- Email: [email protected]
Neuroprotective Effects of Tocilizumab on Experimentally-Induced Spinal Cord Ischemia-Reperfusion Injury
Original Article
Neuroprotective Effects of Tocilizumab on Experimentally-Induced Spinal Cord Ischemia-Reperfusion Injury Yasar Karatas1, Mehmet Fatih Erdi2, Bu¨lent Kaya1, Fatih Keskin2, Go¨khan Cu¨ce3, Ibrahim Kılınc4, Mehmet Uyar5, Emir Kaan Izci6, Erdal Kalkan1
OBJECTIVES: We aimed to evaluate neuroprotective effects of tocilizumab on spinal cord ischemia-reperfusion (I/R) injury. Our study design was an experimental rabbit spinal cord I/R injury model, and the setting was at the Animal Research Laboratory, Necmettin Erbakan University, Meram School of Medicine, Konya, Turkey.
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METHODS: Twenty-four adult New Zealand rabbits were randomly divided into 3 groups: Group 1, control group (n [ 8); Group 2, I/R group, and Group 3 (n [ 8) I/R injury D tocilizumab (4 mg/kg, ip) treatment group. Spinal cord I/R injury repair was performed by infrarenal aortic cross clamping. On neurologic evaluation, spinal cord tissue plasma tumor necrosis factor alpha (TNFa), total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive substances (TBARS), interleukin 6 (IL-6), interleukin 10 (IL-10) levels were analyzed. Spinal cord neuronal damage score and apoptotic cell count were also investigated.
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RESULTS: I/R injury significantly increases the plasma and spinal cord tissue TNFa, TOS, TBARS, and IL-6 levels and decreases the plasma and spinal cord tissue TAS and IL-10 levels. Tocilizumab treatment significantly reduces the plasma and spinal cord tissue TNFa, TOS, TBARS, IL-6 levels and increases plasma and tissue TAS and IL-10 levels. I/R injury significantly increases spinal cord neuronal damage score and apoptotic cell count. Tocilizumab
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treatment significantly reduces spinal cord neuronal damage score and apoptotic cell count. Neurologic examination scores at 24, 48, and 72 hours were significantly better in the treatment group when compared with the I/R group. CONCLUSIONS: This study shows significant neuroprotective effects of tocilizumab on rabbit spinal cord I/R injury.
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INTRODUCTION
P
araplegia is a serious complication of thoracoabdominal aortic surgery with a 2.4%e40% reported incidence.1 Impaired arterial blood flow and ischemia-reperfusion (I/R) injury of the spinal cord owing to collateral supplier damage, perfusion insufficiency, prolonged aortic cross-clamping time, intraoperative proximal hypertension, high cerebrospinal fluid pressure, and postoperative hypotension were previously reported causative factors of this complication.2 The neurologic injury at the time of impact is called the primary injury. Primary injury triggers secondary injury by enhanced inflammation, apoptosis, and free radical formation that has severe worsening effects on outcome. A variety of pharmacologic agents have been used to prevent secondary injury with different success rates,3 but the exact treatment of secondary injury is lacking.
Key words - Ischemia-reperfusion - Neuroprotective - Spinal - Tocilizumab
From the 1Neurosurgery Department, Medova Hospital, Konya; 2Neurosurgery Department, 3 Histology and Embriology Department, 4Medical Biochemitry Department, and 5Public Health Department, Necmettin Erbakan University Meram Medical Faculty, Konya; and 6 Neurosurgery Department, Meram Education Research Hospital, Konya, Turkey
Abbreviations and Acronyms IL-6: Interleukin 6 IL-10: Interleukin 10 I/R: Ischemia-reperfusion TAS: Total antioxidant status TBARS: Thiobarbituric acid reactive substances TNFa: Tumor necrosis factor alpha TOS: Total oxidant status TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling
Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2018.12.069
WORLD NEUROSURGERY -: e1-e6, - 2019
To whom correspondence should be addressed: Yasar Karatas, M.D. [E-mail: [email protected]]
Journal homepage: www.journals.elsevier.com/world-neurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
www.journals.elsevier.com/world-neurosurgery
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ORIGINAL ARTICLE YASAR KARATAS ET AL.
