Netrin-1 Hyperexpression in Mouse Brain PromotesAngiogenesis and Long-Term Neurological Recovery After

Transient Focal IschemiaHaiyan Lu, PhD; Yongting Wang, PhD; Xiaosong He, PhD; Falei Yuan, BS; Xiaojie Lin, BS;

Bohua Xie, BS; Guanghui Tang, BS; Jun Huang, BS; Yaohui Tang, BS; Kunlin Jin, PhD;Shengdi Chen, MD, PhD; Guo-Yuan Yang, MD, PhD

Background and Purpose—Netrin-1 (NT-1) stimulates endothelial cell proliferation and migration in vitro and promotes focalneovascularization in the adult brain in vivo. This in vivo study in mice investigated the effect of NT-1 hyperexpression onfocal angiogenesis and long-term functional outcome after transient middle cerebral artery occlusion (tMCAO).

Methods—Adeno-associated viral vectors carrying either the NT-1 gene (AAV–NT-1) or GFP (AAV-GFP) were generatedand injected into the brains of separate groups of 93 mice. Seven days later, tMCAO followed by 7–28 days ofreperfusion were carried out. Histological outcomes and behavioral deficits were quantified 7–28 days after tMCAO.Small cerebral vessel network and angiogenesis were assessed 28 days after tMCAO, using synchrotron radiationmicroangiography and immunohistochemistry.

Results—Western blot and immunohistochemistry showed that on the day of tMCAO, NT-1 hyperexpression had been achievedin both normal and ischemic hemispheres. Immunofluorescence imaging showed that NT-1 expression was primarily inneurons and astrocytes. Ischemia-induced infarction in the NT-1 hyperexpression group was attenuated in comparison tosaline or AAV-GFP–treated groups (P�0.01). Similarly, neurological deficits were greatly improved in AAV–NT-1–treatedmice compared with mice in saline or AAV-GFP–treated groups (P�0.05). In addition, angiogenesis was increased inAAV–NT-1–treated mice compared with the other 2 groups (P�0.05). In vivo synchrotron radiation microangiography 28days after tMCAO revealed more branches in AAV–NT-1–treated mice than in other groups.

Conclusions—AAV–NT-1 induced NT-1 hyperexpression before tMCAO reduced infarct size, enhanced neovasculariza-tion, and improved long-term functional recovery. (Stroke. 2012;43:00-00.)

Key Words: adeno-associated virus � angiogenesis � gene transfer � ischemia � middle cerebral artery occlusion� netrin-1

Ischemic stroke is the leading cause of disability and asignificant burden on public health worldwide. Because many

drugs that showed neuroprotection in animal stroke modelsfailed to show benefits in clinical trials, new strategies areneeded in developing approaches to stroke therapy. An idealrestorative approach to enhancing long-term functional recoveryafter stroke should promote both postischemic neuronal regen-eration and vascular perfusion in ischemic regions.

Netrin-1 (NT-1), a protein conserved during evolution andinitially purified from chick embryos, was found to providekey guidance cues for the development of commissuralaxons.1 NT-1 receptors include the DCC (Deleted in Colo-rectal Cancer) and UNC5 (uncoordinated-5) families, both ofwhich belong to the transmembrane immunoglobulin super-family.2 NT-1 can either attract or repel axonal growth cones

through binding to its receptors.3,4 Furthermore, NT-1 iscrucial to maintain the survival of DCC-expressed andUNC5H-expressed neurons.5 Because vascular and neuralsystems share similar growth characteristics, there has beenspeculation that NT-1’s role in axon guidance and survivalsuggests it might similarly assist vascular network forma-tion.6 NT-1, a potent vascular mitogen that stimulates in vitroproliferation and adhesion of endothelial and vascular smoothmuscle cells,7 also acts as a survival factor for endothelialcells through the blocking of its UNC5H2 receptor. Suchsilencing of NT-1 during zebrafish development leads tovascular defects.8 In mammalian models, NT-1 promotion ofneovascularization improves limb perfusion in hind limbischemia and reverses vascular and neural pathology indiabetic mice.9 Previous in vivo studies found that NT-1

