ni hms 133911

Atorvastatin induction of VEGF and BDNF promotes brainplasticity after stroke in mice

Jieli Chen1, Chunling Zhang1, Hao Jiang1, Yi Li1, Lijie Zhang1, Adam Robin1, MarkKatakowski1, Mei Lu2, and Michael Chopp1,31 Department of Neurology, Henry Ford Health Sciences Center, Detroit, Michigan, USA2 Department of Biostatistics and Research Epidemiology, Henry Ford Health Sciences Center,Detroit, Michigan, USA3 Department of Physics, Oakland University, Rochester, Michigan, USA

AbstractMolecular mechanisms underlying the role of statins in the induction of brain plasticity andsubsequent improvement of neurologic outcome after treatment of stroke have not been adequatelyinvestigated. Here, we use both in vivo and in vitro studies to investigate the potential roles of twoprominent factors, vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor(BDNF), in mediating brain plasticity after treatment of stroke with atorvastatin. Treatment of strokein adult mice with atorvastatin daily for 14 days, starting at 24 hours after MCAO, shows significantimprovement in functional recovery compared with control animals. Atorvastatin increases VEGF,VEGFR2 and BDNF expression in the ischemic border. Numbers of migrating neurons,developmental neurons and synaptophysin-positive cells as well as indices of angiogenesis weresignificantly increased in the atorvastatin treatment group, compared with controls. In addition,atorvastatin significantly increased brain subventricular zone (SVZ) explant cell migration in vitro.Anti-BDNF antibody significantly inhibited atorvastatin-induced SVZ explant cell migration,indicating a prominent role for BDNF in progenitor cell migration. Mouse brain endothelial cellculture expression of BDNF and VEGFR2 was significantly increased in atorvastatin-treated cellscompared with control cells. Inhibition of VEGFR2 significantly decreased expression of BDNF inbrain endothelial cells. These data indicate that atorvastatin promotes angiogenesis, brain plasticityand enhances functional recovery after stroke. In addition, VEGF, VEGFR2 and BDNF likelycontribute to these restorative processes.

Keywordsangiogenesis; atorvastatin; brain-derived neurotrophic factor; middle cerebral artery occlusion(MCAO); migration; vascular endothelial growth factor

IntroductionWe have previously demonstrated that treatment of stroke in the rat with HMG-CoA reductaseinhibitors (statins), atorvastatin and simvastatin, with treatment initiated 24 hours after onsetof stroke, significantly improves neurologic outcome compared with control animals (Chen etal, 2003b). These agents induce angiogenesis, neurogenesis and synaptogenesis in the ischemic

Correspondence: Dr Michael Chopp, Neurology Research, E&R Bldg., Room #3056, Henry Ford Hospital, 2799 West Grand Boulevard,Detroit, MI 48202, USA. [email protected].

NIH Public AccessAuthor ManuscriptJ Cereb Blood Flow Metab. Author manuscript; available in PMC 2010 January 11.

Published in final edited form as:J Cereb Blood Flow Metab. 2005 February ; 25(2): 281290. doi:10.1038/sj.jcbfm.9600034.

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brain, and this brain remodeling likely contributes to the functional recovery (Chen et al,2003b). However, the molecular triggers for the statin-induced brain plasticity are not known.In addition, our prior observation of statin-induced angiogenesis, neurogenesis andsynaptogenesis raises the question of how these aspects of brain plasticity are coupled, and ifthere are specific molecular factors that can integrate these remodeling events in the brain.

Vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) aretwo important neurotrophic factors that have multiple effects on sustaining and evokingelements of brain plasticity (Jin et al, 2002; Sun et al, 2003). Vascular endothelial growth factoris an angiogenic agent, which promotes neurogenesis and stem-progenitor cell migration (Jinet al, 2002; Sun et al, 2003). Likewise, BDNF regulates neuronal survival, cell migration, andsynaptic function (Aguado et al, 2003; Gorski et al, 2003). Statins increase VEGF in endothelialand osteoblast cells (Chen et al, 2003a; Maeda et al, 2003). Both VEGF and BDNF increasein ischemic brain with other neurorestorative treatments of stroke, such as with bone marrowstromal cells (Chen et al, 2002; Matsuno et al, 2004). It is therefore reasonable to propose thatVEGF and BDNF might be upregulated in the brain after treatment with a statin, and mayorchestrate brain plasticity.