NEUROPROTECTIVE EFFECT OF TOCILIZUMAB
Tocilizumab is an interleukin 6 (IL-6) receptor blocker that has beneficial effects on rheumatoid arthritis,4 Castleman’s disease,5 juvenile rheumatoid arthritis,6 and inflammatory bowel disease7 with it’s antiinflammatory effects. Recently, neuroprotective effects of tocilizumab were investigated in a cerebral ischemia model and beneficial effects were reported.8 In this study, we aimed to investigate the neuroprotective effects of tocilizumab on a rabbit spinal cord I/R injury model. MATERIALS AND METHODS This study was performed in the Experimental Medicine Application and Research Center of Necmettin Erbakan University. The experimental protocol was evaluated and approved by the ethics review committee of Necmettin Erbakan University. The animals were provided (18 Ce21 C) room temperature and fed with a standard diet. A 12-hour light-dark cycle (08:00e20:00 hours light/20:01e07:59 hours dark) was maintained. Free access to food and water was permitted. Groups Twenty-four adult New Zealand rabbits were randomly divided into 3 groups: Group 1, control group (n ¼ 8); Group 2, I/R group; and Group 3 (n ¼ 8) I/R injury þ tocilizumab (4 mg/kg, ip, single dose) treatment group. All rabbits were anesthetized by intramuscular injection of ketamine (50 mg/kg) (Ketalar, Parke-Davis, Eczacıbas¸ı, Istanbul, Turkey) and xylazine (10 mg/kg) (Rompun, Bayer, Istanbul, Turkey) and allowed to breathe spontaneously during the procedure. An intravenous catheter was placed in the ear vein of the animals, and preoperative cefazolin 10 mg/kg (Cefamezin, Eczacıbası, Istanbul, Turkey) was administered as a single dose. As maintenance, 0.9% NaCl (20 mL per hour) was given throughout the experiment. All rabbits underwent laparotomy in supine position. Aortic cross clamping was not applied to Group 1. In Groups 2 and 3, the abdominal aorta was found and dissected carefully from the beginning of the left renal artery by a transperitoneal approach. One hundred IU/kg heparin was given intravenously 5 minutes before occlusion. The aorta was then cross-clamped by using an aneurysm clip with a closing force of 70 g (Yasargil FE 721 Aneurism Clip System, Aesculap Inc., Center Valley, Pennsylvania, USA). The clipping site was just below the left renal artery exit site. After occlusion, pulsation of the femoral artery disappeared. The aneurysm clip was removed after 30 minutes, and aortic pulsation was reachieved. Neither aortic nor caval hemorhage were seen during surgery. Single dose intraperitoneal 4 mg/kg tocilizumab treatment was given to Group 3 rabbits before closure. The dosage of tocilizumab was decided according to the literature.9 After the closure of the laparotomy, all rabbits awakened and returned to their cage. The animals were followed up neurologically, and motor inefficiency and healing rates were noted. Seventy-two hours later, all rabbits were re-anesthetized by an intramuscular injection of ketamine (50 mg/kg) (Ketalar) and xylazine (10 mg/kg) (Rompun). Blood samples were taken from ear vessels for biochemical investigation. Spinal cord samples were taken from lumbar spinal cord segments between L4-L6 by laminectomies for histopathological examination and the animals were sacrificed.
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Biochemical Analysis Venous blood samples were collected by centrifugation at 4 C and 1000 g for 10 minutes to separate plasma. Plasma samples were stored at e80 C until the parameters were studied. Spinal cord tissue samples were taken in pH: 7.4, 50 mM phosphate buffer and stored at e80 C until analysis. The thawed tissue samples were weighed and homogenized in ice using a mechanical homogenizer and an ultrasonic homogenizer in a 10-fold (w/v) cold phosphate buffer (50 mM, pH: 7.4). The supernatants were separated by centrifuging the homogenates for 10 minutes at 4 C and 10,000 g. Plasma and spinal cord tissue total oxidant status (TOS) and total antioxidant status (TAS) (Rel Assay Diagnostics, Gaziantep, Turkey), thiobarbituric acid reactive substances (TBARS) (Oxford Biomedical Research, Kansas City, Missouri, USA) and tissue protein levels were measured spectrophotometrically by using Pierce bicinchoninic acid-BCA (Thermo Scientific, Chicago, Illinois, USA). Plasma and spinal cord tissue IL-6, interleukin 10 (IL-10), and tumor necrosis factor alpha (TNFa) levels were analyzed by using rabbit specific ELISA antigens (Elabscience Biotechnology Co., Wuhan, China). Histopathological Investigations Spinal cord samples were fixed by 10% formaldehyde for 2 days and then embedded in paraffin blocks. After dehydration, coronal sections of the spinal cord segment were cut at a thickness of 4 mm and stained with hematoxylin and eosin for evaluation of structural changes. Gray matter was evaluated in 5 different areas in each section. According to the extent of inflammation, hemorrhage, axonal swelling, congestion, neuronal degeneration, and vacuolization of the spinal cord, the light microscopic findings were graded on a scale from 0e3, corresponding to no change, mild, moderate, and severe changes, respectively. The histopathological score was calculated for each spinal cord specimen.10 Apoptotic cells were labeled using an ApopTag In Situ Apoptosis Detection Kit (Millipore, Burlington, MA, USA). DNA fragments in spinal cord sections were modified via the action of terminal deoxynucleotidyl transferase. All procedures followed the manufacturer’s instructions. Five dark visual fields were randomly selected on every section, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons and the total numbers of neurons in the selective visual fields were counted. TUNEL-positive index (the TUNEL-positive to whole neurons ratio) was calculated. Eight sections of each group were used for measurement, and 5 high-powered visuals from every section were randomly selected to measure the TUNEL-positive indices.11 Neurologic Evaluation Motor function of the rabbits were evaluated by using modified Tarlov’s scale 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.12 Statistical Analysis Data were analyzed by SPSS software version 24.0 (SPSS, Inc., Chicago, Illinois, USA) and expressed as mean SD.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2018.12.069
ORIGINAL ARTICLE YASAR KARATAS ET AL.
