffeic acid phenethyl ester ► lipid peroxidation ► intrathecal
Purpose To examine the effect of intrathecally given caffeic acid phenethyl ester (CAPE) on peroxidation and total oxidant and antioxidant systems, and the effect of intrathecally given methylprednisolone (MP) in spinal cord injury (SCI) models. Materials and Methods Four groups of 10 rats were formed: (1) Laminectomy, intrathecal saline injection, no SCI (sham: S); (2) Laminectomy, intrathecal saline injection, SCI (control: SCI); (3) Laminectomy, intrathecally given single dose of 3 mg/kg MP, SCISCI (SCI þ MP). 4) Laminectomy, intrathecally given single dose of 1 µg/kg CAPE, SCI (SCI þ CAPE). Malondialdehyde (MDA), total oxidant activity (TOA), total antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) values in the spinal cord tissue were evaluated. Results When group S and group SCI were compared, MDA, TOA, and SOD parameters increased post-SCI (p < 0.01). When compared with group SCI, it was observed that CAPE and MP decreased the MDA, TOA, and SOD levels (p < 0.01). This decrease was more pronounced in the SCI þ CAPE group. When group S and group SCI were compared, a statistically substantial decrease was observed in the post-SCI TAC levels. When compared with group SCI, it was shown that CAPE and MP treatment substantially increased TAC levels (p < 0.001). Conclusion Intrathecal injection of both CAPE and MP inhibits lipid peroxidation and increase of oxidants in SCIs.
Introduction Traumatic spinal cord injury (SCI) is composed of primary spinal cord damage and of a series of secondary biochemical mechanisms leading to further damage.1–3 Posttraumatic decomposition in the tissue perfusion increases the generation of oxygen
received March 1, 2013 accepted after revision January 13, 2014 published online May 28, 2014
radicals and gives way to lipid peroxidation. Lipid peroxidation is an important step in secondary spinal cord damage. Increase of lipid peroxidation causes the cell membrane to loose its integrity and increases functional degradation.4–6 Many studies indicate that a high dose of methylprednisolone (MP) has antioxidant effects of and inhibits lipid peroxidation.1,7,8 Despite its side
© 2015 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0034-1371513. ISSN 2193-6315.
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20
Effects of CAPE and Methylprednisolone in Spinal Cord Injuries
Materials and Methods Animals and Study Groups After the approval by the Dicle University Animal trials Ethics Board, 40 adult female Wistar albino rats with normal motor function, weighing 220 to 260 g, were included in the study. Standard laboratory conditions (illumination 12 hours/day and darkness 12 hours/night, with room temperature 22–22°C) were provided for the rats. Four groups of 10 rats were formed. 1. Sham (S): 10 µL intrathecally given isotonic saline after laminectomy. 2. Control (SCI): 10 µL intrathecally given isotonic saline after SCI was generated.3. SCI þ MP: 10 µL (3 mg/kg) intrathecally given MP (Prednol-L, Mustafa Nevzat Pharmaceuticals, Istanbul, Turkey) after SCI was generated. The MP doses were selected according to our previous study.144. SCI þ CAPE: 10 µL (1 µg/kg) intrathecally given CAPE after SCI was generated.
Intrathecal Catheterization All rats were intraperitoneally given 80 mg kg1 ketamine keeping spontaneous respiration. For intrathecal applications, a polyethylene tube (inner diameter: 0.28; mm; outer: diameter 0.61 mm; Becton Dickinson, Philadelphia, Pennsylvania, United States) was used.15,16 The rats that did not experience engine functionality disorder in their front or rear legs after the procedure were injected with 200 µg 2% lidocaine. Within 30 seconds after the injection, paralysis and dragging behavior were observed in the rear legs of the rats, confirming intrathecal implantation of the catheter.
Generation of SCI An area of 3 2 cm on the back was shaved, local antisepsis was achieved with polyvidone iodine, and a single-level laminectomy was performed while being careful not to damage the dura mater at the T5–T12 level. In all but the group S rats, the dura and spinal cord were clipped for 1 minute using a Yasargil aneurysm (Aesculap FE 721 K, B. Braun Company, Hazelwood, Missouri, United States) with a 63-g force around. Following hemostasis, the incision field was closed with 3–0 silk. Drug and saline applications to each group were made using the Hamilton injector (Hamilton
21
Bonaduz AG, Bonaduz, Switzerland) via the intrathecal catheter. Catheters were cleansed with 5 µL saline after the drug administrations. The body temperature of each rat was kept at 37°C during the process via rectal heat probe monitoring. Then, 24 hours later, transcardiac blood samples were taken, the rats were killed, and their spinal cord tissue was extracted.
Biochemical Procedure The excised tissue samples were weighed and immediately stored at 30°C. Tissues were perfused with ice-cold phosphate buffer, minced, and then homogenized in five volumes (w/v) of the same solution. Assays were performed on the supernatant of the homogenate that was prepared at 14,000 rpm for 30 minute at þ 4°C.17 The protein concentration of the tissue was measured by the Lowry method.18 Malonyldialdehyde (MDA) content was measured spectrophotometrically as described previously19. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were measured according to the method described by the manufacturer (Cayman Chemical, Ann Arbor, Michigan, United States). The total antioxidant capacity (TAC) of supernatant fractions was evaluated by using the automated and colorimetric measurement method developed by Erel.20 TAC results are expressed as nmol Trolox equivalent/mg protein. The total oxidant activity (TOA) of supernatant fractions was evaluated by using the automated and colorimetric measurement method developed by Erel.21 TOA results are expressed in terms of nmol hydrogen peroxide (H2O2) equivalent/mg protein.