In this study, we therefore examined the effects of a widely employed hydrophilic statin,atorvastatin, on angiogenesis, neuronal plasticity and neuronal migration in a mouse model ofpermanent middle cerebral artery occlusion (MCAO). We show that atorvastatin increasescerebral expression of VEGF and its receptor VEGFR2 induces endothelial cell proliferationand angiogenesis, and promotes production of neurotrophic factors, BDNF and VEGF, whichmay act in concert to induce brain plasticity and to foster functional recovery after stroke inmice.

Materials and methodsMCAO Model and Atorvastatin Administration

Adult male C57BL/6J mice (age 2 to 3 months, weight 24 to 28 g) were purchased from CharlesRiver (Wilmington, MA, USA). Mice were anesthetized with halothane. Permanent rightMCAO was induced by advancing a 6-0 surgical nylon suture (8.0 to 9.0mm determined bybody weight) with an expanded (heated) tip from the external carotid artery into the lumen ofthe internal carotid artery to block the origin of the MCA (Mao et al, 1999). Atorvastatin wasdissolved in methanol (20mg atorvastatin dissolved in 20mL saline with 10 L methanol) andinjected subcutaneously (s.c.) at 24 hours after MCAO. An atorvastatin dose of 10 mg/kg wasselected based on pretreatment studies of stroke in mice (Gertz et al, 2003; Laufs et al,2000).

After MCAO, mice were randomly divided into the following groups: Group 1, saline (0.25mL) daily for 14 days as a control group (n=12); Group 2, atorvastatin (10 mg/kg) daily for 14days (n=11); Group 3, saline (0.25 mL) daily for 7 days as a control group (n=10); Group 4,atorvastatin (10 mg/kg) daily for 7 days (n=10). To identify newly formed DNA in ischemicbrain, mice received injections of bromodeoxyuridine (BrdU, Sigma Chemical, 100 mg/kg in0.007N NaOH physiologic saline), intraperitoneally (i.p.) starting 1 day after MCAO and dailythereafter until the mice were killed. Functional tests were performed on Groups 1 and 2.Groups 1 and 2 were killed at 14 days after MCAO for immunostaining; mice in Groups 3 and4 were killed at 7 days after MCAO and brain tissue extracts were obtained for enzyme-linkedimmunosorbent (ELISA) assay. In addition, physiologic parameters (heart rate, blood pressure,blood gases) were also measured before killing in Group 3 and 4 mice treated with or withoutatorvastatin for 7 days after stroke (n=4/group). The left femoral artery in animals wascannulated for heart rate and arterial blood pressure (Protocol Systems, Inc.) and blood gasdetermination (Radiometer Copenhagen, Inc.). Arterial blood samples were analyzed for pH,

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arterial oxygen pressure and partial pressure of carbon dioxide by using a blood gas/pH analyzer(Radiometer Copenhagen, Inc.).

Behavioral TestsTo test whether atorvastatin promotes neurologic recovery after stroke, an array of behavioraltests were performed before MCAO, and at 1, 7 and 14 days after MCAO by an investigatorwho was masked to the experimental groups.

Modified neurologic severity scores (mNSS)Neurologic function was graded on ascale of 0 to 14 (normal score 0; maximal deficit score 14, see Table 1; Li et al, 2000). mNSSis a composite of motor, reflex and balance tests. In the severity scores of injury, one scorepoint is awarded for the inability to perform the test or for the lack of a tested reflex; thus, thehigher the score, the more severe is the injury.