Received August 6, 2011; accepted November 17, 2011.From the Department of Neurology (H.L., S.C., G.-Y.Y.), Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China;

Neuroscience and Neuroengineering Research Center (H.L., Y.W., X.H., F.Y., X.L., B.X., G.T., J.H., Y.T., G.-Y.Y.), Med-X Research Institute, Schoolof Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China; and Buck Institute for Age Research (K.J.), Novato, CA.

Correspondence to Guo-Yuan Yang, MD, PhD, Department of Neurology, Ruijin Hospital, Neuroscience and Neuroengineering Research Center,Med-X Research Institute, School of Biomedical Engineering, Shanghai Jiao Tong University, 1954 Hua-shan Rd, Shanghai 200030, China. E-mailgyyang0626@gmail.com

© 2012 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.111.635235

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hyperexpression promotes neovascularization in the adultmouse brain; and earlier in vitro studies demonstrated thatNT-1 hyperexpression induced proliferation, migration, andtube formation of human cerebral endothelial cells andhuman aortic smooth muscle cells.10 However, roles for NT-1in long-term neurological recovery after ischemic brain arelargely unexplored. We suggest that exogenous hyperstimu-lation of NT-1 expression can promote neovascularizationafter stroke from transient middle cerebral artery occlusion(tMCAO) and improve subsequent functional outcomes. Ifso, stimulation of NT-1 expression might serve as a potentialtherapeutic candidate for the treatment of cerebral ischemia.To test this hypothesis in mice, we used an adeno-associatedviral vector with NT-1 (AAV–NT-1) to induce brain NT-1hyperexpression at a later time, when we then caused exper-imental stroke from tMCAO. During and after recovery weinvestigated associations between NT-1 hyperexpression, at-tenuated neural cell death, neovascularization, and improvedneurobehavioral outcomes.

Methods

AAV–NT-1 Viral Vector Production, Purification,and Titer DeterminationpAAV–NT-1 vector was generated by inserting the chicken NT-1cDNA between 2 ITRs of pAAV-MCS plasmid (Invitrogen, Carls-bad, CA). pAAV–NT-1 was cotransfected with pHelper andpAAV-RC plasmids into AAV293 cells by calcium phosphateprecipitation. The viruses were further purified by CsCl densitygradient ultracentrifugation. Viral titer was determined by RT-PCRanalysis of the gene content.11 Adeno-associated virus-IRES-hrGFP(AAV–IRES-hrGFP) was simultaneously prepared as a control.

AAV-Mediated NT-1 InjectionAnimal procedures were carried out according to a protocol ap-proved by the Institutional Animal Care and Use Committee ofShanghai Jiao Tong University, Shanghai, China. Adult male CD-1mice weighing 25 g to 30 g were anesthetized with ketamine (100mg/kg) and xylazine (10 mg/kg, Sigma, San Louis, MO) intraperi-toneally. A burr hole was drilled to the left pericranium 2 mm lateralto sagittal suture and 1 mm posterior to coronal suture. A 10-�Lsyringe (WPI Inc, Sarasota, FL) was slowly inserted into the brain3 mm under the dura. AAV suspension (2.5 �L) with 3.5�109

AAV–NT-1 particles was injected stereotactically at a rate of 0.2�L/min. After half amount of suspension was injected, the needlewas slowly withdrawn to 2 mm under the dura to finish injection ofthe remaining AAV–NT-1 vector. Ten minutes after the completionof injection, the needle was withdrawn from the animal over a courseof 15 minutes. The bone hole was sealed with bone wax and thewound was closed. After sufficient awakening from anesthesia,animals were returned to their cages for long-term recovery.

