Changes of endogenous Spastin expression after sciatic nerve injury in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIN Yaofa , ZONG Haiyang , HU Xianteng , YU Ronghua , SHAO Wanwan , HOU Chunlin ,  LIN Haodong

Department of Orthopaedics, Shanghai Changzheng Hospital, the Second Military Medical University, Shanghai, 200040, P.R.China;

Corresponding?author：

LIN Haodong, Email: haodonglin@hotmail.com

Keywords：

Spastin; sciatic nerve; nerve injury; nerve regeneration; rat

DOI：

10.7507/1002-1892.201610093

Video：

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Objective To investigate the expression change of endogenous Spastin after sciatic nerve injury in rats, and to discuss the role and significance in the peripheral nerve regeneration. Methods Thirty-six adult male Sprague Dawley rats weighing 180–220 g were randomly divided into the experimental group (n=30) and the control group (n=6). Sciatic nerve compression damage model was established in the experimental group, and the sciatic nerve was only exposed in the control group. The L_4-6 spinal cord tissue was obtained to detect Spastin mRNA and protein levels by real-time fluorescence quantitative PCR and Western blot at 1, 3, 7, 14, and 28 days after operation in the experimental group (n=6) and at 7 days in the control group. Meanwhile, the sciatic nerve at 5 mm distal to the injured site was obtained to observe the ultrastructure of the distal axon by transmission electron microscope (TEM). Results The expression trends of Spastin gene and Spastin protein in L_4-6 spinal cord tissue of 2 groups were basically identical. In the experimental group, the expressions of Spastin gene and protein decreased at the beginning, and then increased; the expressions reduced to the minimum at 7 days after operation, and came back to the initial level at 28 days. The expression levels of Spastin mRNA and protein at 3, 7, and 14 days were significantly lower in the experimental group than the control group (P<0.05), but no significant difference was noted between 2 groups at 1 and 28 days (P>0.05). The expression levels of Spastin mRNA and protein at 3, 7, and 14 days were significantly lower than those at 1 and 28 days in the experimental group (P<0.05), but no significant difference was noted between at 1 day and 28 days (P>0.05). At 1, 3, and 7 days after operation, the myelin damage was observed by TEM; at 14 days, there were regenerating Schwann cells; at 28 days, a large number of myelinated nerve fibers were seen, which were closed to normal form. Conclusion In the process of sciatic nerve regeneration after injury, a complex succession of changes take place in the expression of endogenous Spastin protein in rats, indicating that Spastin protein plays an important role in the process.

Citation： LINYaofa, ZONGHaiyang, HUXianteng, YURonghua, SHAOWanwan, HOUChunlin, LINHaodong. Changes of endogenous Spastin expression after sciatic nerve injury in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(1): 80-84. doi: 10.7507/1002-1892.201610093 Copy

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2.	Sadeghian H, Wolfe GI. Therapy update in nerve, neuromuscular junctionandmyopathic disorders. Curr Opin Neurol, 2010, 23(5): 496-501.
3.	朱家愷. 周圍神經損傷診斷與治療進展. 中國修復重建外科雜志, 2006, 20(4): 319-322.
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6.	Ma CH, Omura T, Cobos EJ,et al. Acceleratingaxonal growth promotes motor recovery after peripheral nerve injury in mice. J Clin Invest, 2011, 121(11): 4332-4347.
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8.	Neumann B, Nguyen KC, Hall DH,et al. Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons. Dev Dyn, 2011, 240: 1365-1372.
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13.	Bittner GD, Spaeth CS, Poon AD,et al. Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells. Neural Regen Res, 2016, 11(7): 1033-1042.
14.	Ghergherehchi CL, Bittner GD, Hastings RL,et al. Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves. J Neurosci Res, 2016, 94(3): 231-245.
15.	Stone MC, Rao K, GheresKW,et al. Normal spastin gene dosage is specifically required for axon regeneration. Cell Rep, 2012, 2(5): 1340-1350.
16.	Diaz-Valencia JD, Bailey M, Ross JL. Purification and biophysical analysis of microtubule-severing enzymesin vitro. Methods Cell Biol, 2013, 115: 191-213.
17.	Campbell WW. Evaluation and management of peripheral nerve injury. Clin Neurophysiol, 2008, 119(9): 1951-1965.
18.	林強, 蔡楊庭, 李皓莘. 負載濃度梯度 NGF 的周圍神經導管修復大鼠周圍神經缺損的實驗研究. 中國修復重建外科雜志, 2014, 28(2): 167-172.
19.	宋學文, 魏彥明, 李根, 等. 紅景天苷/膠原蛋白/聚己內酯神經導管修復大鼠坐骨神經缺損的實驗研究. 中國修復重建外科雜志, 2016, 30(5): 634-640.
20.	Sherwood NT, Sun Q, Xue M,et al. Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol, 2004, 2(12): e429.
21.	Yu W, Qiang L, Solowska JM,et al. The microtubule-severing proteins spastinand katanin participate differently in the formation of axonal branches. Mol Biol Cell, 2008, 19(4): 1485-1498.
22.	Lee HH, Jan LY, Jan YN. Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis. Proc Natl Acad Sci U S A, 2009, 106(15): 6363-6368.