Statistical Analysis Statistical analysis was performed by SPSS for Windows v.13.0 (SPSS Inc., Chicago, Illinois, United States). Data were presented as mean values standard deviation for biochemical values. Groups were compared by using the nonparametric Kruskal-Wallis test. The Mann-Whitney U test was used for binary comparisons. The p values < 0.05 were considered significant.
Results When group S and group SCI were compared, a significant increase of MDA, TOA and SOD were found after SCI (p < 0.01). Additionally, a not statistically significant increase in the post-SCI GPx activity was seen (p > 0.05). Details of MDA, TOA, SOC, and GPx values of all groups can be seen in ►Table 1 (►Fig 1). When compared with group SCI, it was observed that CAPE and MP decreased MDA, TOA, and SOD levels in group SCI þ CAPE and SCI þ MP (p < 0.01). This decrease was more pronounced in the SCI þ CAPE group (p < 0.05) (►Table 1). When group S and group SCI were compared, a statistically significant decrease was observed in the post-SCI TAC levels (►Table 1). When compared with group SCI, it was seen that CAPE (group SCI þ CAPE) and MP (group SCI þ MP) treatments statistically increased TAC levels (p < 0.05). This Journal of Neurological Surgery—Part A
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effects, high MP doses have been used widely for the treatment of SCI in the clinic setting. Caffeic acid phenethyl ester (CAPE) is an active component of propolis and known to have anti-inflammatory and antioxidant effects. It has been reported to inhibit lipid peroxidation as well.9–12 CAPE is one of the most powerful lipophilic antioxidants. CAPE has been shown to decrease spinal cord ischemia and to have a protective role in the spinal cord because of its antioxidant, anti-inflammatory, and neuroprotective effects.13 Thus, CAPE has the potential to prevent secondary spinal cord damage after traumatic SCI. However, as far as we know, the protective effect of intrathecally given CAPE in SCI has not been researched. Our purpose was to examine the effect of intrathecal CAPE on peroxidation and total oxidant and antioxidant systems, and also to demonstrate the effects of methylprednisolone (MP) in SCI models.
Göçmez et al.
Effects of CAPE and Methylprednisolone in Spinal Cord Injuries
Göçmez et al.
Table 1 Biochemical parameters according to groups Parameters
S Mean SD
SCI Mean SD
SCI þ MP Mean SD
SCI þ CAPE Mean SD
p
GPx U/g prt
141.54 43.33
166.53 54.60
174.28 94.88
150.50 54.84
NS
MDA (nmol/g prt)
1.81 0.39
3.08 0.69a,
2.00 0.85f
1.60 0.34d
< 0.001
SOD (U/g prt)
5.3145 1.04
8.67 2.39b
5.77 1.03e
3.97 1.33c,d,i
0.001
TAC (nmol Trolox Eq./g prt)
0.40 0.05
0.03 0.02a
0.28 .07a, d
0.12 0.03a,d,g
< 0.001
TOA (nmol H2O2 Eq./g prt)
75.69 16.20
129.12 37.21b
85.10 15.01e
60.01 20.53d,h
0.001
Abbreviations: CAPE, caffeic acid phenethyl ester; Eq., equivalent; GPx, glutathione peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde; MP, methylprednisolone; NS, not significant; prt, protein; S, sham; SCI, spinal cord injury; SD, standard deviation; SOD, superoxide dismutase; TAC, total antioxidant capacity; TOA, total oxidant activity. a Significantly different from sham (S) group (p 0.001). b Significantly different from sham (S) group (p 0.01). c Significantly different from sham (S) group (p 0.05). d Significantly different from control (SCI) group (p 0.001). e Significantly different from control (SCI) group (p 0.05). f Significantly different from control (SCI) group (p 0.01). g Significantly different from treatment (SCI þ MP) group (p 0.001). h Significantly different from treatment (SCI þ MP) group (p 0.05). i Significantly different from treatment (SCI þ MP) group (p 0.01).
increase was more pronounced in the SCI þ MP group (►Table 1). It was seen that the SOD activity in group SCI was statistically higher compared with that in group S. When compared with group SCI, group SCI þ MP and group SCI þ CAPE had statistically lower SOD activity. This decrease was
more pronounced in the SCI þ CAPE group than in the SCI þ MP group (p < 0.001) (►Table 1). After examining GPx activity, all groups were found to have higher GPx activity than group S; however, these values were not statistically significant. CAPE did not substantially affect GPx levels (►Table 1).