Transient MCAO ModelSeven days after the AAV–NT-1 injection, animals were anesthe-tized with 1.5% isoflurane in 70/30 nitrogen/oxygen gas for MCAO.Body temperature was maintained at 37�0.5°C throughout thesurgical procedure, using a thermal blanket. The procedure oftMCAO was performed as described previously with some modifi-cation.12 Briefly, after isolation of the common carotid artery,external and internal carotid arteries, left MCA was occluded by a6–0 nylon suture coated with silica gel. Reperfusion was achievedby partially withdrawing the suture from the internal carotid artery tothe common carotid artery after 60 minutes of occlusion. Thereperfusion was maintained for up to 28 days.

Infarct Volume MeasurementBrains were removed and frozen immediately 7 days after tMCAO.A series of 20-�m-thick coronal sections from anterior commissureto hippocampus were cut and mounted on slides. The sections werefixed and stained, using cresyl violet. Sections were imaged anddigitized, and the border between infarct and noninfarct tissue wasoutlined using Image J (National Institutes of Health). Infarct volumewas calculated by subtracting the volume of intact area in theipsilateral hemisphere from the whole volume of the contralateralhemisphere.

Figure 1. Adeno-associated viral vector (AAV)-mediated Netrin-1(NT-1) hyperexpression in the brain. A, Graphic illustration of amouse brain coronal section and the distribution of AAV-mediated GFP expression. Asterisks indicate 2 different loca-tions of viral injection in the left hemisphere. The ischemic coreis shown as black area, and peri-infarct region is shown as grayarea. Intraparenchymal injection of AAV-GFP resulted in anintense GFP signal (green) at 7 days after injection. Bar�50 �m.B, Western blot analysis of NT-1 expression in the brain. Brainsamples were extracted from the striatum 7 days after adminis-tration of AAV-GFP, normal saline (NS), or AAV–NT-1. L indi-cates left hemisphere, R, right hemisphere. Histogram showsthe quantification of NT-1 level. *P�0.05 vs AAV-GFP or saline,n�5 per group. C, Immunohistochemical staining of NT-1 in thebrain sections after AAV-GFP (a), saline (b), or AAV–NT-1 (c)injection and the phenotype of NT-1–positive cells in AAV–NT-1–injected brain. NT-1–positive cells (green) expressed GFAP (d)and NeuN (e) but not Glut-1 (f). Bar�50 �m. D, Double immu-nostaining of NT-1 receptors. DCC (green in a, d, g) andUNC5H2 (green in a, d, g) receptors are colocalized with GFAP(red in b), NeuN (red in e), and CD31/Glut-1 (red in h), as shownin merged images (yellow in c, f, i). Bar�20 �m.

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Behavioral TestsMice were trained for 3 days before AAV–NT-1 injection. Baselinevalues were generated by averaging 3 trials. Animals were tested at1–4 weeks after tMCAO. Beam-walking and rotarod tests wereperformed as described previously.13

For the beam-walking test, mice were trained to traverse ahorizontally elevated square beam with 5-mm side length to reach anescape platform placed 1 meter away. Mice were placed on one endof the beam, and the latency to traverse 80% of the beam toward theescape platform was recorded. Motor test data were analyzed asmean latency to cross the beam from 3 trials.

For the rotarod test, the task requires the mice to balance on arotating rod. Animals were allowed a 1 minute adaptation period onthe rod at rest, after which the rod was steadily accelerated to 40revolutions per minute over 2 minutes, and time spent on the rod wasrecorded. Motor test data were analyzed as mean duration on therotarod from 3 trials.

Immunohistochemistry and ImmunofluorescenceImmunohistochemistry was performed according to the protocoldescribed previously.10 After blocking with 10% bovine serumalbumin, brain sections were incubated with primary antibodies atthe following dilutions: NT-1 (1:100; Santa Cruz Biotechnology Inc,

Santa Cruz, CA), DCC (1:300; Santa Cruz), UNC5H2 (1:300; R&Dsystems, Tustin, CA), NeuN and GFAP (1:500; Millipore Inc,Billerica, MA), GluT-1 (1:300; Thermo, Waltham, MA), CD31(1:300; R&D systems), and Proliferating Cell Nuclear Antigen(PCNA, 1:1000; Abacm, Cambridge, England) overnight at 4°C.Sections were then incubated with biotinylated or fluorescence-conjugated secondary antibodies. Each experiment had appropriatepositive and negative controls.