Figure 1. The effects of tocilizumab on plasma levels of total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive
Comparisons were made using the Kruskal-Wallis test. Differences among the groups were assessed using the MannWhitney U test. A P value < 0.05 was considered statistically significant. Histopathological score and TUNEL-positive cell count were compared using a one-way analysis of variance with the Tukey test. RESULTS Biochemical Evaluation I/R injury significantly increases the plasma and spinal cord tissue TNFa, TOS, TBARS, IL-6 levels, and decreases the plasma and spinal cord tissue TAS and IL-10 levels. Tocilizumab treatment statistically significantly reduces the plasma and spinal cord tissue TNFa, TOS, TBARS, IL-6 levels, and increases plasma and tissue TAS and IL-10 levels (P ¼ 0) (Figures 1 and 2). Histopathological Evaluation I/R injury significantly increases the spinal cord neuronal damage score and apoptotic cell count. Tocilizumab treatment statistically significantly reduces spinal cord neuronal damage score and apoptotic cell count (P ¼ 0). Large motor cells were seen in the anterior horn of the spinal cord in the control group (Figure 3A). There was no change observed in neurons. The most severe injury was seen in I/R group in the spinal cord in hematoxylin and eosin sections (Figure 3B and C). Necrosis, hemorrhage, and congestion were
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substances (TBARS), interleukin 6 (IL-6), and interleukin 10 (IL-10).
observed in the I/R group. Nissl substances disappeared in necrotic neurons. Also, neuropil vacuolation and tissue loss were seen in the gray matter (Figure 3B and C). Histopathological score increased in the I/R group compared with the control group. Histopathological changes and score significantly decreased in the tocilizumab treatment group (Figure 3DeF). TUNEL-positive cell count increased in the I/R group when compared with the control group (Figure 4A and B). Tocilizumab treatment decreased TUNEL-positive cells count (Figure 4C). Figure 4D shows differences among the groups. Neurologic Evaluation Neurologic evaluation of the control group was normal. Neurologic examination scores of tocilizumab treatment group at 24, 48, and 72 hours after injury were statistically significantly (P < 0.005) better when compared with Group 2. DISCUSSION Neural tissues are very sensitive to ischemia. I/R inury of the spinal cord during thoracoabdominal vascular surgery could lead severe consequences such as paraplegia. Primary injury during the I/R period triggers secondary injury by enhanced inflammation, apoptosis, and free radical formation.13 Secondary injury leads to endothelial dysfunction and enhances vascular permeability that encourage migration and activation of immune cells. These activated immune cells infiltrate the related area and secrete
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ORIGINAL ARTICLE YASAR KARATAS ET AL.
NEUROPROTECTIVE EFFECT OF TOCILIZUMAB
Figure 2. The effects of tocilizumab on tissue levels of total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive substances (TBARS),
proinflammatory cytokines. Enhanced inflammation leads to the production of reactive oxygen species and triggers lipid peroxidation that damage the ultrastructure of neural cell membranes and inhibit it’s critical functions.14 Because of it’s unique anatomic properties, the spinal cord is very sensitive to ischemia. The method used in this study “infrarenal aortic cross clamping” leads to severe spinal cord injury. This method was first decribed by Liang et al. as an experimental model.15-17 It appears that the cause of paraplegia is multifactorial and includes prolonged duration of ischemia,
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tumor necrosis factor alpha (TNFa), interleukin 6 (IL-6), and interleukin 10 (IL-10).
interruption of critical intercostal and lumbar arteries, decrease in spinal cord perfusion pressure, and postoperative reperfusion injury.17 This method is suitable for mimic aortic surgery complications. IL-6 is a proinflammatory cytokine that can be produced by different cell types, such as astrocytes and macrophages. Between the first 6 and 12 hours of spinal cord ischemia, IL-6 levels enhances and IL-6 receptors upregulate. These changes return to its basal levels 4 days after injury.18,19 IL-6 is a key regulator of the inflammatory response, and IL-6 plays an important role in
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2018.12.069
ORIGINAL ARTICLE YASAR KARATAS ET AL.
