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J Cereb Blood Flow Metab. 2005 February ; 25(2): 281290. doi:10.1038/sj.jcbfm.9600034.
Atorvastatin induction of VEGF and BDNF promotes brain

plasticity after stroke in mice
Jieli Chen1, Chunling Zhang1, Hao Jiang1, Yi Li1, Lijie Zhang1, Adam Robin1, Mark
Katakowski1, Mei Lu2, and Michael Chopp1,3
1 Department of Neurology, Henry Ford Health Sciences Center, Detroit, Michigan, USA
2
Department of Biostatistics and Research Epidemiology, Henry Ford Health Sciences Center,
Detroit, Michigan, USA
3
Department of Physics, Oakland University, Rochester, Michigan, USA
Abstract
Molecular mechanisms underlying the role of statins in the induction of brain plasticity and
subsequent improvement of neurologic outcome after treatment of stroke have not been adequately
investigated. Here, we use both in vivo and in vitro studies to investigate the potential roles of two
prominent factors, vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor
(BDNF), in mediating brain plasticity after treatment of stroke with atorvastatin. Treatment of stroke
in adult mice with atorvastatin daily for 14 days, starting at 24 hours after MCAO, shows significant
improvement 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 were
significantly 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 cell
culture expression of BDNF and VEGFR2 was significantly increased in atorvastatin-treated cells
compared with control cells. Inhibition of VEGFR2 significantly decreased expression of BDNF in
brain endothelial cells. These data indicate that atorvastatin promotes angiogenesis, brain plasticity
and enhances functional recovery after stroke. In addition, VEGF, VEGFR2 and BDNF likely
contribute to these restorative processes.
Keywords
angiogenesis; atorvastatin; brain-derived neurotrophic factor; middle cerebral artery occlusion
(MCAO); migration; vascular endothelial growth factor
Introduction
We have previously demonstrated that treatment of stroke in the rat with HMG-CoA reductase
inhibitors (statins), atorvastatin and simvastatin, with treatment initiated 24 hours after onset
of stroke, significantly improves neurologic outcome compared with control animals (Chen et
al, 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. chopp@neuro.hfh.edu.
Chen et al.
Page 2
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 and
synaptogenesis raises the question of how these aspects of brain plasticity are coupled, and if
there are specific molecular factors that can integrate these remodeling events in the brain.
Vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) are
two important neurotrophic factors that have multiple effects on sustaining and evoking
elements of brain plasticity (Jin et al, 2002; Sun et al, 2003). Vascular endothelial growth factor
is an angiogenic agent, which promotes neurogenesis and stem-progenitor cell migration (Jin
et al, 2002; Sun et al, 2003). Likewise, BDNF regulates neuronal survival, cell migration, and
synaptic function (Aguado et al, 2003; Gorski et al, 2003). Statins increase VEGF in endothelial
and osteoblast cells (Chen et al, 2003a; Maeda et al, 2003). Both VEGF and BDNF increase
in ischemic brain with other neurorestorative treatments of stroke, such as with bone marrow
stromal cells (Chen et al, 2002; Matsuno et al, 2004). It is therefore reasonable to propose that
VEGF and BDNF might be upregulated in the brain after treatment with a statin, and may
orchestrate 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 of
permanent middle cerebral artery occlusion (MCAO). We show that atorvastatin increases
cerebral expression of VEGF and its receptor VEGFR2 induces endothelial cell proliferation
and angiogenesis, and promotes production of neurotrophic factors, BDNF and VEGF, which
may act in concert to induce brain plasticity and to foster functional recovery after stroke in
mice.
Materials and methods