Microvessel CountingFrozen coronal sections (20 �m thick) were fixed with 4% parafor-maldehyde at room temperature for 10 minutes and then incubatedwith fluorescein-lycopersicin esculentum lectin (1:400, Vector Lab-oratory, Burlingame, CA) overnight at 4°C. Microvessel density wasquantified by counting the number of microvessels in 3 microscopicfields at the left, right, and bottom of the peri-infarct area of striatum.

Western Blot AnalysisAn equal amount total protein of brain sample was loaded on 10%resolving gel for electrophoresis. Subsequently, proteins were trans-blotted onto a nitrocellulose membrane (Whatman Inc, FlorhamPark, NJ). The membrane was placed in 5% nonfat milk in 0.1%TBST for 1 hour to block nonspecific binding and immunoprobedwith NT-1 primary antibodies overnight at 4°C. After washing with

Figure 2. Netrin-1 (NT-1) hyperexpression attenuated cerebralinfarct volumes after stroke. A, Representative cresyl violetstaining of coronal brain sections treated with saline (NS),adeno-associated viral vector (AAV)-GFP, or AAV–NT-1 for 7days followed by transient middle cerebral artery occlusion. B,Quantification of the infarct volumes. *#P�0.01, AAV–NT-1–treated mice vs saline or AAV-GFP–treated mice, n�5 animalsper group.

Figure 3. The effect of adeno-associated viral vector (AAV)-mediated Netrin-1 (NT-1) hyperexpression on neurobehavioraloutcome after focal ischemia. For neurobehavioral testing, micewere trained before focal ischemia and neurobehavioral deficitswere assessed by beam-walking and rotarod tests 1, 2, and 4weeks after transient middle cerebral artery occlusion (tMCAO).A, Neurological deficits were evaluated by beam-walking test.B, Neurological deficits were evaluated by rotarod test. NS indi-cates saline-treated mice; GFP, AAV-GFP–treated mice; NT-1,AAV–NT-1–treated mice;. B inj, before injection; B surg, beforesurgical procedure of tMCAO; AS, after surgical procedure oftMCAO. Data are presented as mean�SD, n�8 in each group.*#P�0.05, AAV–NT-1–treated mice vs saline or AAV-GFP–treated mice.

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TBST, the blots were incubated with HRP-conjugated secondaryantibodies for 1 hour at room temperature and then reacted with anenhanced ECL substrate (Pierce, Rockford, IL). The result ofchemiluminescence was recorded with an imaging system (Bio-Rad,Hercules, CA).

Synchrotron Radiation Microangiography In VivoSynchrotron radiation microangiography was performed at Beamline 13W of the Shanghai Synchrotron Radiation Facility. A PE-10tube connected with a syringe pump (Longerpump Inc, Baoding,China) was carefully inserted into the proximal external carotidartery of the tMCAO mice after 28 days of reperfusion. The mousewas placed perpendicular to the X-ray beam, lying on its right side.X-ray energy was 33.3 keV, just above the iodine K-edge energy.Nonionic iodine contrast agent (350 mg/mL, Omnipaque, GE,Fairfield, CO) was injected from external carotid artery into internalcarotid artery at a rate of 2 mL/min, with a total volume of 100 �L.The CCD camera was placed 650 mm from the animal. Dynamicimages were obtained every 172 ms, with a resolution of 13 �m perpixel. Six animals per group were subjected to synchrotron radiationimaging.

Statistical AnalysisParametric data in different groups were compared using a 1-wayANOVA followed by Student-Newman-Keuls test. All data were

presented as mean�SD. A probability value of �5% was consideredstatistically significant.

