Effects of individual or combined use of alpha-lipoic acid and methylprednisolone on malondialdehyde , superoxide dismutase , and catalase levels in acute spinal cord injury in rats

Introduction: Recent studies have demonstrated that alpha-lipoic acid (ALA) has a strong antioxidant property and it exerts neurotrophic effects on the peripheral nerves� In this study, we investigated potential effects of ALA on secondary injury mechanisms as well as on apoptosis� Methods: Forty Sprague-Dawley rats were equally divided into 5 groups, as follows: laminectomy (control), laminectomy + trauma (Trauma), laminectomy + posttraumatic methylprednisolone [MP] (Trauma + MP), laminectomy + posttraumatic ALA (Trauma + ALA), laminectomy + posttraumatic MP and ALA (Trauma + MP + ALA)� Yasargil aneurysm clip method was used to induce the spinal cord injury� Twenty-four hours after the procedure the rats were sacrificed� Spinal cord samples were harvested to analyze malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels, as well as histopathological characteristics� Results: The Kruskal-Wallis test (95% confidence level, p < 0�05) showed a statistically significant difference between the groups in MDA (p = 0�006), CAT (p = 0�000), and SOD (p = 0�001) levels� Pairwise comparisons, with Bonferroni correction, of control and Trauma group with the other groups, revealed a significant difference in CAT and SOD levels� Overall, our results showed that ALA administration significantly decreased MDA levels in Trauma + ALA compared to the same effect of MP in Trauma + MP group� RESEARCH ARTICLE Open Access © 2016 Alper Birgul et al�; licensee University of Sarajevo Faculty of Health Studies� This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons�org/licenses/by/2�0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited� *Corresponding author: Gokhan Canaz, Department of Neurosurgery, Istanbul Bakirkoy Prof� Dr� Mazhar Osman Psychiatric Training and Research Hospital, Dr�Tevfik Sağlam Street No:25/2 Post Code: 34147 Bakirkoy/Istanbul� Phone: +90 212 409 15 15� E-mail: gokhancanaz@gmail�com Submitted: 10 November 2016/Accepted: 10 December 2016 DOI: https://doi�org/10�17532/jhsci�2016�409


INTRODUCTION
A spinal cord injury (SCI) remains a challenging clinical entity due to high morbidity and mortality rates.Advancements in technology and dynamics of modern life have markedly increased the rate of SCI and related diseases.For example, among the major causes of SCI today are motor vehicle accidents, falls from height, acts of violence and industrial accidents frequently can be enumerated (1,2).Furthermore, the number of traffic accidents and violent acts has been increasing in recent years and, specifically in Turkey, 1600-2000 severe cases of SCI are reported every year (3).
In SCI, the first trauma occurrs as a result of mechanical stroke and is known as a primary injury.Flexion, extension, dislocation, and penetrating injuries cause strain and rupture of neural elements or spinal cord vessels, which is in direct proportion to the severity of stroke.A pharmacological treatment that could prevent primary SCI is not developed yet.The prevention of injuries by ensuring the stabilization of the body using active and passive safety measures may decrease the severity of damage to some extent.Secondary cellular damage triggered by mechanical injury develops within hours after the primary injury, and is caused by metabolic and biochemical agents.Current studies have been focused on the cessation or slowing down the secondary injury cascade.The development of treatment strategies for SCI includes experimental models consisting of surgical, pharmacological, and physiological methods.In addition, pathophysiologic processes that are initiated after the injury have been explored and neuroprotective treatment options have been investigated (4).A number of chemical agents have been used to prevent existing neural tissue damage, including GM1 ganglioside, naloxone, thyrotropin-releasing hormon (TRH), nimodipine and tirilazad mesylate (5).Some of the agents decrease tissue damage, while others increase functional recovery.With regard to new neuroprotective agents, experimental studies on minocycline and erythropoietin have yielded promising results (5).
However, currently, only methylprednisolone (MP) has been reported as the accepted treatment for acute SCI (6,7).
Alpha-lipoic acid (ALA) is a fatty acid with diverse functions and various mechanisms of action.More specifically, it is a cofactor in an enzyme complex which catalyzes decarboxylation of alpha-keto acids, such as pyruvate and alpha-ketoglutarate (8).Therefore, ALA is required for the regulation of glucose metabolism.In addition, ALA is a very potent antioxidant which functions together with the major antioxidants such as vitamin E, vitamin C, and glutathione [GSH] (8)(9)(10)(11)(12)(13)(14)(15).Oxidative stress due to free oxygen radicals, and associated inflammatory response and apoptosis, play an important role in the process of secondary injury, which occurrs after an acute SCI (16).Cytokines that are released from the central nervous system cells (i.e., IL-1β, IL-6, and TNF-α) induce the process of apoptosis and inflammatory response (17).The two major pathways of apoptosis, namely receptor-dependent extrinsic and receptor-independent intrinsic pathways, are presumably activated in SCI (16).Receptor-dependent apoptosis pathway is stimulated by extracellular signals, especially by tumor necrosis factor (TNF).TNF rapidly accumulates in the injured areas following the onset of SCI.Signaling through Fas receptors in neurons, microglia, and oligo dendrocytes induces successive caspase activation.Among the activated caspases are caspase-8, caspase-3, and caspase-6.The activation of these caspases results in the death of the affected cells (17).In the alternative extrinsic apoptosis pathway, caspase-3 activates inducible nitric oxide synthase (iNOS) which results in programmed cell death (17).In the most recent in vivo and in vitro studies, strong antioxidant and metal-chelating properties of ALA have been demonstrated.Furthermore, ALA increased nerve growth factor (NGF) levels in peripheral nerves, and consequently exerted its neurotrophic effects, while decreasing TNF-alpha levels (7)(8)(9)(10)(11)(12)(13).
Adjustment of dosage and duration of treatment, as well as dose-dependent side effects of ALA should be clarified, before its application in the management of SCI.In this study, we investigated the effects of ALA on the mechanisms of secondary injury and apoptosis pathways, as well as compared the anti-inflammatory and biochemical properties of ALA and ALA-MP in treating SCI.