MCAO Model and Atorvastatin Administration
Adult male C57BL/6J mice (age 2 to 3 months, weight 24 to 28 g) were purchased from Charles
River (Wilmington, MA, USA). Mice were anesthetized with halothane. Permanent right
MCAO was induced by advancing a 6-0 surgical nylon suture (8.0 to 9.0mm determined by
body weight) with an expanded (heated) tip from the external carotid artery into the lumen of
the internal carotid artery to block the origin of the MCA (Mao et al, 1999). Atorvastatin was
dissolved in methanol (20mg atorvastatin dissolved in 20mL saline with 10 L methanol) and
injected subcutaneously (s.c.) at 24 hours after MCAO. An atorvastatin dose of 10 mg/kg was
selected 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 (0.25
mL) daily for 14 days as a control group (n=12); Group 2, atorvastatin (10 mg/kg) daily for 14
days (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 ischemic
brain, mice received injections of bromodeoxyuridine (BrdU, Sigma Chemical, 100 mg/kg in
0.007N NaOH physiologic saline), intraperitoneally (i.p.) starting 1 day after MCAO and daily
thereafter 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 and
4 were killed at 7 days after MCAO and brain tissue extracts were obtained for enzyme-linked
immunosorbent (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 without
atorvastatin for 7 days after stroke (n=4/group). The left femoral artery in animals was
cannulated for heart rate and arterial blood pressure (Protocol Systems, Inc.) and blood gas
determination (Radiometer Copenhagen, Inc.). Arterial blood samples were analyzed for pH,
Chen et al.
Page 3
arterial oxygen pressure and partial pressure of carbon dioxide by using a blood gas/pH analyzer
(Radiometer Copenhagen, Inc.).
Behavioral Tests
To test whether atorvastatin promotes neurologic recovery after stroke, an array of behavioral
tests were performed before MCAO, and at 1, 7 and 14 days after MCAO by an investigator
who was masked to the experimental groups.
Modified neurologic severity scores (mNSS)Neurologic function was graded on a
scale of 0 to 14 (normal score 0; maximal deficit score 14, see Table 1; Li et al, 2000). mNSS
is a composite of motor, reflex and balance tests. In the severity scores of injury, one score
point is awarded for the inability to perform the test or for the lack of a tested reflex; thus, the
higher the score, the more severe is the injury.
Foot-fault testFor locomotor assessment, mice were tested for placement dysfunction of
forelimbs with the modified foot-fault test (Hernandez and Schallert, 1988). The total number
of steps (movement of each forelimb) that the mouse used to cross the grid was counted, and
the total number of foot-faults for left forelimb was recorded. The percentage of foot-fault of
the left paw to the total number of steps is presented.
Corner testMice were placed between two boards with dimensions 30201cm3 for each
in the home cage (Zhang et al, 2002a). The nonischemic animals turn back from either left or
right, randomly. The ischemic animals preferentially turn toward the impaired side. The
number of turns taken on each side was recorded from 10 trials for each test.
Histologic and Immunohistochemical Assessment
Mice 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 with
saline, followed by perfusion and immersion in 4% paraformaldehyde, and the brains were
embedded in paraffin. Coronal sections of tissue were processed and stained with hematoxylin
and eosin (H&E) for calculation of volume of cerebral infarction (Swanson et al, 1990). The
indirect lesion area, in which the intact area of the ipsilateral hemisphere was subtracted from
area of the contralateral hemisphere, was calculated (Swanson et al, 1990). Lesion volume is
presented as a volume percentage of the lesion compared with the contralateral hemisphere.
Immunohistochemical stainingTo measure whether atorvastatin treatment promotes

trophic factor expression, angiogenesis and brain plasticity, BrdU, von Willebrand Factor
(vWF), BDNF, doublecortin (DCX), tubulin isotype III (TUJ-1) and synaptophysin
immunostaining were performed. Briefly, two standard paraffin blocks were obtained from the
center of the lesion, corresponding to coronal coordinates for bregma 1 to 0mm and from 0
to +1mm. A series of 6-m-thick sections were cut from the two blocks. Every 10th coronal
section (6 m thick) for a total of 5 sections from each block was used for immunohistochemical
staining. For immunohistochemical analysis a mouse monoclonal antibody (mAb) against
BrdU, (1:100, Boehringer Mannheim, Indianapolis, IN, USA), vWF (goat polyclonal IgG
antibody, 1:200 dilution, Santa Cruz), BDNF (C-9, 1:200 dilution Santa Cruz); TUJ1, an early
neuron marker, (Monoclonal Anti-B-Tubulin Isotype III, 1:400 dilution, Sigma) and
doublecortin (DCX), a protein expressed in migrating neurons (C-18, goat polyclonal IgG
antibody, 1:200 dilution, Santa Cruz), and synaptophysin (monoclonal antibody, clone SY 38,
1:40 dilution; Boehringer Mannheim Biochemica, Indianapolis, IN) were used. Control
experiments consisted of staining brain coronal tissue sections as outlined above, but omitted
the primary antibodies, as previously described (Li et al, 1998).
Chen et al.
Page 4
After stroke, BrdU-reactive cells are not only expressed in the endothelial cells but also in the
astrocytes, and pericytes around vessels. To specifically identify BrdU-reactive cells
colocalized with endothelial cells, double immunofluorescence staining of BrdU with vWF, a
specific marker for endothelial cells, was performed. To visualize BDNF-reactive cell
colocalization with the neuron marker MAP2, double immunofluorescence of BDNF with
MAP2 was performed. FITC (Calbiochem) and cyanine-5.18 (CY5, Jackson Immunoresearch)
were used for double-label immunoreactivity. Each coronal section was first treated with the
primary anti-BrdU or anti-BDNF antibody with FITC, and then followed by vWF or
microtubule-associated protein 2 (MAP2, monoclonal mouse anti-MAP2 antibody, Chemicon,
Temecula, CA, USA) with Cy5 staining. Control experiments consisted of staining brain
coronal tissue sections as outlined above, but omitted the primary antibodies, as previously
described (Li et al, 1998).
QuantificationFor semiquantitative measurements of BDNF, TUJ1, DCX and

