EFFECT OF TIME-RELATED ADMINISTRATION OF METHOTREXATE ON SPINAL CORD INJURY-INDUCED NEURAL CELL APOPTOSIS IN RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FANGChao ,  XUZhujun , YANGMin , XIEJiabing , LIQuanhui

Department of Orthopedic Trauma, the Affiliated Yijishan Hospital of Wannan Medical College, Wuhu Anhui, 241000, P. R. China;

Corresponding?author：

XUZhujun, Email: WH40352@vip.sina.com

Keywords：

Spinal cord injury; Methotrexate; Low-dose; Prophylaxis; Apoptosis; Rat

DOI：

10.7507/1002-1892.20160094

Video：

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Objective To evaluate the effect of time-related administration of methotrexate (MTX) on neural cell apoptosis in rats after spinal cord injury (SCI) so as to investigate its potential neuroprotective mechanism and appropriate administration time. Method A total of 120 male Sprague Dawley rats, 247-286 g in weight, were randomly divided into 4 groups (n=30) :sham group (group A), control group (group B), MTX treating group (group C), and MTX prophylaxis group (group D). The SCI model was established in the rats of groups B, C, and D by improved Allen method, and just laminectomy was performed in group A. MTX (0.5 mg/kg) was administered with tail vein injection at 1, 6, 12, 18, and 24 hours after injury in group C, and at 30 minutes before injury and at 6, 12, 18, and 24 hours after injury in group D; the equivalence saline was injected at 1, 6, 12, 18, and 24 hours after injury in groups A and B. Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neural function at 1, 3, 7, 14, and 21 days after injury, HE staining to observe histological changes, immunohistochemical staining and TUNEL method to measure the expression of Caspase-3 and neural cells apoptosis, respectively. Results Ten rats died during the experiment in groups B, C, and D; 25 rats in each group were included into the experiments at last. BBB score of group A was significantly higher than that of groups B, C, and D at all time points after injury (P<0.05) . BBB score of groups C and D were significantly higher than that of group B at 3, 7, 14, and 21 days (P<0.05) , and BBB score of group D was significantly higher than that of group C at 3, 7, and 14 days (P<0.05) . The histological observation showed normal structure of spinal cord at all time points after injury in group A. While the degree of SCI in group D was lighter than that in groups B and C, and group C was lighter than group B. At 14 days after injury, the degree of SCI in groups B, C, and D tend to keep the same. The number of Caspase-3 and TUNEL positive cells of groups B, C, and D was significantly more than that of group A at all time points after injury (P<0.05) , group B was significantly more than groups C and D (P<0.05) . The number of Caspase-3 positive cells of group C was significantly more than that of group D at 3, 7, and 14 days (P<0.05) . While the number of TUNEL positive cells of group C was significantly more than that of group D at 3 and 7 days (P<0.05) . And the number of Caspase-3 positive cells and TUNEL positive cells was positively correlated in groups B, C, and D (P<0.05) at 1, 3, 7, 14, and 21 days after injury. Conclusions Low-dose MTX may effectively reduce the degree of the secondary injury of spinal cord by reducing the nerve cell apoptosis. Better effect can be obtained when MTX is used as prevent method than as a way of treatment.

Citation： FANGChao, XUZhujun, YANGMin, XIEJiabing, LIQuanhui. EFFECT OF TIME-RELATED ADMINISTRATION OF METHOTREXATE ON SPINAL CORD INJURY-INDUCED NEURAL CELL APOPTOSIS IN RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(4): 466-472. doi: 10.7507/1002-1892.20160094 Copy

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2.	Becker D, Mcdonald JW 3rd. Approaches to repairing the damaged spinal cord:overview. Handb Clin Neurol, 2012, 109:445-461.
3.	Keith MP, Edison JD, Gilliland WR. Progress toward personalized treatment of rheumatoid arthritis. Clin Pharmacol Ther, 2012, 92(4) :440-442.
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6.	張思, 王爍宇, 顧兵, 等. 低劑量甲氨蝶呤治療大鼠脊髓挫傷對神經細胞凋亡的影響. 神經藥理學報, 2013, 3(6) :1-8.
7.	Meng H, Liu Y, Li K, et al. Comparison of the effects of methotrexate and methylprednisolone in spinal cord Injury in rats. Drug Res (Stuttg), 2015, 65(8) :437-441.
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9.	張思, 顧兵, 李華南, 等. 甲氨蝶呤對大鼠脊髓挫傷急性期氧化損傷相關蛋白的影響. 中國臨床藥理學與治療學, 2015, 20(1) :1-6.
10.	Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open filed testing in rats. J Nearotrauma, 1995, 12(1) :1-21.
11.	Sirbulescu RF, Zupanc GK. Inhibition of caspase-3-mediated apoptosisi improve spinal cord repair in a regeneration-competent vertebrate system. Neuroscience, 2010, 171(2) :599-612.
12.	Kakinohana M, Kida K, Minamishima S, et al. Delayed paraplegia after spinal cord ischemic injury requires caspase-3 activation in mice. Stroke, 2011, 42(8) :2302-2307.
13.	Yilmaz ER, Kertmen H, Dolgun H, et al. Effects of darbepoetin-α in spinal cord ischemia-reperfusion injury in the rabbit. Acta Neurochir (Wien), 2012, 154(6) :1037-1043.
14.	Kertmen H, Gürer B, Yilmaz ER, et al. The protective effect of low-dose methotrexate on ischemia-reperfusion injury of the rabbit spinal cord. Eur J Pharmacol, 2013, 714(1-3) :148-156.
15.	汪紅林, 汪軍玉, 徐祝軍. 環扎法致大鼠脊髓損傷早期減壓后 caspase-3的表達及其與神經細胞凋亡相關性研究. 中國矯形外科雜志, 2012, 20(4) :358-362.
16.	張思, 顧兵, 王爍宇, 等. 甲氨蝶呤對大鼠脊髓挫傷急性期脂質過氧化的影響. 神經藥理學報, 2014, 4(1) :11-16.
17.	Chan ES, Cronstein BN. Methotrexate-how does it really work? Nat Rev Rheumatol, 2010, 6(3) :175-178.
18.	Xinqiang S, Fei L, Nan L, et al. Therapeutic efficacy of experimental rheumatoid arthritis with low-dose methotrexate by increasing partially CD4+ CD25+ Treg cells and inducing Th1 to Th2 shift in both cells and cytokines. Biomed Pharmacother, 2010, 64(7) :463-471.
19.	Tabayashi K, Saiki Y, Kokubo H, et al. Protection from postischem ic spinal cord injury by perfusion cooling of the epidural space during most or all of a descending thoracic or thoracoabdominal aneurysm repair. Gen Thorac Cardiovasc Surg, 2010, 58(5) :228-234.