Fig. 1 The oxidant and antioxidant parameters according to the groups. GPx, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; TAC, total antioxidant capacity; TOA, total oxidant activity. Journal of Neurological Surgery—Part A
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Discussion The secondary damage mechanism which is caused by perfusion decomposition in the post-trauma disintegrated spinal cord play a major role in increasing the extended damage.3 Increasing edema and inflammation, generation of oxygen radicals and lipid peroxidation, and dysfunction of adenosine diphosphate–dependent channels are important components of this vicious cycle.1,2,4,22–24 Lipid peroxidation can be defined as degradation of the lipids in the cell membrane after interaction with oxygen radicals. Free radicals initiate a chain reaction that breaks down the double bonds of unsaturated fatty acids in the presence of oxygen. As a result, cell membrane stability is disturbed, permeability is affected, and the ability to form membrane potential is impaired.25,26 Disintegration of the cell membrane is an important step of irreversible cell damage. Therefore, blocking generation of oxygen radicals and lipid peroxidation are both important to disrupt the vicious cycle in SCIs. MDA and 4-hydroxynonenal (4-HNE) are two markers of lipid peroxidation. MDA is a product of the breakdown of unsaturated fatty acids into their essential chains by way of the oxidation mechanism.27 We found in our study that when compared with group SCI, group SCI þ CAPE had lower MDA levels. This decrease was more prominent when compared with group SCI þ MP. Additionally, the fact that the SCI þ CAPE group had lower MDA levels than group S was interesting. This showed that the potential of intrathecally given CAPE to inhibit lipid peroxidation is very high. When SOD levels were examined, MP and CAPE decreased the SOD level, while the SOD level increased in group SCI. The fact that SOD levels were lower in the SCI þ CAPE group may be a result of the lipid peroxidation inhibiting effect of CAPE. It is not clear through what mechanism CAPE inhibits lipid peroxidation. It is currently being discussed whether the lipid peroxidation inhibiting effects of CAPE are based on an enzymatic or nonenzymatic antioxidant defense system. MDA is produced by both free radical–induced as well as enzymatically induced lipid peroxidation (i.e., arachidonic acid cascade). Thus, in SCI where both processes have long been known to occur acutely and in parallel, the fact that MDA is reduced by CAPE and MP cannot be purely associated with inhibition of free radical–induced peroxidation. In future studies, 4-HNE should be measured by immunoblot because 4-HNE is only produced by free radical–induced peroxidation. Antioxidants, including enzymatic systems such as SOD and GPx, provide the primary antioxidant defense. SOD converts superoxide anion to H2O2. SOD acts against reactive oxygen species and is active in catalyzing detoxification of superoxide radicals. The H2O2 generated in this reaction is restored to water in the presence of GPx.28 The SOD increase in the SCI group in our study indicates a higher SOD activity to eradicate the free oxygen radicals generated after SCI. The increase of GPx enzyme activity in the SCI group was not statistically significant compared with that in group S, showing that oxidative stress in SCI is generated via lipid peroxidation. The fact that SOD and GPx concentration in the SCI þ CAPE group are similar to that in group S is another indicator of
Göçmez et al.
CAPE’s powerful antioxidant effect. Because free oxygen radicals could not have been overproduced, antioxidant enzymes may have remained within normal values in the presence of CAPE. Our results were supported by Ilhan and his colleagues, who investigated the lipid peroxidation inhibiting effect of CAPE in a spinal cord ischemia reperfusion model.9 Previous studies have suggested that oxidative stress and lipid peroxidation play a role in SCI. Serum concentrations of different oxidant species can be measured separately in the laboratory; however, these measurements are time consuming, overly expensive, and require complicated techniques. Measuring different antioxidant and oxidant molecules is not practical, and their antioxidant and oxidant effects are additive. Because there exist plenty of antioxidants and oxidants, measuring total oxidant-antioxidant activity is more valid and credible.19,20,29 When solely a few antioxidant parameters are measured, their levels may be unchanged or decreased, even when the actual oxidant-antioxidant activity is increased. In recent years, total levels of different oxidant compounds have been monitored by determining the TOA.21 Moreover, TAC is a useful estimate of the activity of the total antioxidants in a medium.20 Similar to a previous study, we saw that TAC significantly decreases after SCI.30 This means that the antioxidant defense system degrades following the SCI. In addition, we showed the increase of TOA for the first time, in contradistinction to other experimental SCI studies. This finding proved that SCI causes an increase in total oxidants. Intrathecal injection of both CAPE and MP inhibits lipid peroxidation and the generation of oxidants in SCI. The inhibitory effect of CAPE was more pronounced than that of MP. Additionally, CAPE and MP treatments increased TAC, which was decreased as a result of spinal cord damage. In contrast to the other findings, MP increased TAC levels more significantly than CAPE. Posttraumatic degradation of the anatomical integrity of the spinal cord and the surrounding tissue may prevent systematically applied drugs from reaching a maximally effective dose in the damaged area. Therefore, intrathecal drug application is used as an alternative method in central nervous system diseases. In our study, we administered CAPE intrathecally, unlike in other studies in the literature. The fact that CAPE efficiently inhibited lipid peroxidation may also be a result of the intrathecal application. In the NASCIS II and III trials, continually given MP was shown to have detrimental effects on damaged spinal cord at high doses (30 mg/kg). Thus, we preferred to use MP at low doses in one of our earlier experiments.14,31 We showed that using MP intrathecally at doses of 3 mg/kg provides a neuroprotective effect against SCI.14 According to our information, CAPE was not used intrathecally in previous experiments, but was administered intraperitoneally in doses of 10 µg/kg.13 In our experiments, we administered CAPE intrathecally at lower doses (1 µg/kg) because we aimed to demonstrate the direct effect of CAPE on SCI. It is one of the limitations of our experiment that the study does not show the correlation between the response curve against SCI and CAPE dose. In future experiments, the dose-response curve of using CAPE intrathecally could be researched. Because we have seen CAPE to have an effect if given intrathecally at doses of Journal of Neurological Surgery—Part A
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Effects of CAPE and Methylprednisolone in Spinal Cord Injuries
Effects of CAPE and Methylprednisolone in Spinal Cord Injuries 1 µg/kg, this dose could be taken as a reference in future experiments. In conclusion, intrathecal injection of both CAPE and MP clearly inhibit the lipid peroxidation and the increase of oxidants at 24 hours after SCI. Additionally, CAPE and MP treatment increased TAC, which was decreased as a result of spinal cord damage.