synaptophysin densities, five slides from each block, with each slide containing 8 fields
consisting of cortex and stratum from the ischemic border area as shown in (Figure 1) were
digitized under a 20 objective (Olympus BX40) using a 3-CCD color video camera (Sony
DXC-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 from
background, and a thresholding procedure was established to determine the proportion of
immunoreactive area within each fixed field of view (Calza et al, 2001;Chen et al, 2003b). The
quantification was not analyzed stereologically. Data are presented as a percentage of area, in
which the TUJ1, DCX, synaptophysin immunopositive areas in each field were divided by the
total areas in the field (628480 m2) (Calza et al, 2001;Chen et al, 2003b). Brain-derived
neurotrophic factor (BDNF) quantitative data are presented as the total of number of BDNF
immunoreactive cells within each field.
Brain endothelial cell proliferationBromodeoxyuridine-immunostained sections were
digitized using a 40 objective (Olympus BX40) via the MCID computer imaging analysis
system. The number of endothelial and BrdU-immunoreactive cells (nuclei on the lumen of
vessel) 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;Zhang
et al, 2003). Data are presented as the percentage of number of the BrdU-immunoreactive cells
within vessel/total endothelial cell number.
Vascular perimeterEnlarged and thin-walled vessels, termed mother vessels, are

formed under conditions of cerebral ischemic angiogenesis (Zhang et al, 2002b). For
measurement of vascular perimeters, each vWF immunostained coronal section was digitized
using a 20 objective via the MCID computer imaging analysis system. The total perimeter of
10 enlarged and thin-walled vessels in the ischemic border area (as shown in Figure 1) was
measured in each referenced coronal section, as previously described (Chen et al, 2003b; Wang
et al, 2004; Zhang et al, 2003, 2002b), using MCID computer imaging analysis system (Length
Trace function). The total perimeter of 10 enlarged and thin-walled vessels in the ischemic
border is provided.
ELISA Assay
To test whether atorvastatin promotes the expression of VEGF and VEGFR2, ELISA analysis
was performed on the ischemic brain tissue extracts (Jiang et al, 1997). Mice were subjected
to permanent MCAO and treated daily with saline or atorvastatin (10 mg/kg) starting at 24
hours after stroke for 7 days. Mice were killed at day 7 after stroke. Brain extracts from control
Chen et al.
Page 5
MCAO and atorvastatin-treated animals were obtained from the ischemic border (bregma 1
to 1mm, border region encompassing the ischemic core) and contralateral hemisphere. Brain
tissues were dissected on ice and wet weight was rapidly measured. In 150 mg/ml of tissue
was homogenized in DMEM and centrifuged for 10 mins at 10,000 g at 4C. The brain extracts
were then divided into 200 L triplicate samples. Vascular endothelial growth factor and
VEGFR2 ELISA kit (Cell Signaling Technology) were used to determine the VEGF and
VEGFR2 levels.
Subventricular Zone (SVZ) Explant Cultures and Cell Migration Measurement In Vitro
To measure whether atorvastatin induces neural cell migration, SVZ explant cultures were
prepared from adult male rats subjected to MCAO for 7 days (Dutton and Bartlett, 2000; Lois
and Alvarez-Buylla, 1993). The methods for extracting and employing SVZ explants from rats
have been widely used and are well developed (Katakowski et al, 2003). The SVZ was dissected
from the ipsilateral SVZ of the rat brain. Tissue was minced with scalpels into pieces of ~0.1mm
in each dimension. Explants were cultured within Matrigel (BD Biosciences) in wells with 500
L of Neuralbasal-A Medium containing 2% B27 supplement (Invitrogen). The cultured SVZ
explants were treated in the absence or presence of: (a) atorvastatin (1 mol/L); (b) BDNF (100
ng/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 from
the SVZ explant was measured using a phase contrast microscope and photographed at 4
magnification with a digital camera. The average linear distance of cell migration from the
edge of the SVZ explant was captured and measured at day 7 using the MCID software. This
average distance was assessed in each explant culture.
Brain Endothelial Cell Culture
To measure whether atorvastatin promotes endothelial cell expression of VEGFR2 and BDNF,
mouse brain derived microvessel endothelial cells (MBECs) (1.5104 cells) were cultured in
an 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) with
DC101 (30 g/ml) for 24 hours. VEGFR2 (1:100 dilution, Santa Cruz, USA) and BDNF
immunohistochemistry staining was performed on slides containing cultured endothelial cells.
DAPI was used as a nuclear counterstain. All assays were performed in triplicate. VEGFR2and BDNF-positive cells were counted in five randomly selected microscopic fields under 20
objects. The percentage of VEGFR2 or BDNF immunoreactive cells within the total number
of DAPI positive cells was presented.
Statistical Analysis
Data were evaluated for normality. Data transformation or nonparametric analysis approach
would be considered if data were not normal. As a result, ranked data were used for behavior
test scores. Analysis of variance and covariance (ANCOVA) was used to study treatment and
time effect on functional recovery. Analysis began testing for treatment by time interaction at
the 0.05 significance level, followed by testing for a treatment effect if no interaction was
detected, or testing the treatment effect at each time if an interaction was detected. A significant
interaction (P<0.05) indicates that the effects of the treatment of atorvastatin on behavior test
results depend on time. We also investigated the atorvastatin effects on lesion volume, vWF,
BDNF, TUJ1, DCX, BrdU and synaptophysin expression at 14 days after MCAO, compared
with the controls using two-sample t-test at the significance level of 0.05. Means.d. by
treatment groups are presented as data illustration.
Chen et al.
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Results
Neurologic Outcome and Lesion Volume
Functional tests showed balanced neurologic deficits before treatment between control and
atorvastatin-treated groups. Mice treated with atorvastatin showed significant (P<0.05, n=9/
group) improvement of functional recovery on mNSS, foot-fault and corner tests at days 7 and
14 compared with saline-treated mice (Figure 2). The mortality rate of atorvastatin-treated mice
was two of 11 mice, and three of 12 MCAO control mice. No significant differences of ischemic
lesion volumes were detected between atorvastatin-treated (16.78.7%) and saline-treated
(19.010.7%) mice. These results suggest that atorvastatin promotes functional recovery after
stroke in mice, while it has no effect on reducing the lesion volume. In addition, physiologic
parameters (heart rate, blood pressure and blood gases) do not show significant difference after
ischemia in the atorvastatin-treated group (HR: 562 43/min; BP: 68.83.0mm Hg; pH: 7.31
0.04; PaO2: 143.06.4mm Hg; PaCO2: 41.31.9mm Hg) compared with MCAO control
animals (HR: 568 56/min; BP: 71.33.6mm Hg; pH: 7.350.06; PaO2: 139.510.6mm Hg;
PaCO2: 43.53.1mm Hg).
Angiogenesis and Trophic Factors (Figure 3)
Treatment with atorvastatin significantly increased BrdU-reactive endothelial cell numbers (B

