Journal of Investigative Surgery, 23, 149–155, 2010

Copyright
C Informa Healthcare USA, Inc.

ISSN: 0894-1939 print / 1521-0553 online
DOI: 10.3109/08941930903469482

ORIGINAL RESEARCH

VEGF Promotes Angiogenesis and Functional

Recovery in Stroke Rats
Ji-Ping Yang
Department of Medical Imaging, The
Second Hospital of Hebei Medical
University, Shijiazhuang, Hebei
Province, China and Depatment of
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Huai-Jun Liu
Department of Medical Imaging, The
Second Hospital of Hebei Medical
University, Shijiazhuang, Hebei
Province, China
Xin-Feng Liu
Depatment of Neurology, Jinling
Hospital, Nanjing University School of
Medicine, Nanjing, Jiangsu Province,
China

Neurology, Jinling Hospital, Nanjing

University School of Medicine, Nanjing,
Jiangsu Province, China

ABSTRACT
We evaluated the effects of intranasal vascular endothelial growth factor VEGF on neurological
For personal use only.

function and angiogenesis in ischemic boundary following cerebral ischemia. Sprague–Dawley rats
were randomized into sham operation group (n = 9), VEGF group (n = 18), and control group
(n = 18). The VEGF and control rats were intranasally administered (IN) with VEGF or saline,
starting three days after middle cerebral artery occlusion (MCAO) and daily. Neurological scores
were obtained at 1, 7, and 14 days after MCAO. Rats were sacrificed at 14 days, the von Willebrand
factor (vWF) immunoreactive, BrdU+ /vWF+ cells, and microvessels were evaluated respectively.
Compared to the control rats, intranasal administration of VEGF improved behavioral recovery,
and increased the number of vWF+ , BrdU+ /vWF+ cells, and FITC-dextran perfused microvessels in
ischemic boundary (p < .01). Our data suggest that intranasal administration of VEGF may induce
angiogenesis in ischemic boundary and improve behavioral recovery following cerebral ischemia
in rats, which may provide a powerful strategy for stroke.

Keywords; intranasal administration, middle cerebral artery occlusion, vascular endothelial growth factor,
functional recovery, angiogenesis, cerebral ischemia

INTRODUCTION der zone start to proliferate as early as 12–24 hr follow-

Studies using either mice or rats with transient or
permanent occlusion of middle cerebral artery (MCA)
demonstrated that endothelial cells in the ischemic bor-
Received June 10, 2009; accepted August 3, 2009.
This study was supported by China Postdortor Science Foundation
(grant no. 20070411051, Dr. Yang). Jiangsu Postdoctor Science Foun-
dation (grant no. 0701003A, Dr.Yang).
Address correspondence to Xin-Feng Liu, MD, Department of Neu-
rology, Jinling Hospital, Nanjing University School of Medicine, 305
East Zhongshan Road, Nanjing, Jiangsu Province, 210002 China.
E-mail: xinfliu@yahoo.com.cn
ing vessel occlusion [1–3]. This in turn already leads to
an increase of vessels in the ischemic border zone three
days following cerebral ischemia. Vascular endothelial
growth factor (VEGF) has been extensively character-
ized as an angiogenic polypeptide growth factor and
is known to modulate numerous endothelial functions
that are mediated by its receptor, KDR (kinase insert
domain receptor) [4]. VEGF also plays an important
role in the nervous system [5–7].
Intranasal (IN) administration provides a method to
bypass the blood-brain barrier (BBB), and directly