1. Bozkurt A, Tholl S, Wehner S,et al. Evaluation of functional nerve recoverywithVisual-SSI—a novel computerized approach for the assessment of the staticsciatic index (SSI). J Neurosci Methods, 2008, 170(1): 117-122.
2. Sadeghian H, Wolfe GI. Therapy update in nerve, neuromuscular junctionandmyopathic disorders. Curr Opin Neurol, 2010, 23(5): 496-501.
3. 朱家愷. 周圍神經損傷診斷與治療進展. 中國修復重建外科雜志, 2006, 20(4): 319-322.
4. 劉堅義, 盧德文. 顯微外科技術在周圍神經損傷修復中的應用. 中華顯微外科雜志, 1998, 21(2): 100-101.
5. 謝國均, 勞維藹, 唐國瑜, 等. 應用顯微外科技術修復周圍神經損傷. 中國修復重建外科雜志, 1997, 11(3): 181.
6. Ma CH, Omura T, Cobos EJ,et al. Acceleratingaxonal growth promotes motor recovery after peripheral nerve injury in mice. J Clin Invest, 2011, 121(11): 4332-4347.
7. Deriemer SA, Elliott EJ, Macagno ER,et al. Morphological evidence that regenerating axons can fuse with severed axon segments. Brain Res, 1983, 272(1): 157-161.
8. Neumann B, Nguyen KC, Hall DH,et al. Axonal regeneration proceeds through specific axonal fusion in transected C. elegans neurons. Dev Dyn, 2011, 240: 1365-1372.
9. Eddleman CS, Ballinger ML, Smyers ME,et al. Endocytotic formation of vesiclesand other membranous structures induced by Ca²⁺ and axolemmal injury. J Neurosci, 1998, 18(11): 4029-4041.
10. Yoo S, Bottenstein JE, Bittner GD,et al. Survival of mammalian B104 cellsfollowing neurite transection at different locations depends on somal Ca²⁺ concentration. J Neurobiol, 2004, 60(2): 137-153.
11. Spaeth CS, Boydston EA, Figard LR,et al. A model for sealing plasmalemmal damage in neurons and other eukaryotic cells. J Neurosci, 2010, 30(47): 15790-15800.
12. Bittner GD, Keating CP, Kane JR,et al. Rapid, effective, and long-lasting behavioral recovery produced by microsutures, methylene blue, and polyethylene glycol after completely cutting rat sciatic nerves. J Neurosci Res, 2012, 90(5): 967-980.
13. Bittner GD, Spaeth CS, Poon AD,et al. Repair of traumatic plasmalemmal damage to neurons and other eukaryotic cells. Neural Regen Res, 2016, 11(7): 1033-1042.
14. Ghergherehchi CL, Bittner GD, Hastings RL,et al. Effects of extracellular calcium and surgical techniques on restoration of axonal continuity by polyethylene glycol fusion following complete cut or crush severance of rat sciatic nerves. J Neurosci Res, 2016, 94(3): 231-245.
15. Stone MC, Rao K, GheresKW,et al. Normal spastin gene dosage is specifically required for axon regeneration. Cell Rep, 2012, 2(5): 1340-1350.
16. Diaz-Valencia JD, Bailey M, Ross JL. Purification and biophysical analysis of microtubule-severing enzymesin vitro. Methods Cell Biol, 2013, 115: 191-213.
17. Campbell WW. Evaluation and management of peripheral nerve injury. Clin Neurophysiol, 2008, 119(9): 1951-1965.
18. 林強, 蔡楊庭, 李皓莘. 負載濃度梯度 NGF 的周圍神經導管修復大鼠周圍神經缺損的實驗研究. 中國修復重建外科雜志, 2014, 28(2): 167-172.
19. 宋學文, 魏彥明, 李根, 等. 紅景天苷/膠原蛋白/聚己內酯神經導管修復大鼠坐骨神經缺損的實驗研究. 中國修復重建外科雜志, 2016, 30(5): 634-640.
20. Sherwood NT, Sun Q, Xue M,et al. Drosophila spastin regulates synaptic microtubule networks and is required for normal motor function. PLoS Biol, 2004, 2(12): e429.
21. Yu W, Qiang L, Solowska JM,et al. The microtubule-severing proteins spastinand katanin participate differently in the formation of axonal branches. Mol Biol Cell, 2008, 19(4): 1485-1498.
22. Lee HH, Jan LY, Jan YN. Drosophila IKK-related kinase Ik2 and Katanin p60-like 1 regulate dendrite pruning of sensory neuron during metamorphosis. Proc Natl Acad Sci U S A, 2009, 106(15): 6363-6368.

Chinese Journal of Reparative and Reconstructive Surgery

Changes of endogenous Spastin expression after sciatic nerve injury in rats

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