and C) and vascular perimeter (E and F) in the ischemic border, as compared with the salinetreated animals (A, D). Bromodeoxyuridine with vWF double immunostaining shows that
BrdU-positive endothelial cells (G and I) localize in the vessel (H and I). These data indicate
that atorvastatin enhances angiogenesis in the ischemic brain. Treatment of stroke with
atorvastatin significantly increased BDNF (K, arrow) immunoreactivity cell density in the
ischemic border, as compared with the control mice (J, arrow). Double immunostaining showed
that BDNF immunoreactive cells were colocalized with MAP2 immunoreactive cells in the
cortex (M to O). Brain-derived neurotrophic factor was also detected in the endothelial cells
(P, arrow). These findings suggest that atorvastatin promotes endothelial cell proliferation and
angiogenesis. In addition, atorvastatin promotes BDNF expression. Brain-derived
neurotrophic factor is expressed in neurons and endothelial cells.
Vascular Endothelial Growth Factor and VEGFR2 ELISA Assay
Vascular endothelial growth factor and VEGFR2 were measured by ELISA method in the
ischemic brain extract. Vascular endothelial growth factor and VEGFR2 were significantly
(P<0.05) increased in the ischemic border in the atorvastatin-treated group (VEGF: 30.69.3
pg/g; VEGFR2: 40.212.6 pg/g) compared with the control group (VEGF: 19.2 8.6 pg/g;
VEGFR2: 14.98.0 pg/g). Vascular endothelial growth factor and VEGFR2 showed no
significant difference in the contralateral hemisphere treated with atorvastatin (VEGF: 10.8
2.2 pg/g; VEGFR2: 5.64.0 pg/g) compared with the contralateral hemisphere of MCAO
control mice (VEGF: 12.44.3 pg/g; VEGFR2: 4.93.1 pg/g) (P>0.05). These data suggest
that atorvastatin enhances VEGF and VEGFR2 expression after stroke in the ischemic brain.
Neurogenesis and Synaptic Plasticity
To test whether atorvastatin promotes neuronal migration and plasticity, DCX, TUJ1 and
synaptophysin immunostaining were performed in the brain coronal sections. Figure 4 shows
that atorvastatin treatment after stroke significantly increased DCX (B), TUJ1 (F) and
synaptophysin (I) expression in the ischemic border, as compared with saline-treated mice (A,
E and H). Increased DCX-positive cells were observed around vessels (C). These findings
suggest that atorvastatin promotes cell migration and differentiation into neurons, and
migrating neuronal cells are linked with vessels.
Chen et al.
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Effect of Atorvastatin on Cell Migration in SVZ Explant Cultures
Figure 5 shows that atorvastatin (C) and BDNF (B) promote SVZ explant cell migration,
compared with control (A). Anti-BDNF antibody (D) significantly inhibited cells migration
compared with control (A). Coculture anti-BDNF with atorvastatin significantly inhibited
atorvastatin-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 Brain
Endothelial Cells
Figure 6 shows that atorvastatin increases VEGFR2 (B) and BDNF (E) expression in cultured
endothelial cells, as compared with control (A and D). Inhibition of VEGFR2 using a
neutralizing anti-VEGFR2 antibody (DC101) significantly decreased atorvastatin-induced
BDNF expression in cultured endothelial cells (F). These data indicate that atorvastatin
promotes VEGFR2 and BDNF expression.
Discussion
In the current study, we have demonstrated that a widely and clinically used HMG-CoA
reductase inhibitor (statin), atorvastatin, when administered to mice starting 1 day after MCAO,
evokes significant improvement in functional neurologic recovery. This observation is
consistent with our previous studies using this compound in rats (Chen et al, 2003b). In
addition, we have also shown that atorvastatin promotes: (1) neurogenesis and neuronal
plasticity as well as increases the expression of VEGF, VEGFR2 and BDNF in the ischemic
border after stroke in mice; (2) endothelial cell proliferation and VEGFR2 and BDNF
expression; (3) neural cell migration; (4) BDNF expression, which mediates neural cell
migration in SVZ explants. Collectively, these in vivo and in vitro data strongly support a role
for atorvastatin in promoting brain plasticity and recovery from stroke.
Atorvastatin increases VEGF, VEGFR2 expression as well as promotes endothelial cell