METHODS
This study was performed at the Department of Experimental Animal Biology and Biomedical Application Techniques, Istanbul University.Forty adult Sprague-Dawley rats, produced in that laboratory and weighing between 280 and 300 g (median 283 g), were used.In our experiment, microsurgical instruments, normal size surgical instruments, and surgical microscope were used (D.F.Vasconcellos, Brasil) (Figure 1).

Groups
The rats were randomized equally into 5 groups: 1.Control -Normal spinal cord tissue specimens following laminectomy were used.2. Trauma -Following laminectomy, an extradural injury was induced using Yasargil aneurysm clip (Aesculap, USA) under 0.7 N pressure, applied for 60 seconds.

Surgical procedure
The rats subjected to the surgical procedure to induce SCI were anesthetized using i.p. ketamine HCL (60 mg/kg) and i.p. xylazine (10 mg/kg).The rats were stabilized in prone position, their thoracal regions were disinfected with povidone (PVD)-iodine, and shaved.Following the shaving, the surgical field was again disinfected with PVDiodine.Under a surgical microscope, a 2-cm skin incision was made between T8 and T10 vertebrae.Then, through this skin incision, subcutaneous tissues were passed through, and paravertebral muscles were cleaved.Total laminectomy was perormed between T8 and T10, and the spinal cord was exposed (Figures 2 and 3).Next, Yasargil extradural aneurysm clip (FT217T) was applied for 60 seconds to the rats from Trauma, Trauma + MP, Trauma + ALA, and Trauma + MP + ALA group, under 0.7 N pressure, to induce the SCI.Following hemostasis, the layers were closed with 3/0 silk sutures (Figure 4).

Induction of SCI
In our study, following the total laminectomy performed between T8 and T10 vertebrae, as described by Tator and Rivlin (18), Yasargil extradural aneurysm clip was applied (FT217T) for 60 seconds to induce the SCI (19).

