INTRODUCTION
Spinal cord injury (SCI) is a condition that can have temporary or permanent impact on spinal cord functions and that has physically, psychologically and socially devastating outcomes1. The incidence of SCI varies among geographical regions and over time. Annual incidence rate is reported to be 10.4-83 people per million2.
Despite all therapeutic options, traumatic spinal cord injury (T-SCI)-related morbidity and mortality remain high3. Initially, primary injury occurs with the involvement of the spinal cord and surrounding tissues due to trauma-related mechanical reasons. Thereafter, secondary injury occurs by various mechanisms. Progressive edema, increasing ischemia and triggered pro-apoptotic pathways lead to additional tissue injury and the process results in cell death4. Primary injury usually cannot be prevented and its severity cannot be changed as it occurs due to unexpected reasons during this process. Thus, preventing or reducing and ameliorating secondary injury has been the main goal of the treatment. Nevertheless, there is yet no definite approach for the prevention or management of secondary injury, and numerous agents are under investigation5. Non-steroidal anti-inflammatory drugs (NSAIDs) as well are among the pharmacological agents used in the experimental and clinical studies.
Inflammation is a defense mechanism against injury. However, inflammatory processes may have harmful effects as well as benefits depending on various factors and time6. Anti-inflammatory drugs are used to reduce/ameliorate secondary injury by means of preventing/reducing inflammation. Montelukast sodium (ML) is a leukotriene receptor antagonist that reduces its effects by binding to cysteinyl leukotriene receptors to which leukotrienes, one of the arachidonic acid metabolites released from mast cells, eosinophils and other inflammatory cells that play a role in inflammation7. Diclofenac sodium (DF), on the other hand, has an anti-inflammatory effect by inhibiting cyclooxygenase 1 and 2 (COX-2) enzymes involved in arachidonic acid metabolism, preventing the synthesis of inflammatory mediators8.
The present study aimed to investigate whether the inflammatory agents, DF and/or ML had protective effects on acute inflammation in T-SCI cases. For this purpose, an experimental rat model of T-SCI was created and histopathological examination was performed to investigate the effect of drugs on trauma-related injury.
MATERIALS AND METHODS
In the present study, 40 male Sprague-Dawley rats aged between 10 and 12 weeks and weighing 230-280 gr were used. This study was approved by the Local Ethics Committee for Animal Experiments at the meeting of the University of Health Sciences Turkey, Haydarpaşa Numune Training and Research Hospital (decision number: 2017-05/05, date: 01.11.2017). As anti-inflammatory drugs, DF (Voltaren, Novartis, İstanbul, Turkey) and ML (Singulair, Merck Sharp Dohme, İstanbul, Turkey) were used.
The rats were divided into 5 groups, each comprising 8 rats, as follows:
1. The control group (C): no SCI and no medication
2. The trauma group (T): only spinal cord injury, no medications.
3. The trauma+DF group (tDF): diclofenac administration after spinal cord injury.
4. The trauma+ML group (tML): montelukast administration after spinal cord injury.
5. The trauma+DF+ML group (tDF+ML): diclofenac+montelukast administration after spinal cord injury.
All the rats were kept in rooms provided with adequate air circulation and ambient temperature without food or water restriction. Following spinal cord injury, the subjects were placed in an appropriate environment for the maintenance of care, their daily dressing was done, and the subjects in the treatment groups received relevant medications.
Surgical Procedure
SCI was created in all rats excluding those in the control group. Prior to the surgical procedure, ketamine 60 mg/kg (Ketalar, Parke-Davis, Eczacıbaşı, İstanbul, Turkey) and Xylazine 10 mg/kg (Rompun, Bayer Pharmaceuticals, İstanbul, Turkey) were administered for general anesthesia. Surgical procedures were performed on rats fixed in prone position onto the experimental boards. The surgical area was shaved, and regional antisepsis was provided using povidone iodine solution (Poviod 10% polyvinylpyrrolidone-iodine complex, Saba İlac, İstanbul, Turkey). After covering with sterile drapes, cutaneous and subcutaneous incisions were made at the level of T9-T12 vertebrae. Subperiosteal stripping of the paravertebral muscles was performed. Following T9-T10-T11 laminectomy and flavectomy, the dura was exposed. Extradural SCI was created by one minute compression of the spinal cord using Yaşargil Aneurysm Clip.
Drug Administration
Diclofenac sodium: The subjects in the relevant groups were administered 10 mg/kg DF twice a day by intraperitoneal route for 72 hours after spinal cord injury.
Montelukast sodium: The subjects in the relevant groups were administered 10 mg/kg ML twice a day by intraperitoneal route for 72 hours after spinal cord injury.