proliferation in the ischemic border. Vascular endothelial growth factor is an angiogenic factor.
Vascular endothelial growth factor exerts biologic functions via two closely related receptor
tyrosine kinases VEGFR1 (flt-1) and VEGFR2 (flk-1). Most of the VEGF properties, such as
mitogenicity, chemotaxis and induction of morphologic change, are mediated by its interaction
with VEGFR2 (Waltenberger et al, 1994). Vascular endothelial growth factor/VEGFR2 has
been 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 subsequent
activation of PI3K/Akt and other downstream signaling proteins (Gliki et al, 2002; Nakashio
et al, 2002). Statin-induced capillary-like tube formation is inhibited by a neutralizing antibody
against VEGFR2 and inhibition of PI3K (Chen et al, 2003b). We propose that administration
of atorvastatin promotes VEGF and VEGFR2 expression in the ischemic border, which may
facilitate induction of endothelial cell proliferation and angiogenesis.
The 10 mg/kg dose of atorvastatin used in this study is consistent with the neuroprotective dose
of 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 doseresponse
curve 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 tumor
and in a model of inflammation (Weis et al, 2002). This apparent discrepancy may be attributed
to our use of the stroke model versus tumor model.
In addition to its role in inducing angiogenesis, VEGF also stimulates neurogenesis and axonal
outgrowth, and improves the survival of mouse superior cervical, dorsal root ganglion neurons,
Chen et al.
Page 8
and mesencephalic neurons (Hess et al, 2002; Sondell et al, 1999). Vascular endothelial growth
factor is mitogenic for astrocytes and promotes growth/survival of neurons (Silverman et al,
1999). Our data have shown that atorvastatin promotes angiogenesis, neuronal plasticity as
well as increases VEGF/VEGFR2 expression. We propose that the atorvastatin-induced
increase of VEGF/VEGFR2 may not only cause angiogenesis but also provide a supportive
microenvironment, which can enhance the neuronal and synaptic plasticity.
Neurogenesis and synaptic reorganization are important for functional improvement after
stroke (Gomez-Fernandez, 2000; Hallett, 2001; Shingo et al, 2001). Our data show that
atorvastatin treatment after stroke induces the expression of BDNF and synaptic proteins, and
neuronal migration in the ischemic border, and some neuronal migrating cells are localized to
blood vessels. These data are consistent with other reported studies showing that increasing
synaptic activity elicits compensatory angiogenesis (Black et al, 1989). An index of axonal
sprouting, GAP43, appears to be colocalized with angiogenesis after MCAO (Kawamata et
al, 1997). Neurogenesis occurs in close proximity to blood vessels, where VEGF expression
is high and angiogenesis is ongoing (Palmer et al, 2000). The newly activated and expanded
vasculature substantially increases the production and release of BDNF, whose induction is
both 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 that
neuronal plasticity is closely linked with angiogenesis and BDNF may facilitate atorvastatininduced neuronal plasticity after stroke.
In summary, treatment of stroke at 24 hours after MCAO in mice with a widely used atorvastatin
reduces neurologic deficits associated with stroke. Statin-mediated functional benefit might be
derived from the upregulation of trophic factors such as VEGF/VEGFR2, BDNF and induction
of angiogenesis, neurogenesis, and synaptic plasticity.
Acknowledgments
We wish to thank Cynthia Roberts for technical assistance support. This work was supported by NINDS grants PO1
NS23393, PO1 NS42345 and RO1 NS47682.
References
Aguado F, Carmona MA, Pozas E, Aguilo A, Martinez-Guijarro FJ, Alcantara S, Borrell V, Yuste R,
Ibanez CF, Soriano E. BDNF regulates spontaneous correlated activity at early developmental stages
by increasing synaptogenesis and expression of the K+/Cl co-transporter KCC2. Development
2003;130:126780. [PubMed: 12588844]
Bayas A, Hummel V, Kallmann BA, Karch C, Toyka KV, Rieckmann P. Human cerebral endothelial
cells are a potential source for bioactive BDNF. Cytokine 2002;19:558. [PubMed: 12182839]
Black JE, Polinsky M, Greenough WT. Progressive failure of cerebral angiogenesis supporting neural
plasticity in aging rats. Neurobiol Aging 1989;10:3538. [PubMed: 2478904]
Breier G, Albrecht U, Sterrer S, Risau W. Expression of vascular endothelial growth factor during
embryonic angiogenesis and endothelial cell differentiation. Development 1992;114:52132.
[PubMed: 1592003]
Calza L, Giardino L, Giuliani A, Aloe L, Levi-Montalcini R. Nerve growth factor control of neuronal
expression of angiogenetic and vasoactive factors. Proc Natl Acad Sci USA 2001;98:41605.
[PubMed: 11259645]
Chen J, Zhang ZG, Li Y, Wang L, Xu YX, Gautam SC, Lu M, Zhu Z, Chopp M. Intravenous
administration of human bone marrow stromal cells induces angiogenesis in the ischemic boundary
zone after stroke in rats. Circ Res 2003a;92:6929. [PubMed: 12609969]
Chen et al.
Page 9