Figure 3. (A) Control group, (B) I/R group, chromatolysis (black arrow), *:vacuole, (C) I/R group, necrosis (red arrow), *:vacuole, (D) tocilizumab group, congestion
Figure 4. (A) Group 1, (B) Group 2, terminal deoxynucleotidyl transferase dUTP nick end labeling positive glial cells and neurons (arrows), (C) Group
WORLD NEUROSURGERY -: e1-e6, - 2019
NEUROPROTECTIVE EFFECT OF TOCILIZUMAB
(green arrow), (E) tocilizumab group, white matter hemorrhage (yellow arrow), and (F) normal neurons in tocilizumab group.
3, and (D) statistical differences were determined among the groups.
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ORIGINAL ARTICLE YASAR KARATAS ET AL.
NEUROPROTECTIVE EFFECT OF TOCILIZUMAB
activating and recruiting immune cells, but simultaneously suppresses TNFa and IL1b production.20,21 Tocilizumab is an IL-6 receptor blocker. Strong antiinflammatory effects of tocilizumab was previously shown on rheumatoid arthritis,4 Castleman’s disease,5 juvenile rheumatoid arthritis,6 and inflammatory bowel disease.7 High levels of plasma and spinal cord tissue TNFa, TOS, TBARS, and IL-6 in the I/R group indicates enhanced inflammation and oxidative stress. After I/R ınjury, neuronal damage score and apoptotic cell count were found to be increased. Also neurologic examination scores of the I/R group were significantly worsened. Tocilizumab treatment significantly ameliorates biochemical, histopathological, and neurological parameters in I/R injury. Okada et al.22 investigated the effects of an anti-mouse IL-6 receptor antibody on contusive spinal cord injury. In this study single, intraperitoneal administration of anti-IL-6 receptor antibody prevents IL-6 associated immune response and cause significant motor improvement.
REFERENCES
Wang et al.8 reported that tocilizumab has protective effects in cerebral ischemia. In this study, upregulated Bcl-x and downregulated Caspase 3 prevents apoptosis and promotes tyrosine 705 phosphorylation of signal transducer and activator of transcription 3, which might be the potential regulatory mechanism of tocilizumab in neuronal cells. This study provided evidence for the protective role of tocilizumab against neuronal cell apoptosis in cerebral infarction.8 CONCLUSIONS In this study, we found significant neuroprotective effects of tocilizumab on rabbit spinal cord I/R injury, to our knowlegde, for the first time in the literature. Enhanced inflammation and oxidative stress after I/R ınjury was successfully reversed by tocilizumab and biochemical, histopathological, and neurologic worse parameters was ameliorated. Further studies are needed to implement this treatment into clinical practice.
experimental acute pancreatitis model in rats. Turk J Gastroenterol. 2017;28:485-491.
1. Coselli JS, LeMaire SA, Conklin LD, Koksoy C, Schmittling ZC. Morbidity and mortality after extent II thoracoabdominal aortic aneurysm repair. Ann Thorac Surg. 2002;73:1107-1115.
10. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood. 1995;86:1243-1254.
2. Kocogullari CU, Becit N, Erkut B, et al. Prevention of reperfusion injury of the spinal cord in aortic surgery: an experimental study. Surg Today. 2008; 38:237-244.
11. Gürer B, Kertmen H, Kasim E, et al. Neuroprotective effects of testosterone on ischemia/ reperfusion injury of the rabbit spinal cord. Injury. 2015;46:240-248.
3. Junk AK, Mammis A, Savitz SI, et al. Erythropoietin administration protects retinal neurons from acute ischemia-reperfusion injury. Proc Natl Acad Sci USA. 2002;99:10659-10664.
12. Wang LM, Yan Y, Zou LJ, Jing NH, Xu ZY. Moderate hypothermia prevents neural cell apoptosis following spinal cord ischemia in rabbits. Cell Res. 2005;15:387-393.
4. Dhillon S. Intravenous tocilizumab: a review of its use in adults with rheumatoid arthritis. BioDrugs. 2014;28:75-106.
13. Klune JR, Tsung A. Molecular biology of liver ischemia/reperfusion injury: established mechanisms and recent advancements. Surg Clin North Am. 2001;90:665-677.