Foot-fault testFor locomotor assessment, mice were tested for placement dysfunction offorelimbs with the modified foot-fault test (Hernandez and Schallert, 1988). The total numberof steps (movement of each forelimb) that the mouse used to cross the grid was counted, andthe total number of foot-faults for left forelimb was recorded. The percentage of foot-fault ofthe left paw to the total number of steps is presented.

Corner testMice were placed between two boards with dimensions 30201cm3 for eachin the home cage (Zhang et al, 2002a). The nonischemic animals turn back from either left orright, randomly. The ischemic animals preferentially turn toward the impaired side. Thenumber of turns taken on each side was recorded from 10 trials for each test.

Histologic and Immunohistochemical AssessmentMice subjected to MCAO followed by treatment with saline or atorvastatin daily for 14 days(n=9) were killed at day 14 after MCAO. Brains were fixed by transcardial perfusion withsaline, followed by perfusion and immersion in 4% paraformaldehyde, and the brains wereembedded in paraffin. Coronal sections of tissue were processed and stained with hematoxylinand eosin (H&E) for calculation of volume of cerebral infarction (Swanson et al, 1990). Theindirect lesion area, in which the intact area of the ipsilateral hemisphere was subtracted fromarea of the contralateral hemisphere, was calculated (Swanson et al, 1990). Lesion volume ispresented as a volume percentage of the lesion compared with the contralateral hemisphere.

Immunohistochemical stainingTo measure whether atorvastatin treatment promotestrophic factor expression, angiogenesis and brain plasticity, BrdU, von Willebrand Factor(vWF), BDNF, doublecortin (DCX), tubulin isotype III (TUJ-1) and synaptophysinimmunostaining were performed. Briefly, two standard paraffin blocks were obtained from thecenter of the lesion, corresponding to coronal coordinates for bregma 1 to 0mm and from 0to +1mm. A series of 6-m-thick sections were cut from the two blocks. Every 10th coronalsection (6 m thick) for a total of 5 sections from each block was used for immunohistochemicalstaining. For immunohistochemical analysis a mouse monoclonal antibody (mAb) againstBrdU, (1:100, Boehringer Mannheim, Indianapolis, IN, USA), vWF (goat polyclonal IgGantibody, 1:200 dilution, Santa Cruz), BDNF (C-9, 1:200 dilution Santa Cruz); TUJ1, an earlyneuron marker, (Monoclonal Anti-B-Tubulin Isotype III, 1:400 dilution, Sigma) anddoublecortin (DCX), a protein expressed in migrating neurons (C-18, goat polyclonal IgGantibody, 1:200 dilution, Santa Cruz), and synaptophysin (monoclonal antibody, clone SY 38,1:40 dilution; Boehringer Mannheim Biochemica, Indianapolis, IN) were used. Controlexperiments consisted of staining brain coronal tissue sections as outlined above, but omittedthe primary antibodies, as previously described (Li et al, 1998).

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After stroke, BrdU-reactive cells are not only expressed in the endothelial cells but also in theastrocytes, and pericytes around vessels. To specifically identify BrdU-reactive cellscolocalized with endothelial cells, double immunofluorescence staining of BrdU with vWF, aspecific marker for endothelial cells, was performed. To visualize BDNF-reactive cellcolocalization with the neuron marker MAP2, double immunofluorescence of BDNF withMAP2 was performed. FITC (Calbiochem) and cyanine-5.18 (CY5, Jackson Immunoresearch)were used for double-label immunoreactivity. Each coronal section was first treated with theprimary anti-BrdU or anti-BDNF antibody with FITC, and then followed by vWF ormicrotubule-associated protein 2 (MAP2, monoclonal mouse anti-MAP2 antibody, Chemicon,Temecula, CA, USA) with Cy5 staining. Control experiments consisted of staining braincoronal tissue sections as outlined above, but omitted the primary antibodies, as previouslydescribed (Li et al, 1998).