149
 J.-P. Yang et al.
deliver therapeutic drugs to the central nervous system
(CNS) [8–10]. Results from our previous studies have
documented that intranasally delivered VEGF can by-
pass the BBB via olfactory- and trigeminal-associated
extracellular pathways to directly entry into the CNS
[11].
The present study was designed to evaluate the effects
of IN VEGF on functional recovery and angiogenesis in
ischemic boundary following cerebral ischemia in rats.
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats (220–250 g) were used
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in all experiments. Rats were housed in cages and the
room was kept at 24 ± 1◦ C temperature and 50–60%
humidity, under a 12:12-hr light-dark cycle and with
access to food and water ad libitum. All procedures
were performed under the guideline approved by the
National Institutes of Health Guide for the Care and
Use of Laboratory Animals (NIH Publications No. 80-
23, revised 1996). Every effort was made to minimize
pain and discomfort.
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Middle Cerebral Artery Occlusion (MCAO)
Model
Rats were anesthetized by 10% chloral hydrate
(350 mg/kg, intraperitoneally). The right MCA was
occluded by means of a monofilament suture as de-
scribed previously [12]. Briefly, under aseptic condi-
tions, the wound was anesthetized with Lidocaine 2%
(0.1 ml) and a 4–0 surgical monofilament nylon suture
with rounded tip was introduced into the right inter-
nal carotid artery through the external carotid artery
stump, advanced 17.5–18.5 mm past the carotid artery
bifurcation to occlude the origin of the right MCA. Af-
ter 90 min of occlusion, the suture was withdrawn to
restore blood flow. Rectal temperature was monitored
and maintained at 37◦ C with heating pads throughout
the surgical procedures. Since the procedure was so
short, no analgesics or antibiotics were used.
Experimental Groups and Intranasal
Administration
All rats were randomized into three groups: sham op-
erated group (n = 9), VEGF-treated group (n = 18),
and saline-treated (Control) group (n = 18). VEGF and
control rats were intranasally administered recombi-
nant human VEGF 165 (rhVEGF165, molecular weight
38.2 kDa, PeproTech Corporation, USA) or saline. In-
tranasal administration was performed as described
150
previously [11]. Three days after MCAO rats were
anesthetized with 10% chloral hydrate (350 mg/kg, in-
traperitoneally) and laid on their backs, with their neck
elevated by rolled-up 4 cm × 4 cm gauze, and body tem-
perature was maintained with a heating pad and rectal
thermal probe set at 37◦ C. Under the best aseptic con-
ditions, a total of 100 µl VEGF (200 µg/ml) per rat was
given in olfactory pathway [13], 10 µl at a time, alternat-
ing the nostrils, with an interval of 2 min between each
administration, over a total of 18.5 min. During these
procedures, the mouth and the opposite nostril were
shut. This was repeated once daily until one day before
sacrifice. The same volume of saline served as the treat-
ment for saline-treated group. Animals subjected to
the same surgery without vascular occlusion served as
sham-operated group. All rats were over-anesthetized
with 10% chloral hydrate (400 mg/kg, IP) and sacri-
ficed by decapitation on the 14th day after MCAO.
BrdU Administration
For identification of cell proliferation, BrdU (Sigma,
USA) was injected intraperitoneally at a dosage of
50 mg/kg body weight starting at 24 hr after MCAO
once daily until the day of sacrifice.
Neurological Function
The modified Neurological Severity Scores (mNSS) test
(normal score is 0; maximal deficit score 18) was per-
formed 1, 7, and 14 days after MCAO by an investigator
who was blinded to the experimental groups as previ-
ously described [14].
Infarct Volume Measurement
For quantitative infarct volume, rats (Sham group,
n = 3, VEGF group, n = 6, and Control group, n =
6) were perfused transcardially with saline followed
by 4% paraformaldehyde under deep anesthesia at
14 days after MCAO, and brains were removed. Brains
were postfixed in 4% paraformaldehyde for six hr and
cryoprotected in 30% sucrose solution overnight. In all
20 µm coronal sections were obtained on a cryostat
and stored at −80◦ C. Hematoxylin and eosin-stained
specimens were used to measure infarct volume from
seven coronal sections spaced 2 mm apart per rat, as de-
scribed previously [15]. Infarct volume was presented
as a volume percentage of the infarct compared with
the contralateral hemisphere [15].
Immunohistochemical Assessment
To determine von Willebrand factor (vWF) expression,
rats were fixed in 4% paraformaldehyde then washed
 VEGF Promotes Functional Recovery