Pharmacological applications
ALA thioctacid solution for injection (Meda Pharma GmbH & Co. KG, Bad Homburg, Germany) was administered through i.p. route.
In the SCI-induced rats, the following procedures were applied: Trauma + MP -During the early postoperative period, MP was delivered rapidly through i.p. route at a dose of 3.4 mg/kg within 15 minutes.After a waiting period of 45 minutes, MP was delivered at 4 equal doses of 5.6 mg/kg, within 23 hours.
Trauma + ALA -During the early postoperative period, ALA was delivered at a dose of 50 mg/kg through i.p. route.
Trauma + MP + ALA -During the early postoperative period, MP was delivered rapidly through i.p. route at a dose of 3.4 mg/kg, within 15 minutes.After a waiting period of 45 minutes, MP was delivered at 4 equal doses of 5.6 mg/kg within 23 hours together with 50 mg/kg ALA, via i.p. route.
At 24 hours postoperatively, rats were anesthetized with 60 mg/kg of i.p. ketamine HCL and 10 mg/kg of i.p. xylazine, and sacrificed using 100 mg/kg of i.p. thiopental sodium.Then, spinal cord specimens were harvested.Malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) levels were measured in these specimens.

Preparation of tissue homogenates
The spinal cord of the rats was weighed, mixed at a ratio of 1/9 with 0.1 M phosphate-buffered saline (PBS), and homogenized on ice at 10,000 rpm for one minute using a Miccrahomogenizer.The homogenized samples were centrifuged at 5000 rpm   under + 4 o C ambient temperature for 5 minutes, using a cooling centrifugal device.
The protein levels of supernatants of the homogenized samples were determined using a Siemens Advia immunoassay device (Siemens Healthcare Diagnostics Inc. USA).The tissue protein levels were determined using a CSF measurement kit (Thermo Fisher Scientific.USA).This method is based on the measurement of the changes in absorbance when a pyrogallol red-molybdate complex is bound to protonated basic amine groups of proteins, at acidic pH.
CAT levels were measured using spectrophotometric methods (Bioxytech catalase-520, OxisResearch Product kit -Oxis International.USA).A standard solution prepared by mixing the surfactant of PBS with catalase was placed on the specimen.Then, 10 mM of H 2 O 2 contained in 500 µL PBS was added to this solution and incubated for one minute.After that, sodium azide was added.The prepared solution was placed in screw capped tubes and mixed without vortexing.A 20-µl aliquot of this mixture was pipetted into a tube, and 2 ml of horseradish peroxidase (HRP) in PBS and chromogenic mixture (110 ml chromogen in 110 µl HRP) was added.This mixture was again mixed without vortexing, and its absorbance was read at 520 nm after an incubation period of 10 minutes.All procedures were performed under room temperature.The results were expressed in U/g.MDA levels were measured using spectrophotometric methods (Bioxytech MDA-5861 OxisResearch Product kit).Thiobarbituric acid reactive substances (TBARS) method is a nonspecific reaction which means that it reacts with both, free and protein-bound MDA.Therefore, we aimed to measure free MDA levels using the MDA-586 method after hydrolysis.In that way, other lipid peroxides, such as 4-hydroxy-alkenals, would not affect the results.
The method was based on the reaction between 2 moles of a chromogenic agent N-Methyl-2phenylindole (NMPI) and MDA to produce a stable carbocyanine dye.The measurements were made using spectrophotometric methods and the results were read at 586 nm.Ten microlitres of probucol in methanol was pipetted into a tube and vortexed after the addition of 200 µl of homogenized tissue and 640 µl of diluted reagent-1 (NMPI).Afterwards, 150 µl of reagent-2 (concentrated HCl) was added and re-vortexed.The vortexed solution was incubated at 45 °C for 60 minutes, and centrifuged for 10 minutes at 10,000 rpm.The resultant clear supernatant was transferred into a spectrophotometer cuvette and read at 586 nm.The results were expressed as µmol/g of tissue.
SOD (EC 1.15.1.1.)activity was measured based on the reduction of nitroblue tetrazolium (NBT) by superoxide, which is produced by xanthine-xanthine oxidase system.The produced superoxide radicals reduce NBT, and the result is the formation of colored formazan dye.This complex gives a maximum absorbance at 560 nm (Cayman Chemical Company, Ann Arbor, USA).A colorimetric plate formed as the result of reactions was read at 560 nm using Biotek ELISA plate reader (BioTek.USA).
Results were expressed as U/mg of wet tissue weight.