Results

Fluorescence MicroscopyFluorescence microscopy showed many GFP-positive cells inboth adjacent and distal to the needle tracks of the ipsilateralhemisphere (Figure 1A). Western blot analysis showed thatNT-1 expression was significantly increased in AAV–NT-1–injected mice, compared with AAV-GFP or saline-injectedmice (Figure 1B, P�0.05). Immunocytochemistry showedsubstantial numbers of NT-1–positive cells in the brain 7 daysafter AAV–NT-1 injection. However, NT-1–positive cellswere barely detected in the mouse brain treated with AAV-GFP or saline. Double-labeled immunostaining revealed thatNT-1 proteins were expressed primarily in neurons andastrocytes but not endothelial cells (Figure 1C). In addition,expression of NT-1 receptors (DCC and UNC5H2) was foundin neurons, astrocytes, and endothelial cells (Figure 1D).

Figure 4. Increased angiogenesis in transient middle cerebralartery occlusion (tMCAO) mice after adeno-associated viralvector–Netrin-1 (AAV–NT-1) injection. A, Photomicrographsshow the pattern of lectin-positive microvessels in ischemicpenumbra of striatum in mice treated with saline (NS), AAV-GFP(GFP), and AAV–NT-1 (NT-1) 1, 2, and 4 weeks after tMCAO.Bar�50 �m. B, Quantification of microvessels in the ischemicpenumbra of striatum in mice treated with saline (NS), AAV-GFP(GFP), and AAV–NT-1 (NT-1). Data are presented as mean�SD,n�6 in each group. *#P�0.05, AAV–NT-1–treated vs saline-treated or AAV-GFP–treated groups.

Figure 5. Adeno-associated viral vector (AAV)-mediated Netrin-1(NT-1) hyperexpression increased endothelial cells proliferationafter focal ischemia. A, Representative images of CD31-positive(green) and PCNA-positive (red) cells in ischemic penumbra ofstriatum in mice treated with saline (NS), AAV-GFP (GFP), andAAV–NT-1 (NT-1) 2 weeks after transient middle cerebral arteryocclusion. Bar�50 �m. B, Quantification of CD31/PCNA-positive cells in the ischemic penumbra of striatum after injec-tion of saline (NS), AAV-GFP (GFP), and AAV–NT-1 (NT-1). Dataare presented as mean�SD, n�6 in each group. *#P�0.001,AAV–NT-1 vs saline-treated or AAV-GFP–treated mice.

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Infarct Size After StrokeNext, we examined whether AAV-mediated NT-1 hyperex-pression affected the outcome after focal ischemia. Themodified suture model produced stable infarct that is primar-ily striatal. As shown in Figure 2, AAV–NT-1–treated ische-mia mice reduced infarction volume compared with thatobserved in saline or AAV-GFP–treated ischemia mice(AAV–NT-1�2.6�1.7, saline�9.7�2.5, and AAV-GFP�8.8�2.9 mm3; P�0.01), suggesting that NT-1 affectedhistological outcome after focal ischemia.

Neurological OutcomesWe then determined whether AAV-mediated NT-1 hyperex-pression affected the neurological deficits after focal ische-mia. Neurobehavioral outcome was measured at 1–4 weeksafter tMCAO. Beam-walking and rotarod tests indicated thatthere was not significant difference in performance amongsaline-treated, AAV-GFP–, and AAV–NT-1–treated groupsbefore tMCAO. However, neurological deficits measured bythe beam-walking and rotarod tests were significantly im-proved in AAV–NT-1–treated mice than in saline-treated orAAV-GFP–treated mice at 1, 2, and 4 weeks of reperfusion.(Figure 3, P�0.05).