in phosphate buffered saline (PBS). The coronal paraf-

fin sections were incubated in 3% H2 O2 in PBS for
10 min, in blocking solution (10% goat serum in PBS)
for 30 min at room temperature, and with rabbit
polyclonal anti-vWF (1:75, Santa Cruz Biotechnology
Inc. Santa Cruz, California, USA) at 4◦ C overnight. Sec-
tions were washed and incubated. The reaction was
stopped with diaminobenzidine (DAB). The positive
cells of vWF in nonreplicated fields were measured
with the Meta Morph micro image analysis system un-
der 400-fold light microscope.
For double-label immunocytochemistry, sections were
fixed with 4% paraformadehyde in PBS, and incubated
in 2 mol/l HCl at 37◦ C for 30 min. Sections were incu-
bated in blocking solution with primary antibodies at
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4◦ C overnight and with secondary antibodies at room Figure 1. Quantitative effect of IN VEGF on neurological
temperature for two hr. Primary antibodies were as function after MCAO (mean ±SD). ∗ vs. Control group,
follows: mouse monoclonal anti-BrdU antibody (1:800, p < .01.
Sigma) and rabbit polyclonal anti-vWF antibody (1:200,
Santa Cruz Biotechnology Inc. Santa Cruz, Califor-
nia, USA). The secondary antibodies were fluorescein scores were analyzed by two-way repeated ANOVA.
isothiocyanate (FITC)-conjugated goat antimouse IgG Results were considered significant at p < .05.
(1:100, Jackson Immunoresearch) and tetramethylrho-
damine isothiocyanate (TRITC)-conjugated goat an-
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tirabbit IgG (1:100, Jackson Immunoresearch). BrdU RESULTS

and vWF double labeled cells were detected using a
confocal laser scanning microscope (Leica TCS SP2,
Germany). Results were presented as number of dou-
ble labeled cells.
Vascular Density Detection
At 14 days after MCAO, rats (Sham group, n = 3, VEGF
group, n = 6, and Control group, n = 6) were subjected
to FITC-dextran (2 × 106 molecular weight, Sigma;
1 ml of 50 mg/ml preparation) administration via the
tail vein five min before sacrifice under deep anesthe-
sia with chloral hydrate (350 mg/kg, IP). The brains
were rapidly removed and placed in 4% paraformalde-
hyde at 4◦ C for 48 hr. The green fluorochromes (FITC-
dextran) on the sections were excited by a laser beam at
488 nm. Five 50-µm coronal sections at 2 mm intervals
from each rat and eight views in each section adjacent
to the ischemic lesion were selected to observe by laser-
scanning microscope. The number of microvessels was
measured by Image-Pro Plus Version 6.0 software [16,
17].
Statistical Analysis
Data were presented as mean ± standard deviation
(SD). Histological outcome measures were compared
using one-way analysis of variance (ANOVA) followed
by Tukey’s test for multiple comparisons. Neurological
Neurologic Outcome
No neurological impairments were observed in the
sham-operated group. There was no significant differ-
ence in behavioral recovery between VEGF-treated and
saline-treated group at day 1 after MCAO (p > .05).
Compared to IN saline, VEGF significantly improved
behavioral recovery at days 7 and 14 after MCAO (p <
.01, Figure 1).
Infarct Volume
No infarct volume was observed in the sham-operated
group. Compared to IN saline, VEGF significantly re-
duced infarct volume at day 14 after MCAO (p < .01,
Figure 2).
Angiogenesis in the Ischemic Boundary
Cerebral blood vessels were immunostained with the
antibody against vWF, an endothelial cell marker.
Number of cerebral vessels increased in the ischemic
boundary regions in rats treated with VEGF, compared
to the sham-operated and saline-treated rats (p < .01,
Figure 3).
Three-dimensional measurements of FITC-dextran
perfused cerebral microvessels revealed that VEGF sig-
nificantly increased the number of microvessels in the
boundary regions of ischemia compared to stroke rats
151
 J.-P. Yang et al.

DISCUSSION
Angiogenesis, the growth of new blood vessels, could
be interpreted as a natural defense mechanism help-
ing to restore oxygen and nutrient supply to the is-
chemic brain tissue [18]. Besides, angiogenic vessels
provide neurotrophic support to newly generated neu-
rons [19], and neuroblasts have been found to be con-
centrated around blood vessels following stroke [20].
In the meantime, greater microvessel density in the is-
chemic border correlates with longer survival in stroke
patients [21].
VEGF is the most important mitogen in the process of
angiogenesis. The binding of VEGF to its specific re-
ceptors VEGFR-1(flt-1) and VEGFR-2(flk-1) on the sur-
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face of endothelial cells activates intracellular tyrosine