Statistical analysis
SPSS 22 (Statistical Package for Social Sciences) for Windows 10.0 (IBM -USA), was used for statistical analysis.Parameters with non-normal distribution were compared between the groups using the Kruskal-Wallis H test, and for the determination of the group which differed, Mann Whitney U test was used.Intragroup comparisons were performed using the Wilcoxon signed-rank test.The results were evaluated at a significance level of p < 0.05, and within 95 % confidence interval (CI).Mann -Whitney test with Bonferonni correction was performed between both the control, and the trauma groups versus other groups.The control group was compared with other groups as pairs as follows: (Groups 1, and 2), (Groups 1, and 3), (Groups 1, and 4), (Groups 1, and 5) at an accepted significance level of p < 0.0125.Trauma group was compared with the pairs of other groups, as follows: Trauma versus Trauma + MP, Trauma versus Trauma + ALA, and Trauma versus Trauma + MP + ALA groups, with a significance level set at p < 0.0166.
The results of the Mann-Whitney test (with a Bonferonni correction) did not show a significant difference in MDA levels between the control and treatment groups (Trauma + MP, Trauma + ALA, and Trauma + MP + ALA).
With regard to the CAT levels, a significant difference was not detected only between the control and Trauma + ALA + MP group (p = 0.249).Regarding the SOD activity, a significant difference was not detected only between the control and Trauma + ALA and Trauma + MP + ALA groups (p = 0.016 and p = 0.172, respectively).
A significant difference was detected between Trauma and Trauma + MP + ALA group in MDA levels (p = 0.001).Trauma group was significantly different from the other three groups with respect to CAT levels.As for the SOD levels, a significant difference was detected between Trauma group and Trauma + ALA and Trauma + MP + ALA groups (p = 0.001 and p = 0.012, respectively).The mean SOD level was relatively higher in Trauma + MP + ALA group.

Multiple group comparisons
Since the level of MDA did not differ significantly between the groups (p = 0.085), except between the control and Trauma group (p = 0.001) (Table 3).