NeovascularizationWe confirmed that the number of microvessels in peri-infarctarea was significantly increased in AAV–NT-1–treated micecompared with the saline and AAV-GFP–treated mice (Fig-ure 4, P�0.05). Dual-labeled immunofluorescence showedthat PCNA-positive cells expressed CD31, indicating thepresence of proliferating endothelial cells. Significantly morePCNA/CD31-positive cells were observed in AAV–NT-1–treated mice than in saline and AAV-GFP–treated groups(Figure 5; AAV–NT-1�9.8�2.8, AAV-GFP�2.8�1.2, andsaline�2.7�1.6 cells/field, P�0.001).

Synchrotron Radiation MicroangiographyUsing synchrotron radiation angiography in living animals,we also found that branches of left MCA in AAV–NT–1

treated mice were greater than that in the control groups 28days after tMCAO, suggesting that NT-1 hyperexpressioncould promote local angiogenesis and new vascular systemremodeling after focal ischemia (Figure 6). The number ofMCA branches within the control group or the NT-1–treatedgroup was consistent. However, quantification of branchesusing synchrotron radiation angiography is a challenge sameas in the CTA/MRA quantification. We are currently devel-oping software to quantify angiogenesis based on synchrotronradiation angiography.

DiscussionIncreased AAV-mediated NT-1 protein in the mouse brainsignificantly reduced infarct volume and improved neurobe-havioral outcome compared with the saline and AAV-GFP–treated mice. Immunohistochemistry and synchrotron radia-tion angiography demonstrated that microvessel density andcollateral vessels were increased in AAV–NT-1–treated mice,suggesting that NT-1 promoted local neovascularization andvessel remodeling in injured brain. In the present study, weachieved NT-1 hyperexpression in neurons and astrocytes inthe ischemic brain using AAV–NT-1 virus. NT-1 receptorsincluding DCC and UNC5H2 were expressed in neurons,astrocytes, and endothelial cells of the brain after tMCAO.

AAV–NT-1–mediated gene transfer is a reliable tool forlong-term and steady expression of NT-1 protein. We chosethe AAV–NT-1 vector for delivery into mouse brains becauseit has higher transduction ability with less toxicity.14,15 Ourprevious study showed that NT-1 expression was signifi-cantly increased at mRNA and protein levels in AAV–NT-1–transfected HEK293 cells.10 In the present study, weproved that AAV–NT-1 can effectively transfer into themouse brain neurons and astrocytes.

Netrins are highly conserved laminin-associated secret pro-teins.1 As one of the family members in mammals, NT-1 showsseveral functions in neural system development. NT-1 wouldinduce mouse mammary epithelial cell invasion and migration,mediate pancreatic epithelial cell adhesion, and promote tumor

Figure 6. Live synchrotron radiationangiography revealed newly formed func-tional vessels in transient middle cerebralartery occlusion (tMCAO) mice treatedwith adeno-associated viral vector–Netrin-1 (AAV–NT-1). Photomicrographsshow the perfusion of the middle cerebralartery (MCA) territory in normal mousebrain (a) and in saline-treated (b) or AAV–NT-1–treated (c) mouse brain 28 daysafter tMCAO. MCA branches perfused bycontrast media are illustrated on the right.ICA indicates internal carotid artery.

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epithelial cell survival.16–18 In ex vivo experiments, NT-1induced the aortic endothelial cells proliferation and migration.NT-1 hyperexpression increased the number of neovessels inischemic muscles and reversed neural and vascular pathology ina diabetic rodent model.9 In cardiac ischemia and reperfusion,NT-1 reduced myocardial infarct volume by DCC/ERK1/2feedback mechanism in cardiac endothelial cells and myocardialcells.19 NT-1 also protected renal tubular epithelial cells againstischemia reperfusion-induced injury by increasing proliferationand suppressing apoptosis of the cells.20 NT-1 hyperexpressioninduced angiogenesis in adult mouse brains.10,21 Consistently,our data showed that AAV–NT-1 can induce angiogenesis in theipsilateral hemisphere of an adult mouse model of focal ische-mia. Our data indicated that NT-1 hyperexpression reducedinfarct volume, which was associated with increased functionalmicrovessels in the peri-infarct area. We further demonstratedthat NT-1 hyperexpression effectively improved motor functionas assessed by beam walking and rotarod tests at different timepoints after tMCAO. These findings suggest that NT-1 canparticipate in neovascularization and vessel remodeling process,which may contribute to long-term functional outcome afterischemic brain injury. Further studies are needed to access therescue effects of NT-1 gene or protein transferred after cerebralischemia, which would be more valuable in translation to clinicalpractice. With the development of safer virus-mediated genetherapy techniques, NT-1 gene therapy might provide a prom-ising strategy for stroke treatment.

