NEUROPROTECTIVE EFFECTS OF RECOMBINANT ADENO-ASSOCIATED VIRUS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR ON RAT TRAUMATIC SPINAL CORD INJURY AND ITS MECHANISM_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

QIANG Hui ¹ , ZHANG Chen ² , SHI Zhibin ² , LING Ming ¹

1Department of Orthopaedics, Shaanxi Provincial People’s Hospital (the Third Affiliated Hospital of School of Medicine, Xi’an Jiaotong University), Xi’an Shaanxi, 710068, P.R.China;;
2The First Department of Orthopaedics, the Second Affiliated Hospital of School of Medicine, Xi’an Jiaotong University.;

Corresponding author：

Keywords：

Adeno-associated virus vector; Vascular endothelial growth factor; Spinal cord injury; Nerve regeneration; Rat

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Objective To investigate the neuroprotective effects of recombinant adeno-associated virus (rAAV) expressing vascular endothel ial growth factor (VEGF) on traumatic spinal cord injury (SCI) of rat and its mechanisms. Methods The 144 male Sprague Dawley rats were randomly divided into 4 groups, and each group contained 36 rats. The rats in sham group (group A) received dorsal laminectomy without SCI and microinjection, the rats in model control group (group B), rAAV-green fluorescent protein (GFP) group (group C), and rAAV-hVEGF165-GFP group (group D) received dorsal
laminectomy with SCI and injection of 20 μL sal ine, rAAV-GFP viruses, or rAAV-hVEGF165-GFP viruses, respectively. At 3 and 7 days after operation, Basso-Beattie-Bresnahan (BBB) score was used to evaluate the neurologic function. At 7 days after operation, Nissl’s body staining was used to evaluate the histopathological changes; apoptosis was confirmed by transmission electron microscope examination and TUNEL staining; the expression of aquaporin 4 (AQP-4) was detected by Western blot assay. At 1, 3, 5, and 7 days, ELISA assay was used to detect the VEGF165 protein expression. Results According to BBB scores, the neurologic function in group D was significantly better than those in groups B and C at 3 and 7 days after operation (P lt; 0.05). Nissl’s body staining showed that tissue damage in group D was significantly milder than those in groups B and C at 7 days after operation (P lt; 0.05). ELISA results showed that VEGF165 protein expression was slowly-released in low dose in group D, and the expression in group D was significantly higher than that in groups A, B, and C at 3, 5, and 7 days after operation (P lt; 0.05). The results of transmission electron microscope and TUNEL staining showed that apoptosis rate of spinal cord neurons in group D was significantly lower than that in groups B and C at 7 days after operation (P lt; 0.05). The results of Western blot showed that AQP-4 expression in group D was significantly decreased when compared with that in groups B and C at 7 days after operation (P lt; 0.05). Conclusion The
rAAV expressing VEGF has neuroprotective effects by inhibiting apoptosis of spinal cord neurons and relieving spinal cord edema.

Citation： QIANG Hui,ZHANG Chen,SHI Zhibin,LING Ming. NEUROPROTECTIVE EFFECTS OF RECOMBINANT ADENO-ASSOCIATED VIRUS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR ON RAT TRAUMATIC SPINAL CORD INJURY AND ITS MECHANISM. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(6): 724-730. doi: Copy

