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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med    2011, Vol. 5 Issue (1) : 86-93     DOI: 10.1007/s11684-011-0118-x
RESEARCH ARTICLE |
Overexpression of netrin-1 improves neurological outcomes in mice following transient middle cerebral artery occlusion
Haiyan LU1, Yongting WANG1, Falei YUAN1, Jianrong LIU2, Lili ZENG1,2, Guo-Yuan YANG1,2()
1. Neuroscience and Neuroengineering Research Center, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China; 2. Departments of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China
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Abstract  

Netrin-1 (NT-1) is one of the axon-guiding molecules that are critical for neuronal development. Because of its structural homology to the endothelial mitogens, NT-1 may have similar effects on vascular network formation. NT-1 was shown to be able to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and also promote focal neovascularization in adult brain in vivo. In the present study, we reported the delivery of NT-1 using an adeno-associated virus (AAV) vector (AAV-NT-1) into mouse brain followed by transient middle cerebral artery occlusion (tMCAO). We found that AAV vectors did not elicit a detectable inflammatory response, cell loss or neuronal damage after brain transduction. The level of NT-1 was increased in the AAV-NT-1-transduced tMCAO mice compared with the control mice. Furthermore, the neurobehavioral outcomes were significantly improved in AAV-NT-1-transduced mice compared with the control animals (P<0.05) 7 days after tMCAO. Our data suggests that NT-1 plays a neuronal function recovery role in ischemic brain and that NT-1 gene transfer might present a valuable approach to treat brain ischemic disorders.

Keywords adeno-associated virus      angiogenesis      gene transfer      ischemia      middle cerebral artery occlusion      netrin-1     
Corresponding Authors: YANG Guo-Yuan,Email:gyyang0626@gmail.com   
Issue Date: 05 March 2011
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-011-0118-x     OR     http://academic.hep.com.cn/fmd/EN/Y2011/V5/I1/86
Fig.1  Distribution of NT-1 positive staining following tMCAO. Photomicrographs show the distributions of NT-1 positive staining after 1 h of MCAO following 1 day () and 7 days () of reperfusion in the ipsilateral hemisphere (), contralateral hemisphere (), and the normal control () and sham () animals. Arrows indicate dark brown NT-1 positive staining; suggesting these cells can produce NT-1. Bar= 50 μm.
Fig.2  Distribution of NT-1 positive staining following AAV-NT-1 transduction. Photomicrographs show the distributions of NT-1 positive staining following 3 days (, ) and 7 days (, ) of injection of 2 x10 particles of AAV-NT-1. Arrows indicate that dark brown staining is much more in the AAV-NT-1 injected hemisphere (, ) than in the contralateral hemisphere (, ). Normal () and control () animals show no NT-1 positive staining in the brain. Bar= 50 μm.
Fig.3  NT-1 expression is increased in the brain after AAV-NT-1 gene transfer. Western blot result shows the NT-1 expression in the AAV-NT-1, AAV-GFP, and saline treated mouse brain. β-actin is measured as an internal control. Animals underwent 2×10 particles of AAV-NT-1 in 2.5 μL or the same amount of AAV-GFP or saline injection. L: ipsilateral hemisphere; R: contralateral hemisphere.
Fig.4  NT-1 overexpression improved focal CBF after tMCAO. Bar graph shows the percentage changes of cerebral blood flow at different time points on the surface of ipsilateral hemisphere. The CBF values are calculated and shown as percentages of baseline values. NT-1: NT-1 transduced mice; NS: NS treated mice; and GFP: GFP transduced mice. BS: before surgical procedure of MCAO; AS: after surgical procedure of MCAO. Data are mean?±?standard deviation (SD), = 6 in each group. *<0.05, AAV-NT-1 . saline group, <0.05, AAV-NT-1 . AAV-GFP group.
Fig.5  Increase of the microvessel density in the MCAO mice with Netrin-1 overexpression. Photomicrographs show lectin staining microvessels in the AAV-GFP (), saline (), and AAV-NT-1 () transduced mouse brain after 1 h of tMCAO following 7 day reperfusion, respectively. The images of microvessel are taken from ischemic penumbra in ipsilateral hemisphere () and the same regions of contralateral hemisphere () following 7 days of tMCAO. The number of microvessels is increased in the AAV- NT-1 transduced mice compared to the AAV-GFP and saline treated mice. Bar= 50 μm.
Fig.6  AAV-NT-1-transduced mice showed improved neurobehavioral outcome. Neurobehavioral functions of mice are tested before injection, immediate before and 7 days after tMCAO. Bar graphs show that AAV-NT-1-transduced mice significantly improve performance in crossing the beam walk (, beam test), staying on the rotor-rod (, rotor-rod test) compared with mice treated with saline or AAV-GFP after 7 days of tMCAO. However, there was no significant difference among the three groups of mice before injection and tMCAO. NT-1: NT-1 transduced mice; NS: NS treated mice; and GFP: GFP-transduced mice. Bef. inj.: before injection; Bef. surg.: before surgical procedure of MCAO; Aft. surg.: after surgical procedure of MCAO. Data are mean?±?SD, = 6 in each group. *<0.05, AAV-NT-1-transduced mice . saline or AAV-GFP-transduced mice.
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