Chen J, Zhang ZG, Li Y, Wang Y, Wang L, Jiang H, Zhang C, Lu M, Katakowski M, Feldkamp CS,
Chopp M. Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann Neurol
2003b;53:74351. [PubMed: 12783420]
Chen X, Li Y, Wang L, Katakowski M, Zhang L, Chen J, Xu Y, Gautam SC, Chopp M. Ischemic rat
brain extracts induce human marrow stromal cell growth factor production. Neuropathology 2002;22
(4):2759. [PubMed: 12564767]
Dutton R, Bartlett PF. Precursor cells in the subventricular zone of the adult mouse are actively inhibited
from differentiating into neurons. Dev Neurosci 2000;22:96105. [PubMed: 10657702]
Gertz K, Laufs U, Lindauer U, Nickenig G, Bohm M, Dirnagl U, Endres M. Withdrawal of statin treatment
abrogates stroke protection in mice. Stroke 2003;34:5517. [PubMed: 12574574]
Gliki G, Wheeler-Jones C, Zachary I. Vascular endothelial growth factor induces protein kinase C (PKC)dependent Akt/PKB activation and phosphatidylinositol 3-kinase-mediated PKCdelta
phosphorylation: role of PKC in angiogenesis. Cell Biol Int 2002;26:751. [PubMed: 12377207]
Gomez-Fernandez L. Cortical plasticity and restoration of neurologic functions: an update on this topic.
Rev Neurol 2000;31:74956. [PubMed: 11082885]
Gorski JA, Zeiler SR, Tamowski S, Jones KR. Brain-derived neurotrophic factor is required for the
maintenance of cortical dendrites. J Neurosci 2003;23:685665. [PubMed: 12890780]
Hallett M. Plasticity of the human motor cortex and recovery from stroke. Brain Res Brain Res Rev
2001;36:169174. [PubMed: 11690613]
Hernandez TD, Schallert T. Seizures and recovery from experimental brain damage. Exp Neurol
1988;102:318324. [PubMed: 3197789]
Hess A, Labbe D, Michel O, Teranishi M, Orzechowska O, Schmidt A, Addicks K, Bloch W. In vitro
activation of extracellular signal-regulated kinase1/2 in the inner ear of guinea pigs. Brain Res
2002;956:23645. [PubMed: 12445691]
Jiang H, Movsesyan V, Fink DW Jr, Fasler M, Whalin M, Katagiri Y, Monshipouri M, Dickens G, Lelkes
PI, Guroff G, Lazarovici P. Expression of human p140trk receptors in p140trk-deficient, PC12/
endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis. J
Cell Biochem 1997;66:22944. [PubMed: 9213224]
Jin K, Zhu Y, Sun Y, Mao XO, Xie L, Greenberg DA. Vascular endothelial growth factor (VEGF)
stimulates neurogenesis in vitro and in vivo. Proc Natl Acad Sci USA 2002;99:1194650. [PubMed:
12181492]
Katakowski M, Zhang ZG, Chen J, Zhang R, Wang Y, Jiang H, Zhang L, Robin A, Li Y, Chopp M.
Phosphoinositide 3-kinase promotes adult subventricular neuroblast migration after stroke. J
Neurosci Res 2003;74:494501. [PubMed: 14598293]
Kawamata T, Dietrich WD, Schallert T, Gotts JE, Cocke RR, Benowitz LI, Finklestein SP. Intracisternal
basic fibroblast growth factor enhances functional recovery and up-regulates the expression of a
molecular marker of neuronal sprouting following focal cerebral infarction. Proc Natl Acad Sci USA
1997;94:817984. [PubMed: 9223335]
Laufs U, Gertz K, Huang P, Nickenig G, Bohm M, Dirnagl U, Endres M. Atorvastatin upregulates type
III nitric oxide synthase in thrombocytes, decreases platelet activation, and protects from cerebral
ischemia in normocholesterolemic mice. Stroke 2000;31:24429. [PubMed: 11022078]
Leventhal C, Rafii S, Rafii D, Shahar A, Goldman SA. Endothelial trophic support of neuronal production
and recruitment from the adult mammalian subependyma. Mol Cell Neurosci 1999;13:45064.
[PubMed: 10383830]
Li Y, Chopp M, Chen J, Wang L, Gautam SC, Xu YX, Zhang Z. Intrastriatal transplantation of bone
marrow nonhematopoietic cells improves functional recovery after stroke in adult mice. J Cereb
Blood Flow Metab 2000;20:13119. [PubMed: 10994853]
Li Y, Jiang N, Powers C, Chopp M. Neuronal damage and plasticity identified by microtubule-associated
protein 2, growth-associated protein 43, and cyclin D1 immunoreactivity after focal cerebral ischemia
in rats. Stroke 1998;29:197280. (discussion 19801971). [PubMed: 9731626]
Lois C, Alvarez-Buylla A. Proliferating subventricular zone cells in the adult mammalian forebrain can
differentiate into neurons and glia. Proc Natl Acad Sci USA 1993;90:20747. [PubMed: 8446631]
Chen et al.
Page 10