Follow-Up
After 72-hours of medical treatment and follow-up period, the subjects were sacrificed using 100 mg/kg pentothal sodium (Pental Sodyum, Ulagay İlac, İstanbul, Turkey). The operation site was opened under sterile conditions and the lesion area was exposed. The spinal cord, dura and the surrounding tissues including 5 mm of intact tissue from both proximal and distal segments were excised together. These tissue samples were fixed in 10% formalin solution and then were transferred to the laboratory for histopathological examination. During follow-up period, a total of two subjects, one from the trauma group and one from the trauma+DF group, died.
Histopathological Examination
The sections obtained from the tissue samples were stained with hematoxylin-eosin, and were examined under light microscope for the presence of edema, necrosis, inflammatory cells, apoptosis, neuron injury, and bleeding, and each finding was scored with the grading system which was summarized in Table 1. The histopathological examination findings can be seen in Figure 1.
Statistical Analysis
The Number Cruncher Statistical Software 2007 (NCSS, LLC, Kaysville, Utah, USA) was used for statistical analysis. Descriptive statistics were expressed as numbers and percentages for categorical variables and as median, 25th percentile, and 75th percentile for numerical variables. In multiple group comparisons with non-normal distribution, the Kruskal-Wallis test was used, whereas pairwise comparisons were performed using the Mann-Whitney U test with Bonferroni correction. The level of statistical significance was considered to be p<0.05.
RESULTS
The histopathological scores and statistical differences between the control group and other groups are summarized in Table 2. It was observed that the parameters between the control group and the trauma group were significantly different (p<0.05). Histopathological scores of the trauma group and trauma+drug groups were similar (p>0.05). The edema levels among the groups were examined and it was seen that the group with no difference was the tDF group compared to the control group (p=0.059) and the groups with a significant difference were the tDF+ML group (p=0.034) and the tML group (p=0.007), respectively. It was observed that the least edema was in the tDF group. In the evaluation of necrosis, the group with the least necrosis was tDF+ML (p=0.1), while the groups with more necrosis were tDF (p=0.017) and tML (p=0.008). Considering the number of inflammatory cells, the lowest number of cells was observed in the tDF group (p=0.068), while more cells were observed in the tDF+ML group (p=0.035) and in the tML group (p=0.002), respectively. While the group with the least apoptosis was tDF+ML (p=0.061), it was found to be significantly higher in the tDF group (p=0.039) and tML group (p=0.004), respectively. While neuronal injury was seen at least in the tDF+ML group (p=0.139), significant neural damage was observed in the tDF group (p=0.011) and tML group (p=0.005), respectively. Considering the amount of bleeding in the preparations, it was observed that there was significant bleeding in all groups compared to the control group (p<0.05).
DISCUSSION
T-SCIs are clinical conditions that may lead to devastating physical, psychological and social consequences in the lives of patients. However, a definitive treatment method has not been developed despite all the researches and treatment modalities applied today1. The severity of primary injury is the most critical factor in determining the prognosis of T-SCI. Nevertheless, the mechanisms leading to secondary injury may intensify the injury or may affect healing processes and thus, may affect overall morbidity and mortality.
Inflammation has a critical role in secondary damage. The aim of inflammation is to stabilize and limit the existing damage, and to fulfill the necessary conditions for the repair of the organism, to clean the cell residues and to remove harmful substances in the environment. In case of high severity, neuronal cell damage may increase with the increase of damage, and as a result, functional results may worsen9,10. The mechanisms of secondary injury are complex and include various conditions such as neurogenic shock, bleeding, ischemia-reperfusion, excitotoxicity, calcium-mediated processes, fluid-electrolyte imbalance, immunological processes, apoptosis, and mitochondrial dysfunction11. So far, various medical agents have been investigated for the treatment of SCI. These agents include corticosteroids, vasopressors, minocycline, magnesium, riluzole, glyburide (glibenclamide), thyrotropin-releasing hormone, opioid receptor antagonists (naloxone), granulocyte colony stimulating factor, cethrin, gangliosides, antioxidants, calcium channel blockers, sodium channel blockers, and many others12,13. Corticosteroids are being used for a long time in many experimental and clinical trials due to their anti-inflammatory, edema-resolving and antioxidant characteristics. However, it is currently accepted that corticosteroids have no effect when compared to placebo at 6-12 months of follow-up and therefore have no long-term benefits14.