QuantificationFor semiquantitative measurements of BDNF, TUJ1, DCX andsynaptophysin densities, five slides from each block, with each slide containing 8 fieldsconsisting of cortex and stratum from the ischemic border area as shown in (Figure 1) weredigitized under a 20 objective (Olympus BX40) using a 3-CCD color video camera (SonyDXC-970MD) interfaced with an MCID image analysis system (Imaging Research, St.Catharines, Canada). The ischemic border zone is defined as the area surrounding the lesion,which morphologically differs from the surrounding normal tissue (Nedergaard et al, 1987).The digitalized images were then contrast-enhanced to clearly differentiate positivity frombackground, and a thresholding procedure was established to determine the proportion ofimmunoreactive area within each fixed field of view (Calza et al, 2001;Chen et al, 2003b). Thequantification was not analyzed stereologically. Data are presented as a percentage of area, inwhich the TUJ1, DCX, synaptophysin immunopositive areas in each field were divided by thetotal areas in the field (628480 m2) (Calza et al, 2001;Chen et al, 2003b). Brain-derivedneurotrophic factor (BDNF) quantitative data are presented as the total of number of BDNFimmunoreactive cells within each field.

Brain endothelial cell proliferationBromodeoxyuridine-immunostained sections weredigitized using a 40 objective (Olympus BX40) via the MCID computer imaging analysissystem. The number of endothelial and BrdU-immunoreactive cells (nuclei on the lumen ofvessel) within a total of 10 enlarged and thin-walled vessels located in the ischemic border area(as shown in Figure 1) were counted in each section (Chen et al, 2003b;Wang et al, 2004;Zhanget al, 2003). Data are presented as the percentage of number of the BrdU-immunoreactive cellswithin vessel/total endothelial cell number.

Vascular perimeterEnlarged and thin-walled vessels, termed mother vessels, areformed under conditions of cerebral ischemic angiogenesis (Zhang et al, 2002b). Formeasurement of vascular perimeters, each vWF immunostained coronal section was digitizedusing a 20 objective via the MCID computer imaging analysis system. The total perimeter of10 enlarged and thin-walled vessels in the ischemic border area (as shown in Figure 1) wasmeasured in each referenced coronal section, as previously described (Chen et al, 2003b; Wanget al, 2004; Zhang et al, 2003, 2002b), using MCID computer imaging analysis system (LengthTrace function). The total perimeter of 10 enlarged and thin-walled vessels in the ischemicborder is provided.

ELISA AssayTo test whether atorvastatin promotes the expression of VEGF and VEGFR2, ELISA analysiswas performed on the ischemic brain tissue extracts (Jiang et al, 1997). Mice were subjectedto permanent MCAO and treated daily with saline or atorvastatin (10 mg/kg) starting at 24hours after stroke for 7 days. Mice were killed at day 7 after stroke. Brain extracts from control

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MCAO and atorvastatin-treated animals were obtained from the ischemic border (bregma 1to 1mm, border region encompassing the ischemic core) and contralateral hemisphere. Braintissues were dissected on ice and wet weight was rapidly measured. In 150 mg/ml of tissuewas homogenized in DMEM and centrifuged for 10 mins at 10,000 g at 4C. The brain extractswere then divided into 200 L triplicate samples. Vascular endothelial growth factor andVEGFR2 ELISA kit (Cell Signaling Technology) were used to determine the VEGF andVEGFR2 levels.

Subventricular Zone (SVZ) Explant Cultures and Cell Migration Measurement In VitroTo measure whether atorvastatin induces neural cell migration, SVZ explant cultures wereprepared from adult male rats subjected to MCAO for 7 days (Dutton and Bartlett, 2000; Loisand Alvarez-Buylla, 1993). The methods for extracting and employing SVZ explants from ratshave been widely used and are well developed (Katakowski et al, 2003). The SVZ was dissectedfrom the ipsilateral SVZ of the rat brain. Tissue was minced with scalpels into pieces of ~0.1mmin each dimension. Explants were cultured within Matrigel (BD Biosciences) in wells with 500L of Neuralbasal-A Medium containing 2% B27 supplement (Invitrogen). The cultured SVZexplants were treated in the absence or presence of: (a) atorvastatin (1 mol/L); (b) BDNF (100ng/mL, Sigma); (c) anti-BDNF neutralizing antibody (100 ng/mL, Promega); (d) atorvastatin(1 mol/L) and anti-BDNF neutralizing antibody (100 ng/mL) for 7 days. Cell migration fromthe SVZ explant was measured using a phase contrast microscope and photographed at 4magnification with a digital camera. The average linear distance of cell migration from theedge of the SVZ explant was captured and measured at day 7 using the MCID software. Thisaverage distance was assessed in each explant culture.