Maeda T, Kawane T, Horiuchi N. Statins augment vascular endothelial growth factor expression in
osteoblastic cells via inhibition of protein prenylation. Endocrinology 2003;144:68192. [PubMed:
12538631]
Mao Y, Yang GY, Zhou LF, Stern JD, Betz AL. Focal cerebral ischemia in the mouse: description of a
model and effects of permanent and temporary occlusion. Brain Res Mol Brain Res 1999;63:366
70. [PubMed: 9878831]
Matsuno H, Takei M, Hayashi H, Nakajima K, Ishisaki A, Kozawa O. Simvastatin enhances the
regeneration of endothelial cells via VEGF secretion in injured arteries. J Cardiovasc Pharmacol
2004;43:33340. [PubMed: 15076215]
Nakahashi T, Fujimura H, Altar CA, Li J, Kambayashi J, Tandon NN, Sun B. Vascular endothelial cells
synthesize and secrete brain-derived neurotrophic factor. FEBS Lett 2000;470:1137. [PubMed:
10734218]
Nakashio A, Fujita N, Tsuruo T. Topotecan inhibits VEGF- and bFGF-induced vascular endothelial cell
migration via downregulation of the PI3K-Akt signaling pathway. Int J Cancer 2002;98:3641.
[PubMed: 11857382]
Nedergaard M, Gjedde A, Diemer NH. Hyperglycaemia protects against neuronal injury around
experimental brain infarcts. Neurol Res 1987;9:2414. [PubMed: 2895900]
Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. J Comp Neurol
2000;425:47994. [PubMed: 10975875]
Shingo T, Sorokan ST, Shimazaki T, Weiss S. Erythropoietin regulates the in vitro and in vivo production
of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci 2001;21:973343.
[PubMed: 11739582]
Silverman WF, Krum JM, Mani N, Rosenstein JM. Vascular, glial and neuronal effects of vascular
endothelial growth factor in mesencephalic explant cultures. Neuroscience 1999;90:152941.
[PubMed: 10338318]
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 Neurosci 1999;19:573140. [PubMed: 10407014]
Sun Y, Jin K, Xie L, Childs J, Mao XO, Logvinova A, Greenberg DA. VEGF-induced neuroprotection,
neurogenesis, and angiogenesis after focal cerebral ischemia. J Clin Invest 2003;111:184351.
[PubMed: 12813020]
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method
for measuring brain infarct volume. J Cereb Blood Flow Metab 1990;10:2903. [PubMed: 1689322]
Urbich C, Dernbach E, Zeiher AM, Dimmeler S. Double-edged role of statins in angiogenesis signaling.
Circ Res 2002;90:73744. [PubMed: 11934843]
Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction
properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem
1994;269:2698895. [PubMed: 7929439]
Wang L, Zhang Z, Zhang R, Hafner MS, Wong HK, Jiao Z, Chopp M. Erythropoietin up-regulates SOCS2
in neuronal progenitor cells derived from SVZ of adult rat. Neuroreport 2004;15:12259. [PubMed:
15167538]
Weis M, Heeschen C, Glassford AJ, Cooke JP. Statins have biphasic effects on angiogenesis. Circulation
2002;105:73945. [PubMed: 11839631]
Zhang L, Schallert T, Zhang ZG, Jiang Q, Arniego P, Li Q, Lu M, Chopp M. A test for detecting longterm sensorimotor dysfunction in the mouse after focal cerebral ischemia. J Neurosci Methods 2002a;
117:20714. [PubMed: 12100987]
Zhang R, Wang L, Zhang L, Chen J, Zhu Z, Zhang Z, Chopp M. Nitric oxide enhances angiogenesis via
the synthesis of vascular endothelial growth factor and cGMP after stroke in the rat. Circ Res
2003;92:30813. [PubMed: 12595343]
Zhang ZG, Zhang L, Jiang Q, Chopp M. Bone marrow-derived endothelial progenitor cells participate
in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res 2002b;
90:2848. [PubMed: 11861416]
Chen et al.
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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 (10
mg/kg) starting at 24 hours after stroke daily for 14 days (n=9/group). Functional tests were
performed before MCAO and 1, 7, and 14 days after MCAO. *P<0.05 versus MCAO control.
(A) mNSS test; (B) Foot-fault test; (C) Corner test.
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Figure 3.
Angiogenesis and BDNF expression. Bromodeoxyuridine-reactive endothelial cells were