1.	Dai J, Rabie AB. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res, 2007, 86 (10): 937-950.
2.	Jin KL, Mao XO, Greenberg DA. Vascular endothelial growth factor: direct neuroprotective effect in vitro ischemia. Proc Natl Acad Sci U S A, 2000, 97(18): 10242-10247.
3.	Ding XM, Mao BY, Jiang S, et al. Neuroprotective effect of exogenous vascular endothelial growth factor on rat spinal cord neurons in vitro hypoxia. Chin Med J (Engl), 2005, 118(19): 1644-1650.
4.	Storkebaum E, Lambrechts D, Carmeliet P. VEGF once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays, 2004, 26(9): 943-944.
5.	Schratzberger P, Schratzberger G, Silver M, et al. Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy. Nat Med, 2000, 6(4): 405-413.
6.	Zachary I. Neuroprotective role of vascular endothelial growth factor: signaling mechanisms, biological function, and therapeutic potential. Neurosignals, 2005, 14(5): 207-221.
7.	Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and schwann cell proliferation in the peripheral nervous system. J Neuroscience, 1999, 19 (14): 5731-5740.
8.	Shiote M, Nagano I, Ilieva H, et al. Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia. Neuroscience, 2005, 132(1): 175-182.
9.	Benton RL, Whittemore SR. VEGF165 therapy exacerbates secondary damage following spinal cord injury. Neurochem Res, 2003, 28(11): 1693-1703.
10.	Zhang C, Wang KZ, Qiang H, et al. Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo. Acta Pharmacol Sin, 2010, 31(7): 821-830.
11.	张晨, 马乾鹏, 强辉, 等. hVEGF165及hBMP-7共表达重组腺相关病毒载体体内诱导成骨及成血管研究. 中国修复重建外科杂志, 2010, 24(12): 1449-1454.
12.	Wrathall JR, Pettegrew RK, Harvey F. Spinal cord contusion in the rat: production of graded, reproducible, injury groups. Exp Neurol, 1985, 88(1): 108-122.
13.	刘长江, 孙朝阳. 白藜芦醇对大鼠脊髓损伤的保护作用. 第四军医大学学报, 2009, 30(13): 1199-1201.
14.	Basso DM, Beattie MS, Bresnahan JC, et al. MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J Neurotrauma, 1996, 13(7): 343-359.
15.	Oz Oyar E, Kardeş O, Korkmaz A, et al. Effects of vascular endothelial growth factor on ischemic spinal cord injury caused by aortic cross-clamping in rabbits. J Surg Res, 2009, 151(1): 94-99.
16.	Kim HM, Hwang DH, Lee JE, et al. Ex vivo VEGF delivery by neural stem cells enhances proliferation of glial progenitors, angiogenesis, and tissue sparing after spinal cord injury. PLoS One, 2009, 4(3): e4987.
17.	Tarkowski E, Issa R, Sjogren M, et al. Increased intrathecal levels of the angiogenic factors VEGF and TGF-beta in Alzheimer’s disease and vascular dementia. Neurobiol Aging, 2002, 23(2): 237-243.
18.	Yasuhara T, Shingo T, Kobayashi K, et al. Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson’s disease. Eur J Neurosci, 2004, 19(6): 1494-1504.
19.	Armstrong RJ, Barker RA. Neuro degeneration: a failure of neuroregeneration? Lancet, 2001, 358(9288): 1174-1176.
20.	Benton RL, Maddie MA, Gruenthal MJ, et al. Neutralizing endogenous VEGF following traumatic spinal cord injury modulates microvascular plasticity but not tissue sparing or functional recovery. Curr Neurovasc Res, 2009, 6(2): 124-131.
21.	Hall ED, Springer JE. Neuroprotection and acute spinal cord injury: a reappraisal. NeuroRx, 2004, 1(1): 80-100.
22.	Silverman WF, Krum JM, Mani N, et al. Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures. Neuroscience, 1999, 90(4): 1529-1541.
23.	张晨, 强辉, 党晓谦, 等. hVEGF165及hBMP-7共表达重组腺相关病毒载体介导基因表达的时效性研究. 中国修复重建外科杂志, 2010, 24(9): 1121-1127.
24.	Chen SL, Ma HI, Hands JM, et al. AAV type2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm. Proc Natl Acad Sci U S A, 2007, 104(50): 20096-20101.
25.	Byun J, Heard JM, Huh JE, et al. Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J Mol Cell Cardiol, 2001, 33(2): 295-305.
26.	Samantaray S, Sribnick EA, Das A, et al. Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats. J Pineal Res, 2008, 44(4): 348-357.
27.	Gál P, Kravcuková P, Mokrý M, et al. Chemokines as possible targets in modulation of the secondary damage after acute spinal cord injury: a review. Cell Mol Neurobiol, 2009, 29(6-7): 1025-1035.
28.	Oshio K, Binder DK, Yang B, et al. Expression of aquaporin water channels in mouse spinal cord. Neuroscience, 2004, 127(3): 685-693.
29.	Gunnarson E, Zelenina M, Aperia A. Regulation of brain aquaporins. Neurosci, 2004, 129(4): 947-955.
30.	Rey S, Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovasc Res, 2010, 86(2): 236-242.