Figure 2. Quantitative effect of IN VEGF on infarct volume
after MCAO (mean ±SD). ∗ vs. Control group, p < .01.
with saline treatment and sham-operated rats (p< .01,
Figure 4).
Only few BrdU colocalized with vWF in the sham-
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kinases, triggering multiple downstream signals that
promote angiogenesis [18]. Previous reports showed
an upregulation of VEGF in the penumbra following
experimental cerebral ischemia [3, 22, 23]. An increase
in VEGF expression in the infarcted hemisphere was
confirmed as early as three hr after an ischemic injury
and continued up to three [3] or seven days [22, 23].
The expression level of VEGFR-1 increased in the in-

operated rats. As expected, compared to the sham-
operated and saline-treated rats, a greater number of
BrdU+ /vWF+ immunoreactive cells were detected in
the VEGF-treated rats at day 14 after MCAO (p < .01,
Figure 5).
farct core from the 3rd day up to the 14th day [24] and
in the ischemic border zone at 48 hr following cere-
bral ischemia [3, 23]. VEGFR-2 mediates microvascular
permeability, endothelial cell proliferation, invasion,
migration, and survival [25]. Several reports described

Figure 3. VEGF enhanced the number of microvascular. Cerebral blood vessels immunostained with the antibody against vWF.
Number of cerebral vessels increased in the ischemic boundary regions in rats treated with VEGF (c), compared to those of the
sham-operated group (a), and rats in the saline-treated (Control) group (b). Bar = 40 µm. D, Quantitative data of vWF+ cells
(mean ±SD). ∗ vs. Control group, p < .01. # vs. Sham-operated group, p < .01.
152
 VEGF Promotes Functional Recovery
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Figure 4. FITC-dextran perfused vessels in the ischemic boundary regions of VEGF-treated (c), saline-treated (Control) group
(b), and sham-operated rats (a). VEGF significantly increased the number of microvessels in the boundary regions of ischemia
compared to saline-treated and sham-operated rats. Bar = 20 µm. D, Quantitative data of FITC-dextran perfused microvessels
(mean ±SD). ∗ vs. Control group, p < .01. #vs. Sham-operated group, p < .01.