Brain Endothelial Cell CultureTo measure whether atorvastatin promotes endothelial cell expression of VEGFR2 and BDNF,mouse brain derived microvessel endothelial cells (MBECs) (1.5104 cells) were cultured inan eight-well chamber in the absence (control) or presence of: (a) atorvastatin (1 mol/L); (b)neutralized anti-VEGFR2 antibody (DC101, 30 g/ml); and (c) atorvastatin (1 mol/L) withDC101 (30 g/ml) for 24 hours. VEGFR2 (1:100 dilution, Santa Cruz, USA) and BDNFimmunohistochemistry staining was performed on slides containing cultured endothelial cells.DAPI was used as a nuclear counterstain. All assays were performed in triplicate. VEGFR2-and BDNF-positive cells were counted in five randomly selected microscopic fields under 20objects. The percentage of VEGFR2 or BDNF immunoreactive cells within the total numberof DAPI positive cells was presented.

Statistical AnalysisData were evaluated for normality. Data transformation or nonparametric analysis approachwould be considered if data were not normal. As a result, ranked data were used for behaviortest scores. Analysis of variance and covariance (ANCOVA) was used to study treatment andtime effect on functional recovery. Analysis began testing for treatment by time interaction atthe 0.05 significance level, followed by testing for a treatment effect if no interaction wasdetected, or testing the treatment effect at each time if an interaction was detected. A significantinteraction (P

ResultsNeurologic Outcome and Lesion Volume

Functional tests showed balanced neurologic deficits before treatment between control andatorvastatin-treated groups. Mice treated with atorvastatin showed significant (P

Effect of Atorvastatin on Cell Migration in SVZ Explant CulturesFigure 5 shows that atorvastatin (C) and BDNF (B) promote SVZ explant cell migration,compared with control (A). Anti-BDNF antibody (D) significantly inhibited cells migrationcompared with control (A). Coculture anti-BDNF with atorvastatin significantly inhibitedatorvastatin-induced SVZ neural cell migration, compared with atorvastatin (C) alone group.These data suggest that BDNF amplifies neural cell migration.

VEGFR2 and Brain-Derived Neurotrophic Factor Expression in the Cultured BrainEndothelial Cells

Figure 6 shows that atorvastatin increases VEGFR2 (B) and BDNF (E) expression in culturedendothelial cells, as compared with control (A and D). Inhibition of VEGFR2 using aneutralizing anti-VEGFR2 antibody (DC101) significantly decreased atorvastatin-inducedBDNF expression in cultured endothelial cells (F). These data indicate that atorvastatinpromotes VEGFR2 and BDNF expression.

DiscussionIn the current study, we have demonstrated that a widely and clinically used HMG-CoAreductase inhibitor (statin), atorvastatin, when administered to mice starting 1 day after MCAO,evokes significant improvement in functional neurologic recovery. This observation isconsistent with our previous studies using this compound in rats (Chen et al, 2003b). Inaddition, we have also shown that atorvastatin promotes: (1) neurogenesis and neuronalplasticity as well as increases the expression of VEGF, VEGFR2 and BDNF in the ischemicborder after stroke in mice; (2) endothelial cell proliferation and VEGFR2 and BDNFexpression; (3) neural cell migration; (4) BDNF expression, which mediates neural cellmigration in SVZ explants. Collectively, these in vivo and in vitro data strongly support a rolefor atorvastatin in promoting brain plasticity and recovery from stroke.