14 Celik F, Gocmez C, Kamasak K, et al. The comparison of neuro-
15 16
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References 1 Jia Z, Zhu H, Li J, Wang X, Misra H, Li Y. Oxidative stress in spinal
2
3
4 5
6
7
8 9
10
11
12
13
cord injury and antioxidant-based intervention. Spinal Cord 2012; 50(4):264–274 Duz B, Kaplan M, Bilgic S, Korkmaz A, Kahraman S. Does hypothermic treatment provide an advantage after spinal cord injury until surgery? An experimental study. Neurochem Res 2009;34(3): 407–410 Dalgic A, Okay O, Helvacioglu F, et al. Tobacco-induced neuronal degeneration via cotinine in rats subjected to experimental spinal cord injury. J Neurol Surg A Cent Eur Neurosurg 2013;74(3):136–145 Hall ED. Antioxidant therapies for acute spinal cord injury. Neurotherapeutics 2011;8(2):152–167 Aslan A, Cemek M, Eser O, et al. Does dexmedetomidine reduce secondary damage after spinal cord injury? An experimental study. Eur Spine J 2009;18(3):336–344 Topsakal C, Kilic N, Ozveren F, et al. Effects of prostaglandin E1, melatonin, and oxytetracycline on lipid peroxidation, antioxidant defense system, paraoxonase (PON1) activities, and homocysteine levels in an animal model of spinal cord injury. Spine 2003;28(15): 1643–1652 Cayli SR, Kocak A, Yilmaz U, et al. Effect of combined treatment with melatonin and methylprednisolone on neurological recovery after experimental spinal cord injury. Eur Spine J 2004;13(8):724–732 Bains M, Hall ED. Antioxidant therapies in traumatic brain and spinal cord injury. Biochim Biophys Acta 2012;1822(5):675–684 Ilhan A, Koltuksuz U, Ozen S, Uz E, Ciralik H, Akyol O. The effects of caffeic acid phenethyl ester (CAPE) on spinal cord ischemia/ reperfusion injury in rabbits. Eur J Cardiothorac Surg 1999; 16(4):458–463 Hoşnuter M, Gürel A, Babucçu O, Armutcu F, Kargi E, Işikdemir A. The effect of CAPE on lipid peroxidation and nitric oxide levels in the plasma of rats following thermal injury. Burns 2004;30(2):121–125 Sud’ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 1993;329(1-2):21–24 Kasai M, Fukumitsu H, Soumiya H, Furukawa S. Caffeic acid phenethyl ester reduces spinal cord injury-evoked locomotor dysfunction. Biomed Res 2011;32(1):1–7 Uzar E, Sahin O, Koyuncuoglu HR, et al. The activity of adenosine deaminase and the level of nitric oxide in spinal cord of methotrexate administered rats: protective effect of caffeic acid phenethyl ester. Toxicology 2006;218(2–3):125–133
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19
20
21 22
23 24
25
26
27
28
29
30
31
protective effects of intrathecal dexmedetomidine and methylprednisolone in spinal cord injury. Int J Surg 2013;11(5):414–418 Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17(6):1031–1036 Korkmaz HA, Maltepe F, Erbayraktar S, et al. Antinociceptive and neurotoxicologic screening of chronic intrathecal administration of ketorolac tromethamine in the rat. Anesth Analg 2004;98(1): 148–152 Senoglu M, Nacitarhan V, Kurutas EB, et al. Intraperitoneal alphalipoic acid to prevent neural damage after crush injury to the rat sciatic nerve. J Brachial Plex Peripher Nerve Inj 2009;4:22 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1): 265–275 Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407–421 Erel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 2004;37(4):277–285 Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38(12):1103–1111 Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 1991;75(1):15–26 Collins WF. A review and update of experiment and clinical studies of spinal cord injury. Paraplegia 1983;21(4):204–219 Amar AP, Levy ML. Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery 1999;44(5):1027–1039; discussion 1039–1040 Adibhatla RM, Hatcher JF. Phospholipase A(2), reactive oxygen species, and lipid peroxidation in CNS pathologies. BMB Rep 2008; 41(8):560–567 Muralikrishna Adibhatla R, Hatcher JF. Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia. Free Radic Biol Med 2006;40(3):376–387 Lin Y, Vreman HJ, Wong RJ, Tjoa T, Yamauchi T, Noble-Haeusslein LJ. Heme oxygenase-1 stabilizes the blood-spinal cord barrier and limits oxidative stress and white matter damage in the acutely injured murine spinal cord. J Cereb Blood Flow Metab 2007;27(5): 1010–1021 Temiz C, Solmaz I, Tehli O, et al. The effects of splenectomy on lipid peroxidation and neuronal loss in experimental spinal cord ischemia/reperfusion injury. Turk Neurosurg 2013;23(1):67–74 Buyukhatipoglu H, Sezen Y, Yildiz A, et al. N-acetylcysteine fails to prevent renal dysfunction and oxidative stress after noniodine contrast media administration during percutaneous coronary interventions. Pol Arch Med Wewn 2010;120(10):383–389 Ozgiray E, Serarslan Y, Oztürk OH, et al. Protective effects of edaravone on experimental spinal cord injury in rats. Pediatr Neurosurg 2011;47(4):254–260 Pettiford JN, Bikhchandani J, Ostlie DJ, St Peter SD, Sharp RJ, Juang D. A review: the role of high dose methylprednisolone in spinal cord trauma in children. Pediatr Surg Int 2012;28(3):287–294
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Effects of Intrathecal Caffeic Acid Phenethyl Ester and Methylprednisolone on Oxidant/ Antioxidant Status in Traumatic Spinal Cord Injuries Cuneyt Gocmez1 Feyzi Celik2 Osman Evliyaoglu5
Kagan Kamasak1
1 Department of Neurosurgery, University of Dicle, Diyarbakir, Turkey 2 Department of Anesthesiology, University of Dicle, Diyarbakir, Turkey 3 Department of Neurosurgery, University of Fırat, Elazığ, Turkey 4 Department of Neurology, University of Dicle, Diyarbakir, Turkey 5 Department of Biochemistry, University of Dicle, Diyarbakir, Turkey
Metin Kaplan3
Ertugrul Uzar4
Adalet Arıkanoglu4
Address for correspondence Cuneyt Gocmez, MD, Dicle Üniversitesi, Beyin Cerrahisi AD, Diyarbakir 21280, Turkey (e-mail: [email protected]).