5. Robey RC, Mletzko S, Colley C, Balachandran K, Bower M. The use of monoclonal antibodies to treat Castleman’s disease. Immunotherapy. 2014;6: 211-219. 6. De Benedetti F, Brunner HI, Ruperto N, et al. Randomized trial of tocilizumab in systemic juvenile idiopathic arthritis. N Engl J Med. 2012; 367:2385-2395. 7. Ash Z, Emery P. The role of tocilizumab in the management of rheumatoid arthritis. Expert Opin Biol Ther. 2012;12:1277-1289. 8. Wang S, Zhou J, Kang W, Dong Z, Wang H. Tocilizumab inhibits neuronal cell apoptosis and activates STAT3 in cerebral infarction rat model. Bosn J Basic Med Sci. 2016;16:145-150. 9. Hançerli Y, Kaplan M, Tanoglu A, et al. Efficacy of tocilizumab treatment in cerulein-induced
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18. Shechter R, London A, Varol C, et al. Infiltrating blood-derived macrophages are vital cells playing an anti-inflammatory role in recovery from spinal cord injury in mice. PLoS Med. 2009;6:e1000113. 19. Sahin MA, Onan B, Guler A, et al. Cilostazol, a type III phosphodiesterase inhibitor, reduces ischemia/reperfusion-induced spinal cord injury. Heart Surg Forum. 2011;14:171-177. 20. Klusman I, Schwab ME. Effects of proinflammatory cytokines in experimental spinal cord injury. Brain Res. 1997;762:173-184. 21. Juttler E, Tarabin V, Schwaninger M. Interleukin-6 (IL-6): a possible neuromodulator induced by neuronal activity. Neuroscientist. 2002;8:268. 22. Okada S, Nakamura M, Mikami Y, et al. Blockade of interleukin-6 receptor suppresses reactive astrogliosis and ameliorates functional recovery in experimental spinal cord injury. J Neurosci Res. 2004;76:265-276.
14. Chan PH. Role of oxidants in ischemic brain damage. Stroke. 1996;27:1124-1129. 15. Mukaino M, Nakamura M, Yamada O, et al. AntiIL-6-receptor antibody promotes repair of spinal cord injury by inducing microglia-dominant inflammation. Exper Neurol. 2010;224:403-414. 16. Crawford ES, Crawford JL, Safi HJ, et al. Thoracoabdominal aortic aneurysms: preoperative and intraoperative factors determining immediate and long-term results of operations in 605 patients. J Vasc Surg. 1986;3:389-404. 17. Liang Y, Yang Q-H, Yu X-D, Jiang D-M. Additive effect of tetramethylpyrazine and deferoxamine in the treatment of spinal cord injury caused by aortic crossclamping in rats. Spinal Cord. 2011;49: 302-306.
Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received 23 February 2018; accepted 7 December 2018 Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2018.12.069 Journal homepage: www.journals.elsevier.com/worldneurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2018.12.069
Neuroprotective Effects of Tocilizumab on Experimentally-Induced Spinal Cord Ischemia-Reperfusion Injury Yasar Karatas1, Mehmet Fatih Erdi2, Bu¨lent Kaya1, Fatih Keskin2, Go¨khan Cu¨ce3, Ibrahim Kılınc4, Mehmet Uyar5, Emir Kaan Izci6, Erdal Kalkan1
OBJECTIVES: We aimed to evaluate neuroprotective effects of tocilizumab on spinal cord ischemia-reperfusion (I/R) injury. Our study design was an experimental rabbit spinal cord I/R injury model, and the setting was at the Animal Research Laboratory, Necmettin Erbakan University, Meram School of Medicine, Konya, Turkey.
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METHODS: Twenty-four adult New Zealand rabbits were randomly divided into 3 groups: Group 1, control group (n [ 8); Group 2, I/R group, and Group 3 (n [ 8) I/R injury D tocilizumab (4 mg/kg, ip) treatment group. Spinal cord I/R injury repair was performed by infrarenal aortic cross clamping. On neurologic evaluation, spinal cord tissue plasma tumor necrosis factor alpha (TNFa), total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive substances (TBARS), interleukin 6 (IL-6), interleukin 10 (IL-10) levels were analyzed. Spinal cord neuronal damage score and apoptotic cell count were also investigated.
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RESULTS: I/R injury significantly increases the plasma and spinal cord tissue TNFa, TOS, TBARS, and IL-6 levels and decreases the plasma and spinal cord tissue TAS and IL-10 levels. Tocilizumab treatment significantly reduces the plasma and spinal cord tissue TNFa, TOS, TBARS, IL-6 levels and increases plasma and tissue TAS and IL-10 levels. I/R injury significantly increases spinal cord neuronal damage score and apoptotic cell count. Tocilizumab
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treatment significantly reduces spinal cord neuronal damage score and apoptotic cell count. Neurologic examination scores at 24, 48, and 72 hours were significantly better in the treatment group when compared with the I/R group. CONCLUSIONS: This study shows significant neuroprotective effects of tocilizumab on rabbit spinal cord I/R injury.