Atorvastatin increases VEGF, VEGFR2 expression as well as promotes endothelial cellproliferation in the ischemic border. Vascular endothelial growth factor is an angiogenic factor.Vascular endothelial growth factor exerts biologic functions via two closely related receptortyrosine kinases VEGFR1 (flt-1) and VEGFR2 (flk-1). Most of the VEGF properties, such asmitogenicity, chemotaxis and induction of morphologic change, are mediated by its interactionwith VEGFR2 (Waltenberger et al, 1994). Vascular endothelial growth factor/VEGFR2 hasbeen shown to be essential for endothelial cell proliferation and differentiation (Breier et al,1992). Binding of VEGF to VEGFR2 leads to the receptor phosphorylation and subsequentactivation of PI3K/Akt and other downstream signaling proteins (Gliki et al, 2002; Nakashioet al, 2002). Statin-induced capillary-like tube formation is inhibited by a neutralizing antibodyagainst VEGFR2 and inhibition of PI3K (Chen et al, 2003b). We propose that administrationof atorvastatin promotes VEGF and VEGFR2 expression in the ischemic border, which mayfacilitate induction of endothelial cell proliferation and angiogenesis.

The 10 mg/kg dose of atorvastatin used in this study is consistent with the neuroprotective doseof atorvastatin used in pretreatment of mice after stroke (Gertz et al, 2003; Laufs et al, 2000).We and others have shown that in vitro and in vivo, atorvastatin has a U-shaped doseresponsecurve on inducing angiogenesis (Chen et al, 2003b; Urbich et al, 2002; Weis et al, 2002).However, a 2.5 mg/kg dose of atorvastatin was shown to decrease angiogenesis in brain tumorand in a model of inflammation (Weis et al, 2002). This apparent discrepancy may be attributedto our use of the stroke model versus tumor model.

In addition to its role in inducing angiogenesis, VEGF also stimulates neurogenesis and axonaloutgrowth, and improves the survival of mouse superior cervical, dorsal root ganglion neurons,

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and mesencephalic neurons (Hess et al, 2002; Sondell et al, 1999). Vascular endothelial growthfactor is mitogenic for astrocytes and promotes growth/survival of neurons (Silverman et al,1999). Our data have shown that atorvastatin promotes angiogenesis, neuronal plasticity aswell as increases VEGF/VEGFR2 expression. We propose that the atorvastatin-inducedincrease of VEGF/VEGFR2 may not only cause angiogenesis but also provide a supportivemicroenvironment, which can enhance the neuronal and synaptic plasticity.

Neurogenesis and synaptic reorganization are important for functional improvement afterstroke (Gomez-Fernandez, 2000; Hallett, 2001; Shingo et al, 2001). Our data show thatatorvastatin treatment after stroke induces the expression of BDNF and synaptic proteins, andneuronal migration in the ischemic border, and some neuronal migrating cells are localized toblood vessels. These data are consistent with other reported studies showing that increasingsynaptic activity elicits compensatory angiogenesis (Black et al, 1989). An index of axonalsprouting, GAP43, appears to be colocalized with angiogenesis after MCAO (Kawamata etal, 1997). Neurogenesis occurs in close proximity to blood vessels, where VEGF expressionis high and angiogenesis is ongoing (Palmer et al, 2000). The newly activated and expandedvasculature substantially increases the production and release of BDNF, whose induction isboth spatially and temporally associated with recruitment of new neurons (Leventhal et al,1999). Cerebral endothelial cells are a potential source for bioactive BDNF (Bayas et al,2002). Vascular endothelial cells may contribute to circulating BDNF (Nakahashi et al,2000). Atorvastatin and BDNF induce neuronal migration in the SVZ explant culture.Inhibition of BDNF decreases neuronal migration in the SVZ explant. These data suggest thatneuronal plasticity is closely linked with angiogenesis and BDNF may facilitate atorvastatin-induced neuronal plasticity after stroke.