J Neurol Surg A 2015;76:20–24.
Abstract
Keywords
► spinal cord injury ► caffeic acid phenethyl ester ► lipid peroxidation ► intrathecal
Purpose To examine the effect of intrathecally given caffeic acid phenethyl ester (CAPE) on peroxidation and total oxidant and antioxidant systems, and the effect of intrathecally given methylprednisolone (MP) in spinal cord injury (SCI) models. Materials and Methods Four groups of 10 rats were formed: (1) Laminectomy, intrathecal saline injection, no SCI (sham: S); (2) Laminectomy, intrathecal saline injection, SCI (control: SCI); (3) Laminectomy, intrathecally given single dose of 3 mg/kg MP, SCISCI (SCI þ MP). 4) Laminectomy, intrathecally given single dose of 1 µg/kg CAPE, SCI (SCI þ CAPE). Malondialdehyde (MDA), total oxidant activity (TOA), total antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) values in the spinal cord tissue were evaluated. Results When group S and group SCI were compared, MDA, TOA, and SOD parameters increased post-SCI (p < 0.01). When compared with group SCI, it was observed that CAPE and MP decreased the MDA, TOA, and SOD levels (p < 0.01). This decrease was more pronounced in the SCI þ CAPE group. When group S and group SCI were compared, a statistically substantial decrease was observed in the post-SCI TAC levels. When compared with group SCI, it was shown that CAPE and MP treatment substantially increased TAC levels (p < 0.001). Conclusion Intrathecal injection of both CAPE and MP inhibits lipid peroxidation and increase of oxidants in SCIs.
Introduction Traumatic spinal cord injury (SCI) is composed of primary spinal cord damage and of a series of secondary biochemical mechanisms leading to further damage.1–3 Posttraumatic decomposition in the tissue perfusion increases the generation of oxygen
received March 1, 2013 accepted after revision January 13, 2014 published online May 28, 2014
radicals and gives way to lipid peroxidation. Lipid peroxidation is an important step in secondary spinal cord damage. Increase of lipid peroxidation causes the cell membrane to loose its integrity and increases functional degradation.4–6 Many studies indicate that a high dose of methylprednisolone (MP) has antioxidant effects of and inhibits lipid peroxidation.1,7,8 Despite its side
© 2015 Georg Thieme Verlag KG Stuttgart · New York
DOI http://dx.doi.org/ 10.1055/s-0034-1371513. ISSN 2193-6315.
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
20
Effects of CAPE and Methylprednisolone in Spinal Cord Injuries
Materials and Methods Animals and Study Groups After the approval by the Dicle University Animal trials Ethics Board, 40 adult female Wistar albino rats with normal motor function, weighing 220 to 260 g, were included in the study. Standard laboratory conditions (illumination 12 hours/day and darkness 12 hours/night, with room temperature 22–22°C) were provided for the rats. Four groups of 10 rats were formed. 1. Sham (S): 10 µL intrathecally given isotonic saline after laminectomy. 2. Control (SCI): 10 µL intrathecally given isotonic saline after SCI was generated.3. SCI þ MP: 10 µL (3 mg/kg) intrathecally given MP (Prednol-L, Mustafa Nevzat Pharmaceuticals, Istanbul, Turkey) after SCI was generated. The MP doses were selected according to our previous study.144. SCI þ CAPE: 10 µL (1 µg/kg) intrathecally given CAPE after SCI was generated.
Intrathecal Catheterization All rats were intraperitoneally given 80 mg kg1 ketamine keeping spontaneous respiration. For intrathecal applications, a polyethylene tube (inner diameter: 0.28; mm; outer: diameter 0.61 mm; Becton Dickinson, Philadelphia, Pennsylvania, United States) was used.15,16 The rats that did not experience engine functionality disorder in their front or rear legs after the procedure were injected with 200 µg 2% lidocaine. Within 30 seconds after the injection, paralysis and dragging behavior were observed in the rear legs of the rats, confirming intrathecal implantation of the catheter.
Generation of SCI An area of 3 2 cm on the back was shaved, local antisepsis was achieved with polyvidone iodine, and a single-level laminectomy was performed while being careful not to damage the dura mater at the T5–T12 level. In all but the group S rats, the dura and spinal cord were clipped for 1 minute using a Yasargil aneurysm (Aesculap FE 721 K, B. Braun Company, Hazelwood, Missouri, United States) with a 63-g force around. Following hemostasis, the incision field was closed with 3–0 silk. Drug and saline applications to each group were made using the Hamilton injector (Hamilton
21
Bonaduz AG, Bonaduz, Switzerland) via the intrathecal catheter. Catheters were cleansed with 5 µL saline after the drug administrations. The body temperature of each rat was kept at 37°C during the process via rectal heat probe monitoring. Then, 24 hours later, transcardiac blood samples were taken, the rats were killed, and their spinal cord tissue was extracted.
Biochemical Procedure The excised tissue samples were weighed and immediately stored at 30°C. Tissues were perfused with ice-cold phosphate buffer, minced, and then homogenized in five volumes (w/v) of the same solution. Assays were performed on the supernatant of the homogenate that was prepared at 14,000 rpm for 30 minute at þ 4°C.17 The protein concentration of the tissue was measured by the Lowry method.18 Malonyldialdehyde (MDA) content was measured spectrophotometrically as described previously19. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were measured according to the method described by the manufacturer (Cayman Chemical, Ann Arbor, Michigan, United States). The total antioxidant capacity (TAC) of supernatant fractions was evaluated by using the automated and colorimetric measurement method developed by Erel.20 TAC results are expressed as nmol Trolox equivalent/mg protein. The total oxidant activity (TOA) of supernatant fractions was evaluated by using the automated and colorimetric measurement method developed by Erel.21 TOA results are expressed in terms of nmol hydrogen peroxide (H2O2) equivalent/mg protein.