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INTRODUCTION
P
araplegia is a serious complication of thoracoabdominal aortic surgery with a 2.4%e40% reported incidence.1 Impaired arterial blood flow and ischemia-reperfusion (I/R) injury of the spinal cord owing to collateral supplier damage, perfusion insufficiency, prolonged aortic cross-clamping time, intraoperative proximal hypertension, high cerebrospinal fluid pressure, and postoperative hypotension were previously reported causative factors of this complication.2 The neurologic injury at the time of impact is called the primary injury. Primary injury triggers secondary injury by enhanced inflammation, apoptosis, and free radical formation that has severe worsening effects on outcome. A variety of pharmacologic agents have been used to prevent secondary injury with different success rates,3 but the exact treatment of secondary injury is lacking.
Key words - Ischemia-reperfusion - Neuroprotective - Spinal - Tocilizumab
From the 1Neurosurgery Department, Medova Hospital, Konya; 2Neurosurgery Department, 3 Histology and Embriology Department, 4Medical Biochemitry Department, and 5Public Health Department, Necmettin Erbakan University Meram Medical Faculty, Konya; and 6 Neurosurgery Department, Meram Education Research Hospital, Konya, Turkey
Abbreviations and Acronyms IL-6: Interleukin 6 IL-10: Interleukin 10 I/R: Ischemia-reperfusion TAS: Total antioxidant status TBARS: Thiobarbituric acid reactive substances TNFa: Tumor necrosis factor alpha TOS: Total oxidant status TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling
Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2018.12.069
WORLD NEUROSURGERY -: e1-e6, - 2019
To whom correspondence should be addressed: Yasar Karatas, M.D. [E-mail: [email protected]]
Journal homepage: www.journals.elsevier.com/world-neurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
www.journals.elsevier.com/world-neurosurgery
e1
ORIGINAL ARTICLE YASAR KARATAS ET AL.
NEUROPROTECTIVE EFFECT OF TOCILIZUMAB
Tocilizumab is an interleukin 6 (IL-6) receptor blocker that has beneficial effects on rheumatoid arthritis,4 Castleman’s disease,5 juvenile rheumatoid arthritis,6 and inflammatory bowel disease7 with it’s antiinflammatory effects. Recently, neuroprotective effects of tocilizumab were investigated in a cerebral ischemia model and beneficial effects were reported.8 In this study, we aimed to investigate the neuroprotective effects of tocilizumab on a rabbit spinal cord I/R injury model. MATERIALS AND METHODS This study was performed in the Experimental Medicine Application and Research Center of Necmettin Erbakan University. The experimental protocol was evaluated and approved by the ethics review committee of Necmettin Erbakan University. The animals were provided (18 Ce21 C) room temperature and fed with a standard diet. A 12-hour light-dark cycle (08:00e20:00 hours light/20:01e07:59 hours dark) was maintained. Free access to food and water was permitted. Groups Twenty-four adult New Zealand rabbits were randomly divided into 3 groups: Group 1, control group (n ¼ 8); Group 2, I/R group; and Group 3 (n ¼ 8) I/R injury þ tocilizumab (4 mg/kg, ip, single dose) treatment group. All rabbits were anesthetized by intramuscular injection of ketamine (50 mg/kg) (Ketalar, Parke-Davis, Eczacıbas¸ı, Istanbul, Turkey) and xylazine (10 mg/kg) (Rompun, Bayer, Istanbul, Turkey) and allowed to breathe spontaneously during the procedure. An intravenous catheter was placed in the ear vein of the animals, and preoperative cefazolin 10 mg/kg (Cefamezin, Eczacıbası, Istanbul, Turkey) was administered as a single dose. As maintenance, 0.9% NaCl (20 mL per hour) was given throughout the experiment. All rabbits underwent laparotomy in supine position. Aortic cross clamping was not applied to Group 1. In Groups 2 and 3, the abdominal aorta was found and dissected carefully from the beginning of the left renal artery by a transperitoneal approach. One hundred IU/kg heparin was given intravenously 5 minutes before occlusion. The aorta was then cross-clamped by using an aneurysm clip with a closing force of 70 g (Yasargil FE 721 Aneurism Clip System, Aesculap Inc., Center Valley, Pennsylvania, USA). The clipping site was just below the left renal artery exit site. After occlusion, pulsation of the femoral artery disappeared. The aneurysm clip was removed after 30 minutes, and aortic pulsation was reachieved. Neither aortic nor caval hemorhage were seen during surgery. Single dose intraperitoneal 4 mg/kg tocilizumab treatment was given to Group 3 rabbits before closure. The dosage of tocilizumab was decided according to the literature.9 After the closure of the laparotomy, all rabbits awakened and returned to their cage. The animals were followed up neurologically, and motor inefficiency and healing rates were noted. Seventy-two hours later, all rabbits were re-anesthetized by an intramuscular injection of ketamine (50 mg/kg) (Ketalar) and xylazine (10 mg/kg) (Rompun). Blood samples were taken from ear vessels for biochemical investigation. Spinal cord samples were taken from lumbar spinal cord segments between L4-L6 by laminectomies for histopathological examination and the animals were sacrificed.