significantly increased in the atorvastatin-treated group (B) compared with control (A, P<0.05).
Arrows (A and B) show BrdU-positive endothelial cells. Cerebral vascular perimeters (vWF)
were significantly increased in the atorvastatin-treated group (E) compared with the control
group (D, P<0.05). Arrows (D and E) show vWF-positive vessels. (C) and (F) show
quantification data of BrdU and vWF, respectively. (G) to (H) show double immunostaining
BrdU (G, arrow) reactive cells colocalized with vWF (H, arrow) in the vessel. (J) to (K) show
BDNF immunostaining in the ischemic boundary area at 14 days after MCAO treated with
saline (J) and atorvastatin (K), respectively. (L) shows quantitative data of BDNF density in
Chen et al.
Page 14
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) shows
merged from (J) and (K). Brain-derived neurotrophic factor immunoreactive cells were also
present 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 in
the 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, in
control 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) show
quantitative data of DCX (D), TUJ1 (G) and synaptophysin (J) density in the ischemic
boundary area. (C) shows DCX immunoreactive cells around vessel (arrow: DCX-positive
cells; 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 with
control 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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Page 18
Table 1
Modified neurologic severity scores (mNSS)
Motor tests
Points
Raising the mouse by the tail
1 Flexion of forelimb
1 Flexion of hindlimb
1 Head moved more than 10 to the vertical axis within 30 seconds
Walking on the floor (normal=0; maximum=3)
0 Normal walk
1 Inability to walk straight
2 Circling toward the paretic side
3 Falling down to the paretic side
Beam balance tests (normal=0; maximum=6)
0 Balances with steady posture

1 Grasps side of beam
2 Hugs the beam and one limb falls down from the beam
3 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 (<10 seconds)
Reflexes absence
1 Pinna reflex (a head shake when touching the auditory meatus)

1 Corneal reflex (an eye blink when lightly touching the cornea with cotton)
Maximum points
14


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Atorvastatin induction of VEGF and BDNF promotes brain

Department of Physics, Oakland University, Rochester, Michigan, USA

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Materials and methods

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Immunohistochemical stainingTo measure whether atorvastatin treatment promotes

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QuantificationFor semiquantitative measurements of BDNF, TUJ1, DCX and

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Vascular perimeterEnlarged and thin-walled vessels, termed mother vessels, are

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Treatment with atorvastatin significantly increased BrdU-reactive endothelial cell numbers (B

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Effect of Atorvastatin on Cell Migration in SVZ Explant Cultures

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Atorvastatin increases VEGF, VEGFR2 expression as well as promotes endothelial cell

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Quantification field of view in the ischemic border.

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Angiogenesis and BDNF expression. Bromodeoxyuridine-reactive endothelial cells were

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Modified neurologic severity scores (mNSS)

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Raising the mouse by the tail

0 Balances with steady posture

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1 Pinna reflex (a head shake when touching the auditory meatus)

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