Statistical Analysis Statistical analysis was performed by SPSS for Windows v.13.0 (SPSS Inc., Chicago, Illinois, United States). Data were presented as mean values standard deviation for biochemical values. Groups were compared by using the nonparametric Kruskal-Wallis test. The Mann-Whitney U test was used for binary comparisons. The p values < 0.05 were considered significant.
Results When group S and group SCI were compared, a significant increase of MDA, TOA and SOD were found after SCI (p < 0.01). Additionally, a not statistically significant increase in the post-SCI GPx activity was seen (p > 0.05). Details of MDA, TOA, SOC, and GPx values of all groups can be seen in ►Table 1 (►Fig 1). When compared with group SCI, it was observed that CAPE and MP decreased MDA, TOA, and SOD levels in group SCI þ CAPE and SCI þ MP (p < 0.01). This decrease was more pronounced in the SCI þ CAPE group (p < 0.05) (►Table 1). When group S and group SCI were compared, a statistically significant decrease was observed in the post-SCI TAC levels (►Table 1). When compared with group SCI, it was seen that CAPE (group SCI þ CAPE) and MP (group SCI þ MP) treatments statistically increased TAC levels (p < 0.05). This Journal of Neurological Surgery—Part A
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effects, high MP doses have been used widely for the treatment of SCI in the clinic setting. Caffeic acid phenethyl ester (CAPE) is an active component of propolis and known to have anti-inflammatory and antioxidant effects. It has been reported to inhibit lipid peroxidation as well.9–12 CAPE is one of the most powerful lipophilic antioxidants. CAPE has been shown to decrease spinal cord ischemia and to have a protective role in the spinal cord because of its antioxidant, anti-inflammatory, and neuroprotective effects.13 Thus, CAPE has the potential to prevent secondary spinal cord damage after traumatic SCI. However, as far as we know, the protective effect of intrathecally given CAPE in SCI has not been researched. Our purpose was to examine the effect of intrathecal CAPE on peroxidation and total oxidant and antioxidant systems, and also to demonstrate the effects of methylprednisolone (MP) in SCI models.
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Effects of CAPE and Methylprednisolone in Spinal Cord Injuries
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Table 1 Biochemical parameters according to groups Parameters
S Mean SD
SCI Mean SD
SCI þ MP Mean SD
SCI þ CAPE Mean SD
p
GPx U/g prt
141.54 43.33
166.53 54.60
174.28 94.88
150.50 54.84
NS
MDA (nmol/g prt)
1.81 0.39
3.08 0.69a,
2.00 0.85f
1.60 0.34d
< 0.001
SOD (U/g prt)
5.3145 1.04
8.67 2.39b
5.77 1.03e
3.97 1.33c,d,i
0.001
TAC (nmol Trolox Eq./g prt)
0.40 0.05
0.03 0.02a
0.28 .07a, d
0.12 0.03a,d,g
< 0.001
TOA (nmol H2O2 Eq./g prt)
75.69 16.20
129.12 37.21b
85.10 15.01e
60.01 20.53d,h
0.001
Abbreviations: CAPE, caffeic acid phenethyl ester; Eq., equivalent; GPx, glutathione peroxidase; H2O2, hydrogen peroxide; MDA, malondialdehyde; MP, methylprednisolone; NS, not significant; prt, protein; S, sham; SCI, spinal cord injury; SD, standard deviation; SOD, superoxide dismutase; TAC, total antioxidant capacity; TOA, total oxidant activity. a Significantly different from sham (S) group (p 0.001). b Significantly different from sham (S) group (p 0.01). c Significantly different from sham (S) group (p 0.05). d Significantly different from control (SCI) group (p 0.001). e Significantly different from control (SCI) group (p 0.05). f Significantly different from control (SCI) group (p 0.01). g Significantly different from treatment (SCI þ MP) group (p 0.001). h Significantly different from treatment (SCI þ MP) group (p 0.05). i Significantly different from treatment (SCI þ MP) group (p 0.01).
increase was more pronounced in the SCI þ MP group (►Table 1). It was seen that the SOD activity in group SCI was statistically higher compared with that in group S. When compared with group SCI, group SCI þ MP and group SCI þ CAPE had statistically lower SOD activity. This decrease was
more pronounced in the SCI þ CAPE group than in the SCI þ MP group (p < 0.001) (►Table 1). After examining GPx activity, all groups were found to have higher GPx activity than group S; however, these values were not statistically significant. CAPE did not substantially affect GPx levels (►Table 1).