1. Trivedi A, Olivas AD, Noble-Haeusslein LJ. Inflammation and spinal cord ?njury:infiltrating leukocytes as determinants of injury and repair processes. Clin Neurosci Res, 2006, 6(5) :283-292.
2. Becker D, Mcdonald JW 3rd. Approaches to repairing the damaged spinal cord:overview. Handb Clin Neurol, 2012, 109:445-461.
3. Keith MP, Edison JD, Gilliland WR. Progress toward personalized treatment of rheumatoid arthritis. Clin Pharmacol Ther, 2012, 92(4) :440-442.
4. 錢列, 陳雄生, 譚軍, 等. 不同時間應用甲基強的松龍對大鼠急性脊髓損傷的作用. 中國脊柱脊髓雜志, 2009, 19(4) :298-303.
5. Bakar B, K?se EA, Kupana Ayva S, et al. Effects of low-dose methotrexate in spinal cord injury in rats. Ulus Travma Acil Cerrahi Derg, 2013, 19 (4) :285-293.
6. 張思, 王爍宇, 顧兵, 等. 低劑量甲氨蝶呤治療大鼠脊髓挫傷對神經細胞凋亡的影響. 神經藥理學報, 2013, 3(6) :1-8.
7. Meng H, Liu Y, Li K, et al. Comparison of the effects of methotrexate and methylprednisolone in spinal cord Injury in rats. Drug Res (Stuttg), 2015, 65(8) :437-441.
8. Sanli AM, Serbes G, Sargon MF, et al. Methothrexate attenuates early neutrophil infiltration and the associated lipid peroxidation in the injured spinal cord but does not induce neurotoxicity in the uninjured spinal cord in rats. Acta Neurochir (Wien), 2012, 154(6) :1045-1054.
9. 張思, 顧兵, 李華南, 等. 甲氨蝶呤對大鼠脊髓挫傷急性期氧化損傷相關蛋白的影響. 中國臨床藥理學與治療學, 2015, 20(1) :1-6.
10. Basso DM, Beattie MS, Bresnahan JC. A sensitive and reliable locomotor rating scale for open filed testing in rats. J Nearotrauma, 1995, 12(1) :1-21.
11. Sirbulescu RF, Zupanc GK. Inhibition of caspase-3-mediated apoptosisi improve spinal cord repair in a regeneration-competent vertebrate system. Neuroscience, 2010, 171(2) :599-612.
12. Kakinohana M, Kida K, Minamishima S, et al. Delayed paraplegia after spinal cord ischemic injury requires caspase-3 activation in mice. Stroke, 2011, 42(8) :2302-2307.
13. Yilmaz ER, Kertmen H, Dolgun H, et al. Effects of darbepoetin-α in spinal cord ischemia-reperfusion injury in the rabbit. Acta Neurochir (Wien), 2012, 154(6) :1037-1043.
14. Kertmen H, Gürer B, Yilmaz ER, et al. The protective effect of low-dose methotrexate on ischemia-reperfusion injury of the rabbit spinal cord. Eur J Pharmacol, 2013, 714(1-3) :148-156.
15. 汪紅林, 汪軍玉, 徐祝軍. 環扎法致大鼠脊髓損傷早期減壓后 caspase-3的表達及其與神經細胞凋亡相關性研究. 中國矯形外科雜志, 2012, 20(4) :358-362.
16. 張思, 顧兵, 王爍宇, 等. 甲氨蝶呤對大鼠脊髓挫傷急性期脂質過氧化的影響. 神經藥理學報, 2014, 4(1) :11-16.
17. Chan ES, Cronstein BN. Methotrexate-how does it really work? Nat Rev Rheumatol, 2010, 6(3) :175-178.
18. Xinqiang S, Fei L, Nan L, et al. Therapeutic efficacy of experimental rheumatoid arthritis with low-dose methotrexate by increasing partially CD4+ CD25+ Treg cells and inducing Th1 to Th2 shift in both cells and cytokines. Biomed Pharmacother, 2010, 64(7) :463-471.
19. Tabayashi K, Saiki Y, Kokubo H, et al. Protection from postischem ic spinal cord injury by perfusion cooling of the epidural space during most or all of a descending thoracic or thoracoabdominal aneurysm repair. Gen Thorac Cardiovasc Surg, 2010, 58(5) :228-234.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF TIME-RELATED ADMINISTRATION OF METHOTREXATE ON SPINAL CORD INJURY-INDUCED NEURAL CELL APOPTOSIS IN RATS

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