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Biochemical Analysis Venous blood samples were collected by centrifugation at 4 C and 1000 g for 10 minutes to separate plasma. Plasma samples were stored at e80 C until the parameters were studied. Spinal cord tissue samples were taken in pH: 7.4, 50 mM phosphate buffer and stored at e80 C until analysis. The thawed tissue samples were weighed and homogenized in ice using a mechanical homogenizer and an ultrasonic homogenizer in a 10-fold (w/v) cold phosphate buffer (50 mM, pH: 7.4). The supernatants were separated by centrifuging the homogenates for 10 minutes at 4 C and 10,000 g. Plasma and spinal cord tissue total oxidant status (TOS) and total antioxidant status (TAS) (Rel Assay Diagnostics, Gaziantep, Turkey), thiobarbituric acid reactive substances (TBARS) (Oxford Biomedical Research, Kansas City, Missouri, USA) and tissue protein levels were measured spectrophotometrically by using Pierce bicinchoninic acid-BCA (Thermo Scientific, Chicago, Illinois, USA). Plasma and spinal cord tissue IL-6, interleukin 10 (IL-10), and tumor necrosis factor alpha (TNFa) levels were analyzed by using rabbit specific ELISA antigens (Elabscience Biotechnology Co., Wuhan, China). Histopathological Investigations Spinal cord samples were fixed by 10% formaldehyde for 2 days and then embedded in paraffin blocks. After dehydration, coronal sections of the spinal cord segment were cut at a thickness of 4 mm and stained with hematoxylin and eosin for evaluation of structural changes. Gray matter was evaluated in 5 different areas in each section. According to the extent of inflammation, hemorrhage, axonal swelling, congestion, neuronal degeneration, and vacuolization of the spinal cord, the light microscopic findings were graded on a scale from 0e3, corresponding to no change, mild, moderate, and severe changes, respectively. The histopathological score was calculated for each spinal cord specimen.10 Apoptotic cells were labeled using an ApopTag In Situ Apoptosis Detection Kit (Millipore, Burlington, MA, USA). DNA fragments in spinal cord sections were modified via the action of terminal deoxynucleotidyl transferase. All procedures followed the manufacturer’s instructions. Five dark visual fields were randomly selected on every section, and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons and the total numbers of neurons in the selective visual fields were counted. TUNEL-positive index (the TUNEL-positive to whole neurons ratio) was calculated. Eight sections of each group were used for measurement, and 5 high-powered visuals from every section were randomly selected to measure the TUNEL-positive indices.11 Neurologic Evaluation Motor function of the rabbits were evaluated by using modified Tarlov’s scale 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.12 Statistical Analysis Data were analyzed by SPSS software version 24.0 (SPSS, Inc., Chicago, Illinois, USA) and expressed as mean SD.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2018.12.069
ORIGINAL ARTICLE YASAR KARATAS ET AL.
Figure 1. The effects of tocilizumab on plasma levels of total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive
Comparisons were made using the Kruskal-Wallis test. Differences among the groups were assessed using the MannWhitney U test. A P value < 0.05 was considered statistically significant. Histopathological score and TUNEL-positive cell count were compared using a one-way analysis of variance with the Tukey test. RESULTS Biochemical Evaluation I/R injury significantly increases the plasma and spinal cord tissue TNFa, TOS, TBARS, IL-6 levels, and decreases the plasma and spinal cord tissue TAS and IL-10 levels. Tocilizumab treatment statistically significantly reduces the plasma and spinal cord tissue TNFa, TOS, TBARS, IL-6 levels, and increases plasma and tissue TAS and IL-10 levels (P ¼ 0) (Figures 1 and 2). Histopathological Evaluation I/R injury significantly increases the spinal cord neuronal damage score and apoptotic cell count. Tocilizumab treatment statistically significantly reduces spinal cord neuronal damage score and apoptotic cell count (P ¼ 0). Large motor cells were seen in the anterior horn of the spinal cord in the control group (Figure 3A). There was no change observed in neurons. The most severe injury was seen in I/R group in the spinal cord in hematoxylin and eosin sections (Figure 3B and C). Necrosis, hemorrhage, and congestion were
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substances (TBARS), interleukin 6 (IL-6), and interleukin 10 (IL-10).