Fig. 1 The oxidant and antioxidant parameters according to the groups. GPx, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; TAC, total antioxidant capacity; TOA, total oxidant activity. Journal of Neurological Surgery—Part A
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Discussion The secondary damage mechanism which is caused by perfusion decomposition in the post-trauma disintegrated spinal cord play a major role in increasing the extended damage.3 Increasing edema and inflammation, generation of oxygen radicals and lipid peroxidation, and dysfunction of adenosine diphosphate–dependent channels are important components of this vicious cycle.1,2,4,22–24 Lipid peroxidation can be defined as degradation of the lipids in the cell membrane after interaction with oxygen radicals. Free radicals initiate a chain reaction that breaks down the double bonds of unsaturated fatty acids in the presence of oxygen. As a result, cell membrane stability is disturbed, permeability is affected, and the ability to form membrane potential is impaired.25,26 Disintegration of the cell membrane is an important step of irreversible cell damage. Therefore, blocking generation of oxygen radicals and lipid peroxidation are both important to disrupt the vicious cycle in SCIs. MDA and 4-hydroxynonenal (4-HNE) are two markers of lipid peroxidation. MDA is a product of the breakdown of unsaturated fatty acids into their essential chains by way of the oxidation mechanism.27 We found in our study that when compared with group SCI, group SCI þ CAPE had lower MDA levels. This decrease was more prominent when compared with group SCI þ MP. Additionally, the fact that the SCI þ CAPE group had lower MDA levels than group S was interesting. This showed that the potential of intrathecally given CAPE to inhibit lipid peroxidation is very high. When SOD levels were examined, MP and CAPE decreased the SOD level, while the SOD level increased in group SCI. The fact that SOD levels were lower in the SCI þ CAPE group may be a result of the lipid peroxidation inhibiting effect of CAPE. It is not clear through what mechanism CAPE inhibits lipid peroxidation. It is currently being discussed whether the lipid peroxidation inhibiting effects of CAPE are based on an enzymatic or nonenzymatic antioxidant defense system. MDA is produced by both free radical–induced as well as enzymatically induced lipid peroxidation (i.e., arachidonic acid cascade). Thus, in SCI where both processes have long been known to occur acutely and in parallel, the fact that MDA is reduced by CAPE and MP cannot be purely associated with inhibition of free radical–induced peroxidation. In future studies, 4-HNE should be measured by immunoblot because 4-HNE is only produced by free radical–induced peroxidation. Antioxidants, including enzymatic systems such as SOD and GPx, provide the primary antioxidant defense. SOD converts superoxide anion to H2O2. SOD acts against reactive oxygen species and is active in catalyzing detoxification of superoxide radicals. The H2O2 generated in this reaction is restored to water in the presence of GPx.28 The SOD increase in the SCI group in our study indicates a higher SOD activity to eradicate the free oxygen radicals generated after SCI. The increase of GPx enzyme activity in the SCI group was not statistically significant compared with that in group S, showing that oxidative stress in SCI is generated via lipid peroxidation. The fact that SOD and GPx concentration in the SCI þ CAPE group are similar to that in group S is another indicator of
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CAPE’s powerful antioxidant effect. Because free oxygen radicals could not have been overproduced, antioxidant enzymes may have remained within normal values in the presence of CAPE. Our results were supported by Ilhan and his colleagues, who investigated the lipid peroxidation inhibiting effect of CAPE in a spinal cord ischemia reperfusion model.9 Previous studies have suggested that oxidative stress and lipid peroxidation play a role in SCI. Serum concentrations of different oxidant species can be measured separately in the laboratory; however, these measurements are time consuming, overly expensive, and require complicated techniques. Measuring different antioxidant and oxidant molecules is not practical, and their antioxidant and oxidant effects are additive. Because there exist plenty of antioxidants and oxidants, measuring total oxidant-antioxidant activity is more valid and credible.19,20,29 When solely a few antioxidant parameters are measured, their levels may be unchanged or decreased, even when the actual oxidant-antioxidant activity is increased. In recent years, total levels of different oxidant compounds have been monitored by determining the TOA.21 Moreover, TAC is a useful estimate of the activity of the total antioxidants in a medium.20 Similar to a previous study, we saw that TAC significantly decreases after SCI.30 This means that the antioxidant defense system degrades following the SCI. In addition, we showed the increase of TOA for the first time, in contradistinction to other experimental SCI studies. This finding proved that SCI causes an increase in total oxidants. Intrathecal injection of both CAPE and MP inhibits lipid peroxidation and the generation of oxidants in SCI. The inhibitory effect of CAPE was more pronounced than that of MP. Additionally, CAPE and MP treatments increased TAC, which was decreased as a result of spinal cord damage. In contrast to the other findings, MP increased TAC levels more significantly than CAPE. Posttraumatic degradation of the anatomical integrity of the spinal cord and the surrounding tissue may prevent systematically applied drugs from reaching a maximally effective dose in the damaged area. Therefore, intrathecal drug application is used as an alternative method in central nervous system diseases. In our study, we administered CAPE intrathecally, unlike in other studies in the literature. The fact that CAPE efficiently inhibited lipid peroxidation may also be a result of the intrathecal application. In the NASCIS II and III trials, continually given MP was shown to have detrimental effects on damaged spinal cord at high doses (30 mg/kg). Thus, we preferred to use MP at low doses in one of our earlier experiments.14,31 We showed that using MP intrathecally at doses of 3 mg/kg provides a neuroprotective effect against SCI.14 According to our information, CAPE was not used intrathecally in previous experiments, but was administered intraperitoneally in doses of 10 µg/kg.13 In our experiments, we administered CAPE intrathecally at lower doses (1 µg/kg) because we aimed to demonstrate the direct effect of CAPE on SCI. It is one of the limitations of our experiment that the study does not show the correlation between the response curve against SCI and CAPE dose. In future experiments, the dose-response curve of using CAPE intrathecally could be researched. Because we have seen CAPE to have an effect if given intrathecally at doses of Journal of Neurological Surgery—Part A
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Effects of CAPE and Methylprednisolone in Spinal Cord Injuries
Effects of CAPE and Methylprednisolone in Spinal Cord Injuries 1 µg/kg, this dose could be taken as a reference in future experiments. In conclusion, intrathecal injection of both CAPE and MP clearly inhibit the lipid peroxidation and the increase of oxidants at 24 hours after SCI. Additionally, CAPE and MP treatment increased TAC, which was decreased as a result of spinal cord damage.