Figure 5. IN VEGF enhanced the angiogenesis in the boundary regions of ischemia. Part a through c shows colocalization of
BrdU and vWF in the ischemic boundary of rats treated with VEGF. Bar = 40 µm. D, Quantitative data of BrdU+ /vWF+
immunoreactive cells (mean ±SD). ∗ vs. Control group, p < .01. #vs. Sham-operated group, p < .01.
153
 J.-P. Yang et al.
an upregulation of VEGFR-2 following experimental
cerebral ischemia [3, 22, 26].
In hypoxic and ischemic brain, the temporal-spatial ex-
pression of VEGF has been shown to correlate with vas-
culogenesis [27, 28]. VEGF expression in the ischemic
border zone increased progressively between 2 and
14 days after MCAO, and evidence of new vessel forma-
tion was present during days 7 to 28 [28]. Intracerebral
infusion of VEGF stimulates angiogenesis detectable by
laminin immunostaining in rat cerebral cortex 3–7 days
later [29, 30]. In the ischemic rat brain, intravenously
administered VEGF also triggers angiogenesis, as man-
ifested by an increase in the number and volume of
FITC-dextran-perfused cerebral cortical microvessels
7 days later [31]. Sun et al. [32] also found an increase
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in the density of vWF-immunoreactive blood vessels
7–28 days after focal ischemia plus VEGF treatment in
the striatal ischemic penumbra.
Increases in expression of VEGF/VEGF receptors at the
penumbra region after ischemia plus VEGF treatment
are coincident with the time and distribution of neovas-
cularization in ischemic brain, suggesting that VEGF is
temporally and spatially correlated with angiogenesis
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[28]. Therefore, exogenous VEGF may promote thera-
peutic angiogenesis in the ischemic rats, and restoration
of cerebral microvascular circulation is important in the
ischemic brain for functional recovery after stroke.
In our study, an increase of microvessels in the
ischemic border zone was confirmed by detecting
more FITC-dextran-perfused microvessels, vWF+ cells,
and BrdU+ / vWF+ cells in the rats treated with IN
rhVEGF165 when compared to IN saline rats. Thus,
these data indicated that IN rhVEGF165 significantly
enhanced angiogenesis in ischemic brain.
In the literature on stroke, much attention has been
focused on the penumbra [33]. Excitotoxic damage, in-
flammation, and ultimately cell death in the penum-
bra are possible targets for therapy [34]. Reports from
stroke models and patients document the proliferation
of vascular cells and expression of markers for angio-
genesis surrounding infarcts [21, 35–37]. However, the
relationship between angiogenesis and functional re-
covery in the brain after focal ischemia has not been
characterized. Our data demonstrated that late admin-
istration of IN rhVEGF165 to ischemic rats significantly
improved neurological function and reduced infarct
size. Therefore, treatment with VEGF to potentiate new
vessel formation may be an important therapy for be-
havioral recovery during the repair process after stroke.
The effect of VEGF depends on the dose, route, and time
of administration in relation to brain ischemia. When
VEGF was delivered in the acute stage after stroke, the
BBB was damaged and this damage may have compro-
154
mised the beneficial neuroprotective actions of VEGF
[38]. However, late administration of VEGF, i.e., on
days 1–3 of reperfusion improved neurological out-
come [31, 32]. In our study, VEGF was intranasally de-
livered on days 3–13 of ischemia which could enhance
angiogenesis and improve functional recovery.
CONCLUSION
In summary, IN VEGF can improve angiogenesis in the
ischemic border zone and functional recovery follow-
ing cerebral ischemia in rats. IN VEGF may provide a
powerful strategy for stroke.
DECLARATION OF INTEREST
The authors report no conflict of interest. The authors
alone are responsible for the content and writing of this
paper.
REFERENCES
1. Beck H, Acker T, Wiessner C, et al. Expression of angiopoietin-
1, angiopoietin-2, and tie receptors after middle cerebral artery
occlusion in the rat. Am J Pathol. 2000;157:1473–1483.
2. Hayashi T, Noshita N, Sugawara T, et al. Temporal profile of
angiogenesis and expression of related genes in the brain after
ischemia. J Cereb Blood Flow Metab. 2003;23:166–180.
3. Marti HJ, Bernaudin M, Bellail A, et al. Hypoxia-induced vas-
cular endothelial growth factor expression precedes neovascu-
larization after cerebral ischemia. Am J Pathol. 2000;156:965–976.
4. Petrova TV, Makinen T, Alitalo K. Signaling via vascular en-
dothelial growth factor receptors. Exp Cell Res. 1999;253:117–130.
5. Raab S, Plate KH. Different networks, common growth factors:
shared growth factors and receptors of the vascular and the
nervous system. Acta Neuropathol. 2007;113:607–626.
6. Yang JP, Liu XF, Liu HJ, et al. Extracellular signal-regulated
kinase involved in NGF/VEGF-induced neuroprotective effect.
Neurosci Lett. 2008;434:212–217.
7. Yang JP, Liu HJ, Li Y. Effect of endoplasmic reticulum stress in
VEGF-induced neuroprotection. J Invest Surg. 2009;22:29–34.
8. Thorne RG, Pronk GJ, Padmanabhan V, et al. Delivery of insulin-
like growth factor-I to the rat brain and spinal cord along olfac-
tory and trigeminal pathways following intranasal administra-
tion. Neuroscience. 2004;127:481–496.
9. Liu XF, Fawcett JR, Thorne RG, et al. Intranasal administration
of insulin-like growth factor-I bypasses the blood-brain barrier
and protects against focal cerebral ischemic damage. J Neurol
Sci. 2001;187:91–97.
10. Thorne RG, Frey WH. 2nd. Delivery of neurotrophic factors
to the central nervous system: pharmacokinetic considerations.
Clin Pharmacokinet. 2001;40:907–946.
11. Yang JP, Liu HJ, Cheng SM, et al. Direct transport of VEGF from
the nasal cavity to brain. Neurosci Lett. 2009;449:108–111
12. Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle
cerebral artery occlusion without craniectomy in rats. Stroke.
1989;20:84–91.
13. Yang JP, Liu HJ, Wang ZL, et al. The dose-effectiveness of in-
tranasal VEGF in treatment of experimental stroke. Neurosci Lett.
2009;461:212–216.

