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Catalpol Increases Brain Angiogenesis and Up-Regulates VEGF and EPO in the Rat after Permanent Middle Cerebral Artery Occlusion

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To investigate the role and mechanism of catalpol in brain angiogenesis in a rat model of stroke, the effect of catalpol (5 mg/kg; i.p) or vehicle administered 24 hours after permanent middle cerebral artery occlusion (pMCAO) on behavior, angiogenesis, ultra-structural integrity of brain capillary endothelial cells, and expression of EPO and VEGF were assessed. Repeated treatments with Catalpol reduced neurological deficits and significantly improved angiogenesis, while significantly increasing brain levels of EPO and VEGF without worsening BBB edema. These results suggested that catalpol might contribute to infarcted-brain angiogenesis and ameliorate the edema of brain capillary endothelial cells (BCECs) by upregulating VEGF and EPO coordinately.
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Int. J. Biol. Sci. 2010, 6
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2010; 6(5):443-453
© Ivyspring International Publisher. All rights reserved
Research Paper
Catalpol Increases Brain Angiogenesis and Up-Regulates VEGF and EPO in
the Rat after Permanent Middle Cerebral Artery Occlusion
Hui-Feng Zhu
1
, Dong Wan
2
, Yong Luo
3
, Jia-Li Zhou
4
, Li Chen
5
, Xiao-Yu Xu
1
1. School of Pharmaceutical Sciences & School of Chinese Medicine, Southwest University, Chongqing 400715, China;
2. Department of Emergency Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016,
China;
3. Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;
4. Chongqing Medical And Pharmaceutical College;
5. Southwest University 2nd Hospital, Chongqing 400715, China
Corresponding author: Professor Xiao-Yu Xu, Laboratory of Molecular Pharmacology, School of Pharmaceutical Science
& School of Chinese Medicine, Southwest University, Chongqing 400715, China. Tel: +8 6 -23-6825-0761; Fax:
+86-23-6825-1225; E-mail: zhfbsci@yahoo.cn; xxy0618 @ s i n a .com
Received: 2010.05.24; Accepted: 2010.08.01; Publish ed: 2010.08.20
Abstract
To investigate the role and mechanism of catalpol in brain angiogenesis in a rat model of
stroke , th e ef fect of catal pol (5 mg/kg; i.p) or veh icle administered 24 hours aft er p erma nent
middle cerebral artery occlusion (pMCAO) on behavior, angiogenesis, ultra-structural inte-
grity of brain capillary endothelial cells, and expression of EPO and VEGF were assessed.
Repeated treatments with Catalpol reduced neurological deficits and significantly improved
angiogenesis, while significantly increasing brain levels of EPO and VEGF without worsening
BBB edema. These results suggested that catalpol might contribute to infarcted-brain angi-
ogenesis and ameliorate the edema of brain capillary endothelial cells (BCECs) by upregulating
VEGF and EPO coordinately.
Key words: Catalpol, VEGF, EPO, Permanent occlusion of middle cerebral artery, Angiogenesis
1. Introduction
Stroke has been emerging as one of the most
c o m m o n c a u s e s o f m o r t a l i t y a n d m o r b i d i t y i n m o d e r n
society. A l t h o u g h m u c h p r o g r e s s h a s b e e n m a d e t o-
ward understanding the mechanistic basis of stroke,
the effectiveness of dru gs availa ble for stroke patients
is limited. Tissue plasminogen a ct i v a t o r ( T P A) , w h i c h
di ss olves bl ood c lots in the b rain, is pre se ntly th e on ly
approved treatment for stroke; however, it is effective
only in the first 3 h after the stroke and may lead to
cerebral hemorrhage [1]. Many drugs focus on the
isc he mic pe numb ra and c asc ad e of d ama ge , in cl uding
anti-N-meth y l -D-aspartate receptor (Aptiganel and
gavestinel), potassium channel agonists (MaxiPost),
and GABA modulators (Zendra). However, the uses
of these drugs for stroke have been abandoned be-
cause they are not effective and even harmful to
stroke patients, despite their apparent effectiveness in
animal models of brain ischemia [2]. Therefore, new
drugs a r e in demand an d need to be developed t o
treat stroke.
The neurovascular unit concept emphasizes not
only the neuron but also the brain vascular structure
[3-4]. Previous research on stroke has largely focused
on neuroprotection, but neglected the ischemic vas-
cular structure and the possible benefits of its func-
tional reconstruction [1,4,5]. Brain vascular structures
are coupled with brain neurons in structure and
function [6]. Angiogenesis is associated with neuro-
genesis [7]. Mounting evidence has shown that vas-
cular-remodeling occurs after st ro k e [ 8 -9]. It has been
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shown that higher blood vessel counts correlate with
longer survival in stroke patients [ 10 ] . T h e
three-dimensional images of angiogenesis in the tis-
sue surrounding focal brain infarcts are demonstrated
by scanning electron microscopy with corrosion cast-
ing [11]. These studies suggest that targeting brain
vascular-remodeling for drug discovery is necessary
for stroke and even for ischemia disease.
Rehmannia Root is the main natural herbal
medicine that plays an important role in treat in g
stroke. Recently, catalpol, a main active component of
Rehmannia Root, was determined to pose extensive
ischemic neural protection, such as preventing the
loss of hippocampal CA1 neurons and reducing
working errors [12], modulating the expressions of
Bcl-2 and Bax [13], attenuating apoptosis in the
ischemic brain [13], and increasing hippocampal
neuroplasticity by up-regulating PKC and BDNF in
aged rats [14]. Catalpol improves Y-maze perfor-
mance and the survival of neurons in the CA1 sub-
field after transient global ischemia in gerbils [15-16] .