14 Celik F, Gocmez C, Kamasak K, et al. The comparison of neuro-
15 16
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References 1 Jia Z, Zhu H, Li J, Wang X, Misra H, Li Y. Oxidative stress in spinal
2
3
4 5
6
7
8 9
10
11
12
13
cord injury and antioxidant-based intervention. Spinal Cord 2012; 50(4):264–274 Duz B, Kaplan M, Bilgic S, Korkmaz A, Kahraman S. Does hypothermic treatment provide an advantage after spinal cord injury until surgery? An experimental study. Neurochem Res 2009;34(3): 407–410 Dalgic A, Okay O, Helvacioglu F, et al. Tobacco-induced neuronal degeneration via cotinine in rats subjected to experimental spinal cord injury. J Neurol Surg A Cent Eur Neurosurg 2013;74(3):136–145 Hall ED. Antioxidant therapies for acute spinal cord injury. Neurotherapeutics 2011;8(2):152–167 Aslan A, Cemek M, Eser O, et al. Does dexmedetomidine reduce secondary damage after spinal cord injury? An experimental study. Eur Spine J 2009;18(3):336–344 Topsakal C, Kilic N, Ozveren F, et al. Effects of prostaglandin E1, melatonin, and oxytetracycline on lipid peroxidation, antioxidant defense system, paraoxonase (PON1) activities, and homocysteine levels in an animal model of spinal cord injury. Spine 2003;28(15): 1643–1652 Cayli SR, Kocak A, Yilmaz U, et al. Effect of combined treatment with melatonin and methylprednisolone on neurological recovery after experimental spinal cord injury. Eur Spine J 2004;13(8):724–732 Bains M, Hall ED. Antioxidant therapies in traumatic brain and spinal cord injury. Biochim Biophys Acta 2012;1822(5):675–684 Ilhan A, Koltuksuz U, Ozen S, Uz E, Ciralik H, Akyol O. The effects of caffeic acid phenethyl ester (CAPE) on spinal cord ischemia/ reperfusion injury in rabbits. Eur J Cardiothorac Surg 1999; 16(4):458–463 Hoşnuter M, Gürel A, Babucçu O, Armutcu F, Kargi E, Işikdemir A. The effect of CAPE on lipid peroxidation and nitric oxide levels in the plasma of rats following thermal injury. Burns 2004;30(2):121–125 Sud’ina GF, Mirzoeva OK, Pushkareva MA, Korshunova GA, Sumbatyan NV, Varfolomeev SD. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 1993;329(1-2):21–24 Kasai M, Fukumitsu H, Soumiya H, Furukawa S. Caffeic acid phenethyl ester reduces spinal cord injury-evoked locomotor dysfunction. Biomed Res 2011;32(1):1–7 Uzar E, Sahin O, Koyuncuoglu HR, et al. The activity of adenosine deaminase and the level of nitric oxide in spinal cord of methotrexate administered rats: protective effect of caffeic acid phenethyl ester. Toxicology 2006;218(2–3):125–133
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18
19
20
21 22
23 24
25
26
27
28
29
30
31
protective effects of intrathecal dexmedetomidine and methylprednisolone in spinal cord injury. Int J Surg 2013;11(5):414–418 Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17(6):1031–1036 Korkmaz HA, Maltepe F, Erbayraktar S, et al. Antinociceptive and neurotoxicologic screening of chronic intrathecal administration of ketorolac tromethamine in the rat. Anesth Analg 2004;98(1): 148–152 Senoglu M, Nacitarhan V, Kurutas EB, et al. Intraperitoneal alphalipoic acid to prevent neural damage after crush injury to the rat sciatic nerve. J Brachial Plex Peripher Nerve Inj 2009;4:22 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1): 265–275 Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 1990;186:407–421 Erel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem 2004;37(4):277–285 Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem 2005;38(12):1103–1111 Tator CH, Fehlings MG. Review of the secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms. J Neurosurg 1991;75(1):15–26 Collins WF. A review and update of experiment and clinical studies of spinal cord injury. Paraplegia 1983;21(4):204–219 Amar AP, Levy ML. Pathogenesis and pharmacological strategies for mitigating secondary damage in acute spinal cord injury. Neurosurgery 1999;44(5):1027–1039; discussion 1039–1040 Adibhatla RM, Hatcher JF. Phospholipase A(2), reactive oxygen species, and lipid peroxidation in CNS pathologies. BMB Rep 2008; 41(8):560–567 Muralikrishna Adibhatla R, Hatcher JF. Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia. Free Radic Biol Med 2006;40(3):376–387 Lin Y, Vreman HJ, Wong RJ, Tjoa T, Yamauchi T, Noble-Haeusslein LJ. Heme oxygenase-1 stabilizes the blood-spinal cord barrier and limits oxidative stress and white matter damage in the acutely injured murine spinal cord. J Cereb Blood Flow Metab 2007;27(5): 1010–1021 Temiz C, Solmaz I, Tehli O, et al. The effects of splenectomy on lipid peroxidation and neuronal loss in experimental spinal cord ischemia/reperfusion injury. Turk Neurosurg 2013;23(1):67–74 Buyukhatipoglu H, Sezen Y, Yildiz A, et al. N-acetylcysteine fails to prevent renal dysfunction and oxidative stress after noniodine contrast media administration during percutaneous coronary interventions. Pol Arch Med Wewn 2010;120(10):383–389 Ozgiray E, Serarslan Y, Oztürk OH, et al. Protective effects of edaravone on experimental spinal cord injury in rats. Pediatr Neurosurg 2011;47(4):254–260 Pettiford JN, Bikhchandani J, Ostlie DJ, St Peter SD, Sharp RJ, Juang D. A review: the role of high dose methylprednisolone in spinal cord trauma in children. Pediatr Surg Int 2012;28(3):287–294
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