14. Chen J, Sanberg PR, Li Y, et al. Intravenous administration of
human umbilical cord blood reduces behavioral deficits after
stroke in rats. Stroke. 2001;32:2682–2688.
15. Swanson RA, Morton MT, Tsao-Wu G, et al. A semiautomated
method for measuring brain infarct volume. J Cereb Blood Flow
Metab. 1990;10:290–293.
16. Barros LF, Belfort R Jr. The effects of the subconjunctival injec-
tion of bevacizumab (Avastin) on angiogenesis in the rat cornea.
An Acad Bras Cienc. 2007;79:389–394.
17. Blatt RJ, Clark AN, Courtney J, et al. Automated quantitative
analysis of angiogenesis in the rat aorta model using Image-Pro
Plus 4.1. Comput Methods Programs Biomed. 2004;75:75–79.
18. Beck H, Plate KH. Angiogenesis after cerebral ischemia. Acta
Neuropathol. 2009;117:481–496.
19. Leventhal C, Rafii S, Rafii D, et al. Endothelial trophic support
of neuronal production and recruitment from the adult mam-
malian subependyma. Mol Cell Neurosci. 1999;13:450–464.
20. Yamashita T, Ninomiya M, Hernández Acosta P, et al. Subven-
J Invest Surg Downloaded from informahealthcare.com by Mcgill University on 11/17/14
tricular zone-derived neuroblasts migrate and differentiate into
mature neurons in the post-stroke adult striatum. J Neurosci.
2006;26:6627–6636.
21. Krupinski J, Kaluza J, Kumar P, et al. Role of angiogenesis in pa-
tients with cerebral ischemic stroke. Stroke. 1994;25:1794–1798.
22. Hayashi T, Abe K, Suzuki H, et al. Rapid induction of vascular
endothelial growth factor gene expression after transient middle
cerebral artery occlusion in rats. Stroke. 1997;28:2039–2044.
23. Plate KH, Beck H, Danner S, et al. Cell type specific up-
regulation of vascular endothelial growth factor in an MCA-
occlusion model of cerebral infarct. J Neuropathol Exp Neurol.
For personal use only.
1999;58:654–666.
24. Kovács Z, Ikezaki K, Samoto K, et al. VEGF and flt. Expression
time kinetics in rat brain infarct. Stroke. 1996;27:1865–1872.
25. Hicklin DJ, Ellis LM. Role of the vascular endothelial growth
factor pathway in tumor growth and angiogenesis. J Clin Oncol.
2005;23:1011–1027.
26. Lennmyr F, Ata KA, Funa K, et al. Expression of vascular en-
dothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1)
following permanent and transient occlusion of the middle cere-
bral artery in the rat. J Neuropathol Exp Neurol. 1998;57:874–882.
VEGF Promotes Functional Recovery
27. Ogunshola OO, Stewart WB, Mihalcik V, et al. Neuronal
VEGF expression correlates with angiogenesis in postnatal
developing rat brain. Brain Res Dev Brain Res. 2000;119:139–
153.
28. Zhang ZG, Zhang L, Tsang W, et al. Correlation of VEGF and
angiopoietin expression with disruption of blood-brain barrier
and angiogenesis after focal cerebral ischemia. J Cereb Blood Flow
Metab. 2002;22:379–392.
29. Rosenstein JM, Mani N, Silverman WF, et al. Patterns of
brain angiogenesis after vascular endothelial growth factor ad-
ministration in vitro and in vivo. Proc Nat Acad Sci U S A.
1998;95:7086–7091.
30. Krum JM, Mani N, Rosenstein JM. Angiogenic and astroglial
responses to vascular endothelial growth factor administration
in adult rat brain. Neuroscience. 2002;110:589–604.
31. Zhang ZG, Zhang L, Jiang Q, et al. VEGF enhances angiogenesis
and promotes blood–brain barrier leakage in the ischemic brain.
J Clin Invest. 2000;106:829–838.
32. Sun Y, Jin K, Xie L, et al. VEGF-induced neuroprotection, neu-
rogenesis, and angiogenesis after focal cerebral ischemia. J Clin
Invest. 2003;111:1843–1851.
33. Astrup J, Siesjo BK, Symon L. Thresholds in cerebral ischemia:
the ischemic penumbra. Stroke. 1981;12:723–725.
34. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of
ischaemic stroke: an integrated view. Trends Neurosci.
1999;22:391–397.
35. Chen HH, Chien CH, Liu M. Correlation between angiogenesis
and basic fibroblast growth factor expression in experimental
brain infarct. Stroke. 1994;25:1651–1657.
36. Krupinski J, Kumar P, Kumar S, et al. Increased expression
of TGF-beta 1 in brain tissue after ischemic stroke in humans.
Stroke. 1996;27:852–857.
37. Okada Y, Copeland BR, Hamann GF, et al. Integrin alphav-
beta3 is expressed in selected microvessels after focal cerebral
ischemia. Am J Pathol. 1996;149:37–44.
38. van Bruggen N, Thibodeaux H, Palmer JT, et al. VEGF an-
tagonism reduces edema formation and tissue damage after
ischemia/reperfusion injury in the mouse brain. J Clin Invest.
1999;104:1613–1620.
155