DISCUSSION
The protection from secondary injury in cases with acute SCI is called neuroprotection.Numerous medical approaches, including surgeries, drug treatments, regulation of tissue oxygenization, spinal decompression, and stabilization of the spine have  Secondary injury starts within minutes or hours following the primary injury, and persists for weeks.Because it is not possible to treat primary injury, the rationale of investigating secondary injuries is to discover pharmacologic agents that will preserve the vitality of tissues, their connections with distal neurons, protect and increase the "endurance" of neurons in the injured area, or halt the pathological processes (20).Dramatic pathological changes in the spinal cord lesion within first few days after the injury, represent the most important subject of clinical and experimental observations (13).The concept of dual mechanism in the spinal cord injury was introduced at the beginning of 1900s by Allen who demonstrated that progressive damage occurrs in patients with spinal cord injuries (21).Following SCI, a series of pathological changes occurs in the spinal cord, including bleeding, edema, demyelinization, axonal and neuronal necrosis, and cavity.Ducker demonstrated gradual worsening of these pathological changes up to 6 days after the injury (22).Nemecek defined this necrotic process as autodestruction (23).Attempts to prevent the secondary injury in SCI target glutamatergic, cholinergic and catecholaminergic neurotransmission systems, production of free radicals, lipid peroxidation, calcium and other anion channels, growth factors, inflammatory processes, endogenous opioid receptors, enzymes, apoptotic cell death, and regeneration mechanisms.
The cell death occurring after SCI has been explained by necrosis, developed as a consequence of ischemic and inflammatory reactions associated with tissue damage (24).In addition, the important role of apoptosis (previously thought to be present only during the development of central nervous system) and neuronal damage in posttraumatic spinal cord degeneration has already been recognized (25,26).been used.Although, for the last 20 years, a number of studies investigating SCI have been performed, a standardized treatment that could be used in humans has not been established yet.(27).Anderson et al. demonstrated that neurological damage emerging after an acute SCI is related to necrosis which occurs after the primary and subsequent secondary injury; furthermore, apoptosis is observed at a later stage (28).Liu et al. revealed that neuronal and glial apoptosis occurs within 4 and 9 days after SCI (28).According to Lu et al., neuronal and glial apoptotic cells were not observed within the first 5 minutes after a contusion injury; nevertheless, the apoptotic process was detected in the injured area between the first 4 hours and 14 days after the injury, and it peaked at the 24 th hour (29).It has also been showed that in SCI, the activation of caspase-1 and caspase-3 induces apoptosis (30).In the study by Emery et al., the caspase-3 activation was demonstrated in the human spinal cord, following SCI (29).Li et al. emphasized that the inhibition of caspase-3 after an induced SCI in rats, markedly decreased the injured area and improved the motor functions (31).Recently, therapeutic prophylaxis targeted at the apoptotic cascade in cells, after SCI, has been in focus (32).
In this study, we investigated the neuroprotective effects of ALA alone or in combination with MP on MDA, SOD, and CAT levels in SCI-induced rats.
Lipid peroxidation levels can be determined via the intermediate products produced during lipid peroxidation, such as MDA (33).In the body, different antioxidant mechanisms prevent the production of free radicals or eliminate existing free radicals (34).In cells, polyunsaturated fatty acids in the membrane phospholipids can be easily oxidized spontaneously or by binding to oxidative metabolites, and transformed to peroxide derivatives.The most potent antioxidant is vitamin E, which prevents this cascade accompanied by the production of free oxygen radicals at the level of cell membrane, and neutralizes the existing free oxygen radicals.Other antioxidant systems in the body (vitamin C, glutathione peroxidase, beta-carotene, etc.) are not as effective as vitamin E in this pathway.However, in general, due to beneficial effects on the membrane lipids as well as on subcellular structures, vitamins protect the membrane against oxidative damage (35).
In this study, we biochemically investigated the neuroprotective effects of ALA on SOD, CAT, and MDA levels.
Barut et al. measured lipid peroxidation and MDA levels during the early period after SCI (at posttraumatic 1 and 15 minutes, and 1, 2 and 4 hours), and detected that MDA levels increased starting from 15 minutes after the injury, then peaked at 1 hour, and decreased afterwards (33).
Kaynar et al. evaluated the efficacy of nimodipine and N-acetyl cysteine (NAC) by measuring tissue lipid peroxidation levels, one hour after an induced SCI.Based on the MDA measurements, a single dose of NAC had no effect on the peroxidation of lipid membranes in the experimentally induced SCI (36).Kaptanoglu et al. investigated the antioxidant effects of thiopental and propofol in experimental cases with SCI, as well as the microstructural effects of the SCI.They demonstrated increased levels of MDA, the indicator of lipid peroxidation, in rats subjected to cerebral contusion.They also demonstrated that thiopental and propofol decreased lipid peroxidation; however, propofol could not reverse the microstructural pathological changes (37).
Hanci et al. compared the efficacy of MP, NAC, and prednisolone + NAC treatment in an experimental SCI.The mean MDA levels were significantly lower in the MP, NAC, and MP + NAC groups when compared with the control group (p < 0.05).However, the mean SOD levels were significantly higher in MP, NAC, and MP + NAC groups compared to their control group (p < 0.05).On contrary, a significant difference was not observed between MP and NAC group in the mean MDA and SOD values (38).

CONCLUSION
In our study, the administration of ALA alone decreased significantly the MDA levels compared to the effect of MP and, at the same time, ALA increased the SOD and CAT levels.A combined use of ALA and MP demonstrated synergistic effects, and yielded even more significant results.In our opinion, the combined use of ALA and MP could provide the benefit for patients with SCI.However, additional studies are required to confirm our results.

FIGURE 2 .
FIGURE 2. Macroscopic appearance of spinal cord before exposure to trauma.

FIGURE 3 .
FIGURE 3. Macroscopic appearance of spinal cord before exposure to trauma.

FIGURE 6 .
FIGURE 6. Catalase (CAT) values in experimental and control groups.

FIGURE 7 .
FIGURE 7. Superoxide dismutase (SOD) values in experimental and control groups.

TABLE 1 .
Data set.MDA, CAT and SOD levels of groups

TABLE 2 .
MDA, CAT and SOD levels of groups.Data are given as mean±standard deviation

TABLE 3 .
Results of Kruskal-Wallis H test (95% CI and p < 0.05) Crowe et al. reported that the mechanisms of secondary injury, triggered following a primary SCI, induce apoptosis and progressive neurodegeneration