In summary, treatment of stroke at 24 hours after MCAO in mice with a widely used atorvastatinreduces neurologic deficits associated with stroke. Statin-mediated functional benefit might bederived from the upregulation of trophic factors such as VEGF/VEGFR2, BDNF and inductionof angiogenesis, neurogenesis, and synaptic plasticity.

AcknowledgmentsWe wish to thank Cynthia Roberts for technical assistance support. This work was supported by NINDS grants PO1NS23393, PO1 NS42345 and RO1 NS47682.

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Figure 1.Quantification field of view in the ischemic border.

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Figure 2.Atorvastatin promotes functional recovery. Mice were treated with or without atorvastatin (10mg/kg) starting at 24 hours after stroke daily for 14 days (n=9/group). Functional tests wereperformed before MCAO and 1, 7, and 14 days after MCAO. *P

Figure 3.Angiogenesis and BDNF expression. Bromodeoxyuridine-reactive endothelial cells weresignificantly increased in the atorvastatin-treated group (B) compared with control (A, P

the ischemic boundary area. (M) to (O) show double immunostaining BDNF-reactive cell(M; arrow, BDNF-positive cell) colocalized with MAP2 (N; MAP2-positive cell). (O) showsmerged from (J) and (K). Brain-derived neurotrophic factor immunoreactive cells were alsopresent in endothelial cells (P; arrow, BDNF-positive endothelial cell). Scale bar (A), (D) and(J)=100 m. Scale bar (I) and (N)=50 m and P=20 m.

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Figure 4.DCX, TUJ1 and synaptophysin expression. DCX, TUJ1 and synaptophysin are expressed inthe ischemic boundary area at 14 days after MCAO treated with atorvastatin (10 mg/kg). (A),(E) and (H) show DCX (A), TUJ1 (E) and synpatophysin (H) expression, respectively, incontrol saline-treated animal. (B), (F) and (I) show DCX (B), TUJ1 (F) and synpatophysin(I) expression, respectively, in an atorvastatin-treated animal. (D), (G) and (J) showquantitative data of DCX (D), TUJ1 (G) and synaptophysin (J) density in the ischemicboundary area. (C) shows DCX immunoreactive cells around vessel (arrow: DCX-positivecells; arrow head: vessel). Scale bar (A), (E), (H)=100 m. Scale bar (C)=20 m.

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Figure 5.SVZ explant cell migration. (A) Control; (B) BDNF treatment; (C) atorvastatin treatment;(D) anti-BDNF antibody treatment; (E) atorvastatin with anti-BDNF antibody treatment; (F)quantitative data of SVZ cell migration. Scale bar (A)=200 m.

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Figure 6.VEGFR2 and BDNF expression in the cultured endothelial cells. VEGFR2 (B) and BDNF(E) immunoreactive cells were increased in the atorvastatin-treated group compared withcontrol group (A: VEGFR2; D: BDNF). (C) and (F) quantitative data of percent of VEGFR2(C) and BDNF (F) immunoreactive cells in cultured mouse brain endothelial cell. Scale bar(A)=50 m.

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Table 1

Modified neurologic severity scores (mNSS)

Motor tests Points

Raising the mouse by the tail 3

1 Flexion of forelimb1 Flexion of hindlimb1 Head moved more than 10 to the vertical axis within 30 secondsWalking on the floor (normal=0; maximum=3) 3

0 Normal walk1 Inability to walk straight2 Circling toward the paretic side3 Falling down to the paretic sideBeam balance tests (normal=0; maximum=6) 6

0 Balances with steady posture1 Grasps side of beam2 Hugs the beam and one limb falls down from the beam3 Hugs the beam and two limbs fall down from the beam, or spins on beam (>30 seconds)4 Attempts to balance on the beam but falls off (>20 seconds)5 Attempts to balance on the beam but falls off (>10 seconds)6 Falls off: No attempt to balance or hang on to the beam (

ni hms 133911

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