NT-1 receptors include DCC and UNC5 family members.22

NT-1 attracts axons through interaction with DCC and repelsaxons by UNC5 homodimer or UNC5-DCC heterodimer.3,4,23

The involvement of UNC5H2 and DCC in NT-1–inducedangiogenesis is still a topic of debate.8,19,24–26 We found DCCand UNC5H2 receptors expressed in neurons, astrocytes, andendothelial cells after tMCAO. They may participate inNT-1–induced angiogenesis in mouse brains after tMCAO.Further studies are needed to identify their roles and mecha-nisms underlying NT-1–induced angiogenesis.

AcknowledgmentsWe thank Dr Yongjing Guan (Med-X Research Institute), DrHonglan Xie, and Dr Guohao Du (BM13W, Shanghai SynchrotronRadiation Facility) for technical advice on synchrotronradiation microangiography.

Sources of FundingThis study was supported by the National Natural Science Founda-tion of China (Project No. 30973097), the National Basic ResearchPrograms (973 Programs 2010CB834306 and 2011CB504405), andKC Wong Foundation.

DisclosuresNone.

References1. Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-

Lavigne M. The netrins define a family of axon outgrowth-promotingproteins homologous to c Elegans UNC-6. Cell. 1994;78:409–424.

2. Huber AB, Kolodkin AL, Ginty DD, Cloutier JF. Signaling at the growthcone: ligand-receptor complexes and the control of axon growth andguidance. Annu Rev Neurosci. 2003;26:509–563.

3. Fazeli A, Dickinson SL, Hermiston ML, Tighe RV, Steen RG, Small CG,et al. Phenotype of mice lacking functional deleted in colorectal cancer(DCC) gene. Nature. 1997;386:796–804.

4. Hong K, Hinck L, Nishiyama M, Poo MM, Tessier-Lavigne M, Stein E.A ligand-gated association between cytoplasmic domains of UNC5 andDCC family receptors converts netrin-induced growth cone attraction torepulsion. Cell. 1999;97:927–941.

5. Llambi F, Causeret F, Bloch-Gallego E, Mehlen P. Netrin-1 acts as asurvival factor via its receptors UNC5h and DCC. EMBO J. 2001;20:2715–2722.

6. Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve andblood vessel wiring. Nature. 2005;436:193–200.

7. Park KW, Crouse D, Lee M, Karnik SK, Sorensen LK, Murphy KJ, et al.The axonal attractant netrin-1 is an angiogenic factor. Proc Natl Acad SciU S A. 2004;101:16210–16215.

8. Castets M, Coissieux MM, Delloye-Bourgeois C, Bernard L, Delcros JG,Bernet A, et al. Inhibition of endothelial cell apoptosis by netrin-1 duringangiogenesis. Dev Cell. 2009;16:614–620.

9. Wilson BD, Ii M, Park KW, Suli A, Sorensen LK, Larrieu-Lahargue F, etal. Netrins promote developmental and therapeutic angiogenesis. Science.2006;313:640–644.

10. Fan Y, Shen F, Chen Y, Hao Q, Liu W, Su H, et al. Overexpression ofnetrin-1 induces neovascularization in the adult mouse brain. J CerebBlood Flow Metab. 2008;28:1543–1551.