1. Dai J, Rabie AB. VEGF: an essential mediator of both angiogenesis and endochondral ossification. J Dent Res, 2007, 86 (10): 937-950.
2. Jin KL, Mao XO, Greenberg DA. Vascular endothelial growth factor: direct neuroprotective effect in vitro ischemia. Proc Natl Acad Sci U S A, 2000, 97(18): 10242-10247.
3. Ding XM, Mao BY, Jiang S, et al. Neuroprotective effect of exogenous vascular endothelial growth factor on rat spinal cord neurons in vitro hypoxia. Chin Med J (Engl), 2005, 118(19): 1644-1650.
4. Storkebaum E, Lambrechts D, Carmeliet P. VEGF once regarded as a specific angiogenic factor, now implicated in neuroprotection. Bioessays, 2004, 26(9): 943-944.
5. Schratzberger P, Schratzberger G, Silver M, et al. Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy. Nat Med, 2000, 6(4): 405-413.
6. Zachary I. Neuroprotective role of vascular endothelial growth factor: signaling mechanisms, biological function, and therapeutic potential. Neurosignals, 2005, 14(5): 207-221.
7. Sondell M, Lundborg G, Kanje M. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and schwann cell proliferation in the peripheral nervous system. J Neuroscience, 1999, 19 (14): 5731-5740.
8. Shiote M, Nagano I, Ilieva H, et al. Reduction of a vascular endothelial growth factor receptor, fetal liver kinase-1, by antisense oligonucleotides induces motor neuron death in rat spinal cord exposed to hypoxia. Neuroscience, 2005, 132(1): 175-182.
9. Benton RL, Whittemore SR. VEGF165 therapy exacerbates secondary damage following spinal cord injury. Neurochem Res, 2003, 28(11): 1693-1703.
10. Zhang C, Wang KZ, Qiang H, et al. Angiopoiesis and bone regeneration via co-expression of the hVEGF and hBMP genes from an adeno-associated viral vector in vitro and in vivo. Acta Pharmacol Sin, 2010, 31(7): 821-830.
11. 张晨, 马乾鹏, 强辉, 等. hVEGF165及hBMP-7共表达重组腺相关病毒载体体内诱导成骨及成血管研究. 中国修复重建外科杂志, 2010, 24(12): 1449-1454.
12. Wrathall JR, Pettegrew RK, Harvey F. Spinal cord contusion in the rat: production of graded, reproducible, injury groups. Exp Neurol, 1985, 88(1): 108-122.
13. 刘长江, 孙朝阳. 白藜芦醇对大鼠脊髓损伤的保护作用. 第四军医大学学报, 2009, 30(13): 1199-1201.
14. Basso DM, Beattie MS, Bresnahan JC, et al. MASCIS evaluation of open field locomotor scores: effects of experience and teamwork on reliability. Multicenter Animal Spinal Cord Injury Study. J Neurotrauma, 1996, 13(7): 343-359.
15. Oz Oyar E, Kardeş O, Korkmaz A, et al. Effects of vascular endothelial growth factor on ischemic spinal cord injury caused by aortic cross-clamping in rabbits. J Surg Res, 2009, 151(1): 94-99.
16. Kim HM, Hwang DH, Lee JE, et al. Ex vivo VEGF delivery by neural stem cells enhances proliferation of glial progenitors, angiogenesis, and tissue sparing after spinal cord injury. PLoS One, 2009, 4(3): e4987.
17. Tarkowski E, Issa R, Sjogren M, et al. Increased intrathecal levels of the angiogenic factors VEGF and TGF-beta in Alzheimer’s disease and vascular dementia. Neurobiol Aging, 2002, 23(2): 237-243.
18. Yasuhara T, Shingo T, Kobayashi K, et al. Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson’s disease. Eur J Neurosci, 2004, 19(6): 1494-1504.
19. Armstrong RJ, Barker RA. Neuro degeneration: a failure of neuroregeneration? Lancet, 2001, 358(9288): 1174-1176.
20. Benton RL, Maddie MA, Gruenthal MJ, et al. Neutralizing endogenous VEGF following traumatic spinal cord injury modulates microvascular plasticity but not tissue sparing or functional recovery. Curr Neurovasc Res, 2009, 6(2): 124-131.
21. Hall ED, Springer JE. Neuroprotection and acute spinal cord injury: a reappraisal. NeuroRx, 2004, 1(1): 80-100.
22. Silverman WF, Krum JM, Mani N, et al. Vascular, glial and neuronal effects of vascular endothelial growth factor in mesencephalic explant cultures. Neuroscience, 1999, 90(4): 1529-1541.
23. 张晨, 强辉, 党晓谦, 等. hVEGF165及hBMP-7共表达重组腺相关病毒载体介导基因表达的时效性研究. 中国修复重建外科杂志, 2010, 24(9): 1121-1127.
24. Chen SL, Ma HI, Hands JM, et al. AAV type2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm. Proc Natl Acad Sci U S A, 2007, 104(50): 20096-20101.
25. Byun J, Heard JM, Huh JE, et al. Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector. J Mol Cell Cardiol, 2001, 33(2): 295-305.
26. Samantaray S, Sribnick EA, Das A, et al. Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats. J Pineal Res, 2008, 44(4): 348-357.
27. Gál P, Kravcuková P, Mokrý M, et al. Chemokines as possible targets in modulation of the secondary damage after acute spinal cord injury: a review. Cell Mol Neurobiol, 2009, 29(6-7): 1025-1035.
28. Oshio K, Binder DK, Yang B, et al. Expression of aquaporin water channels in mouse spinal cord. Neuroscience, 2004, 127(3): 685-693.
29. Gunnarson E, Zelenina M, Aperia A. Regulation of brain aquaporins. Neurosci, 2004, 129(4): 947-955.
30. Rey S, Semenza GL. Hypoxia-inducible factor-1-dependent mechanisms of vascularization and vascular remodelling. Cardiovasc Res, 2010, 86(2): 236-242.

Chinese Journal of Reparative and Reconstructive Surgery

NEUROPROTECTIVE EFFECTS OF RECOMBINANT ADENO-ASSOCIATED VIRUS EXPRESSING VASCULAR ENDOTHELIAL GROWTH FACTOR ON RAT TRAUMATIC SPINAL CORD INJURY AND ITS MECHANISM

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