observed in the I/R group. Nissl substances disappeared in necrotic neurons. Also, neuropil vacuolation and tissue loss were seen in the gray matter (Figure 3B and C). Histopathological score increased in the I/R group compared with the control group. Histopathological changes and score significantly decreased in the tocilizumab treatment group (Figure 3DeF). TUNEL-positive cell count increased in the I/R group when compared with the control group (Figure 4A and B). Tocilizumab treatment decreased TUNEL-positive cells count (Figure 4C). Figure 4D shows differences among the groups. Neurologic Evaluation Neurologic evaluation of the control group was normal. Neurologic examination scores of tocilizumab treatment group at 24, 48, and 72 hours after injury were statistically significantly (P < 0.005) better when compared with Group 2. DISCUSSION Neural tissues are very sensitive to ischemia. I/R inury of the spinal cord during thoracoabdominal vascular surgery could lead severe consequences such as paraplegia. Primary injury during the I/R period triggers secondary injury by enhanced inflammation, apoptosis, and free radical formation.13 Secondary injury leads to endothelial dysfunction and enhances vascular permeability that encourage migration and activation of immune cells. These activated immune cells infiltrate the related area and secrete
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Figure 2. The effects of tocilizumab on tissue levels of total antioxidant status (TAS), total oxidant status (TOS), thiobarbituric acid reactive substances (TBARS),
proinflammatory cytokines. Enhanced inflammation leads to the production of reactive oxygen species and triggers lipid peroxidation that damage the ultrastructure of neural cell membranes and inhibit it’s critical functions.14 Because of it’s unique anatomic properties, the spinal cord is very sensitive to ischemia. The method used in this study “infrarenal aortic cross clamping” leads to severe spinal cord injury. This method was first decribed by Liang et al. as an experimental model.15-17 It appears that the cause of paraplegia is multifactorial and includes prolonged duration of ischemia,
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tumor necrosis factor alpha (TNFa), interleukin 6 (IL-6), and interleukin 10 (IL-10).
interruption of critical intercostal and lumbar arteries, decrease in spinal cord perfusion pressure, and postoperative reperfusion injury.17 This method is suitable for mimic aortic surgery complications. IL-6 is a proinflammatory cytokine that can be produced by different cell types, such as astrocytes and macrophages. Between the first 6 and 12 hours of spinal cord ischemia, IL-6 levels enhances and IL-6 receptors upregulate. These changes return to its basal levels 4 days after injury.18,19 IL-6 is a key regulator of the inflammatory response, and IL-6 plays an important role in
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ORIGINAL ARTICLE YASAR KARATAS ET AL.
Figure 3. (A) Control group, (B) I/R group, chromatolysis (black arrow), *:vacuole, (C) I/R group, necrosis (red arrow), *:vacuole, (D) tocilizumab group, congestion
Figure 4. (A) Group 1, (B) Group 2, terminal deoxynucleotidyl transferase dUTP nick end labeling positive glial cells and neurons (arrows), (C) Group
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(green arrow), (E) tocilizumab group, white matter hemorrhage (yellow arrow), and (F) normal neurons in tocilizumab group.
3, and (D) statistical differences were determined among the groups.
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activating and recruiting immune cells, but simultaneously suppresses TNFa and IL1b production.20,21 Tocilizumab is an IL-6 receptor blocker. Strong antiinflammatory effects of tocilizumab was previously shown on rheumatoid arthritis,4 Castleman’s disease,5 juvenile rheumatoid arthritis,6 and inflammatory bowel disease.7 High levels of plasma and spinal cord tissue TNFa, TOS, TBARS, and IL-6 in the I/R group indicates enhanced inflammation and oxidative stress. After I/R ınjury, neuronal damage score and apoptotic cell count were found to be increased. Also neurologic examination scores of the I/R group were significantly worsened. Tocilizumab treatment significantly ameliorates biochemical, histopathological, and neurological parameters in I/R injury. Okada et al.22 investigated the effects of an anti-mouse IL-6 receptor antibody on contusive spinal cord injury. In this study single, intraperitoneal administration of anti-IL-6 receptor antibody prevents IL-6 associated immune response and cause significant motor improvement.
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Wang et al.8 reported that tocilizumab has protective effects in cerebral ischemia. In this study, upregulated Bcl-x and downregulated Caspase 3 prevents apoptosis and promotes tyrosine 705 phosphorylation of signal transducer and activator of transcription 3, which might be the potential regulatory mechanism of tocilizumab in neuronal cells. This study provided evidence for the protective role of tocilizumab against neuronal cell apoptosis in cerebral infarction.8 CONCLUSIONS In this study, we found significant neuroprotective effects of tocilizumab on rabbit spinal cord I/R injury, to our knowlegde, for the first time in the literature. Enhanced inflammation and oxidative stress after I/R ınjury was successfully reversed by tocilizumab and biochemical, histopathological, and neurologic worse parameters was ameliorated. Further studies are needed to implement this treatment into clinical practice.
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Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received 23 February 2018; accepted 7 December 2018 Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2018.12.069 Journal homepage: www.journals.elsevier.com/worldneurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2018.12.069