Due to the strong anti-inflammatory effects of NSAIDs, it is expected that they may have anti-inflammatory effects in secondary damage. In experimental studies, inhibition of the RhoA pathway and neuroprotective effect by reducing apoptosis and providing histological improvement bring these drugs to a promising position in terms of treatment15. In our study, when the edema level among the groups was examined, it was seen that the group that did not differ from the control group was the tDF group. There was no significant difference in the ML group in terms of edema. In addition, when the tDF+tML group and other drug groups were compared, it was found that combined drug administration reduced the anti-edema effect compared to single applications. In the study of Saiwai et al.16, the importance of leukotrienes in the injury mechanisms after SCI was emphasized. In the study conducted by Cavus et al.17, the effects were compared with montelukast and methylprednisolone and it was reported to be neuroprotective. Genovese et al.18 compared the effects of montelukast and zilueton and found the anti-edema effects to be significant.
Inflammation is the basic defense mechanism of the organism and begins to appear in the trauma area in the 3rd post-traumatic hour and can remain at the maximum level until the 3rd day19. Schwartz20 reported that the presence of neutrophils was necessary for repair after axonal damage and that T lymphocytes in the environment were necessary for defensive and repair events. Schwab et al.21 stated that the amount of LTC4 and TXA2, which are arachidonic acid metabolites in CSF, increased 5-9 times higher than normal after the injury. In our study, when the number of inflammatory cells in the groups was compared, it was found that the number of cells was the lowest in the tDF group. No significant difference was found in the other groups. Hains et al.22 reported that COX-2 inhibitors improved histological and motor function results. In the literature, studies in which indomethacin and ketorolac were applied intrathecally have been reported to have a protective effects23,24. Genovese et al.18 also showed that zileuton and montelukast sodium, which are 5-lipoxygenase inhibitors, decreased the number of inflammatory cells and myeloperoxidase activity, which increased in the 24 hours after trauma.
As a result of the comparisons between the groups, it was seen that necrosis, apoptosis and neural injury were the least in the group, in which tDF+ML was used in combination, compared to the other groups. It has been shown that caspas-8 and 9 are activated in the lesion center at the 6th hour after trauma and this activity lasts for 7 days25. Inflammatory cytokines, especially TNF- released from neurons, activate the RhoA pathway and cause apoptosis24. Mills et al.26 showed that activated Rho pathway induced Bcl-2 dependent apoptosis. Xing et al.27 stated that the inhibition of RhoA in rats, which they applied NSAID treatment for 5 post-traumatic days, especially protected oligodenrocytes from apoptosis, and increased axonal myelination in the rostral and caudal of the lesion. In the experimental study on rats, Wang et al.28 reported that the treatment of ibuprofen given at a dose of 60-70 mg/kg for 1-28 days had a protective effect on the rostral axons of the spinal cord, but this effect was not observed in the caudal fibers. As a result, they stated that ibuprofen could be a potential agent. Sharp et al.29 reported that 60 mg/kg ibuprofen treatment for 42 days after trauma resulted in improvement in Basso-Beattie-Bresnahan score and protection in motor functions in rats as a result of treatment.
Post-traumatic bleeding is a pathology that begins to occur after primary injury and continues with secondary injury. In their study, Noble and Wrathall30 stated that the localization of bleeding after SCI was related to the severity and direction of the blow that caused the primary injury, and they reported that bleeding localizations could be seen simultaneously in the rostral and caudal regions of the spinal cord and in more than one region. Erşahin et al.31 reported that ML reduced bleeding in the spinal cord white matter. Saiwai et al.16 reported that neutrophils that migrated to the environment at the 12th hour after trauma increased the bleeding status and this situation decreased due to the decreased cell migration after the use of ONO-4057, which is an LTB4 antagonist. In our study, we detected that there was no significant difference between the groups in terms of the amount of bleeding.
Study Limitations
The most important limitation of our study is that it is not supported by functional recovery and biochemical parameters such as apoptosis and inflammation markers, as well as investigating the patological changes.
CONCLUSION
In our study, it was determined that the application of DF alone in traumatic SCI had a reducing effect in the development of edema and the number of inflammatory cells, while the application of DF+ML had a reducing effect on the development of necrosis, apoptosis, and neural damage.
Acknowledgment
We thank Assoc. Prof. Meltem Öznur from Namık Kemal University, Department of Pathology for her supports during the writing of our article.
Ethics
Ethics Committee Approval: The study were approved by the Local Ethics Committee for Animal Experiments of University of Health Sciences Turkey, Haydarpaşa Numune Training and Research Hospital (decision number: 2017-05/05, date: 01.11.2017).
Informed Consent: Animal experiments.
Peer-review: Externally peer-reviewed.
Authorship Contributions
Surgical and Medical Practices: T.E., D.C., F.V.A., Concept: T.E., B.E., Design: T.E., M.İ., F.V.A., Data Collection or Processing: T.E., D.C., F.V.A., Analysis or Interpretation: T.E., F.V.A., T.T., T.Ç., Literature Search: T.E., M.İ., B.E., Writing: T.E., M.İ., B.E., T.T., T.Ç.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.