11. Rohr UP, Wulf MA, Stahn S, Steidl U, Haas R, Kronenwett R. Fast andreliable titration of recombinant adeno-associated virus type-2 usingquantitative real-time PCR. J Virol Methods. 2002;106:81–88.

12. Yang G, Chan PH, Chen J, Carlson E, Chen SF, Weinstein P, et al.Human copper-zinc superoxide dismutase transgenic mice are highlyresistant to reperfusion injury after focal cerebral ischemia. Stroke. 1994;25:165–170.

13. Liu J, Tang TS, Tu H, Nelson O, Herndon E, Huynh DP, et al. Derangedcalcium signaling and neurodegeneration in spinocerebellar ataxia type 2.J Neurosci. 2009;29:9148–9162.

14. Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscletissue of immunocompetent mice by adeno-associated virus vector.J Virol. 1996;70:8098–8108.

15. McCown TJ. Adeno-associated virus (AAV) vectors in the CNS. CurrGene Ther. 2005;5:333–338.

16. Strizzi L, Bianco C, Raafat A, Abdallah W, Chang C, Raafat D, et al.Netrin-1 regulates invasion and migration of mouse mammary epithelialcells overexpressing cripto-1 in vitro and in vivo. J Cell Sci. 2005;118:4633–4643.

17. Hebrok M, Reichardt LF. Brain meets pancreas: netrin, an axon guidancemolecule, controls epithelial cell migration. Trends Cell Biol. 2004;14:153–155.

18. Delloye-Bourgeois C, Fitamant J, Paradisi A, Cappellen D, Douc-Rasy S,Raquin MA, et al. Netrin-1 acts as a survival factor for aggressiveneuroblastoma. J Exp Med. 2009;206:833–847.

19. Zhang J, Cai H. Netrin-1 prevents ischemia/reperfusion-induced myo-cardial infarction via a DCC/erk1/2/enos s1177/no/DCC feed-forwardmechanism. J Mol Cell Cardiol. 2010;48:1060–1070.

20. Wang W, Reeves WB, Pays L, Mehlen P, Ramesh G. Netrin-1 overex-pression protects kidney from ischemia reperfusion injury by suppressingapoptosis. Am J Pathol. 2009;175:1010–1018.

21. Sun H, Le T, Chang TT, Habib A, Wu S, Shen F, et al. AAV-mediatednetrin-1 overexpression increases peri-infarct blood vessel density andimproves motor function recovery after experimental stroke. NeurobiolDis. 2011;44:73–83.

22. Freitas C, Larrivee B, Eichmann A. Netrins and UNC5 receptors inangiogenesis. Angiogenesis. 2008;11:23–29.

23. Keleman K, Dickson BJ. Short- and long-range repulsion by the dro-sophila UNC5 netrin receptor. Neuron. 2001;32:605–617.

24. Lu X, Le Noble F, Yuan L, Jiang Q, De Lafarge B, Sugiyama D, et al. Thenetrin receptor UNC5b mediates guidance events controlling morpho-genesis of the vascular system. Nature. 2004;432:179–186.

25. Navankasattusas S, Whitehead KJ, Suli A, Sorensen LK, Lim AH, ZhaoJ, et al. The netrin receptor UNC5b promotes angiogenesis in specificvascular beds. Development. 2008;135:659–667.

26. Nguyen A, Cai H. Netrin-1 induces angiogenesis via a DCC-dependenterk1/2-enos feed-forward mechanism. Proc Natl Acad Sci U S A. 2006;103:6530–6535.

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Jun Huang, Yaohui Tang, Kunlin Jin, Shengdi Chen and Guo-Yuan YangHaiyan Lu, Yongting Wang, Xiaosong He, Falei Yuan, Xiaojie Lin, Bohua Xie, Guanghui Tang,

Neurological Recovery After Transient Focal IschemiaNetrin-1 Hyperexpression in Mouse Brain Promotes Angiogenesis and Long-Term

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