Our previous studies have demonstrated that catalpol
at doses of 10 and 5 mg/kg can improve neurobeha-
vioral outcome following permanent focal cerebral
ischemia in Sprague Dawley rats, and upregulate the
expression of growth-associated protein 43 (GAP-43)
[17]. These findings suggest that catalpol contributes
to neuroplasticity after stoke. However, whether cat-
alpol can modulate brain angiogenesis after focal
ischemia is unclear.
Erythropoietin (EPO) and Vascular en dothel ial
g r o w t h f a c t o r ( V E G F ) h a v e p l e i o t r o p i c e f f e c t s o n b r a i n
function, including neuroprotection, and promotion
of angiogenesis and neurogenesis [18]. Notably, EPO
enhances angiogenesis, without aggravating brain
edema, e ven u se d wi th VEG F [19].
In this study, we have investigated the effect of
catalpol on angiogenesis following permanent middle
cerebral artery occlusion (pMCAO) in rats. In order to
explore the cellular and molecular mechanism by
which catalpol may regulate the vascular plasticity of
the brain, we examined the expression of VEGF and
EPO by immunohistochemistry and western blotting.
2. Materials and Methods
2.1 Animals and diets
Healthy male Sprague-Dawley (SD) rats (220
280 g) were obtained from the Experimental Animal
Center, Chongqing University of Medicine, China.
Animals were housed under conditions of natural
illumination with food and water available ad libi-
tum. These experiments were performed in accor-
dance with Chinas guidelines for care and use of la-
boratory animals. An i m a l s w e r e d i v i d e d i n t o 3 g r o u p s
r a n d o m l y ( 1 ) t h e s h a m o p e r a t e d g r o u p ( n = 2 4 ) ; ( 2 ) t h e
vehicle group (n = 24); (3) the catalpol-treated group
(n = 24).
2.2 The pMCAO model
Strokes were induced by electrocoagulation of
the right middle cerebral artery as d es c ri b ed p r e-
viously with minor modifications [20]. B rie fly, rats
were anesthetized and placed in a stereotaxic instru-
ment (Shanghai Jiangwan) in the prone position. The
scalp was opened and brain was exposed, held up
lightly with a glass retractor, inferior cerebral vein
and olfactory bundle were seen perspicuously and the
right middle cerebral artery was along the brain sur-
face verticality striding over inferior cerebral vein and
olfactory bundle. The middle cerebral artery ventral
to the olfactory tract was electrocoagulated (power
35W), resulting in infarction of the right dorsolateral
cerebral cortex. Rats were prescreened to select those
in line with the criteria described as Bederson [21].
2.3 Drug administration
Catalpol was dissolved in physiological saline,
which was purchased from National Institute for the
Control of Pharmaceutical and Biological Products
(China) and its purity was more than 98%. Catalpol
(5mg/kg, ip) were administered 24h after stroke and
then daily for 7 days. Likewise, the sham-operation
group and the vehicle group received equal volumes
of physiological saline by ip injection. The dose of
catalpol was based on our previous study [17] and
Li’s study [15-16].
2.4 Bedersons Score
After operation, the neurological function of all
animals was evaluated daily with a 4-point scale as
previously described [21]: (0) no apparent deficit, (1)
contr ala teral f ore limb flexion , (2) lowe red r esista nce
to lateral push without circling, and (3) circling to
ipsilateral stroke if spontaneous activity.
2.5 B e a m -Walking test
Beam walking test [22-23] was used to evaluate
sensorimotor reflexes, motor strength and coordina-
tion. The testing apparatus was a 2.5 cm in diameter
and 80 cm in length wooden beam elevated 100 cm
a b o v e t h e f l o o r w i t h w o o d e n supports as described by
Stanley et al, and a 5cm thickness foam pillow placed
under the beam avoid getting wound in a fall.
Rats were allowed to walk to a platform located
at the end of the beam and their behaviors were rec-
orded based on the following six cri t e ri a : (0) the rat
traverses the beam without falling down; (1) the rat
traverses the beam but footslips less than 50%; (2) the
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rat crosses the beam but footslips more than 50%; (3)
t h e r a t c r o s s e s t h e b e a m w i t h o u t t h e a i d o f t h e a f f e c t e d
hindlimb; (4) the rat cant traverses the beam but can
sit on the horizontal surface of the beam; (5) the rat
will fall down when placed on the beam. Each trial
consisted of five repetitions of this assay.
2.6 Examination of the healing Ischemic brain
cortex and the surface vessels
Stroke model rats in each group were sacrificed
15 days after the operation. After removing the sur-
face meninges, the surface vessels, distribution pat-
terns, and the healing state of ischemic cortex were
examined using a microscope attached to a digital
camera as described previously [24].
2.7 T is sue p re pa ra tio n
Fifteen days after operation/stroke induction,
the rats were deeply anesthetized with an overdose of
Chloral Hydrate (35 mg/mL, i.p), and transcardially
perfused with 0.9 % NaCl solution to rinse out the
blood, followed by 250 mL of 4% formalin (4°C) to fix
the brain tissue. After extraction from the skull, the
b ra i ns we re p os t -fixed in 4% formalin solution and
subsequently cut into 30 µm coronal sections on a
cryostat (Leica). For EPO analyses, imbedded brain
tissue was used. Ipsilateral ischemic cortex (0.1 g per
brain) in each group was also weighed for western
blotting a n aly ses .
2.8 Immunohistochemistry
Animal br a in tissues were transcardially per-
fuse d and fixed with 4% paraformaldehyde, cryopre-
served in 30% sucrose, and cut into three series of
consecutive sections (30 μm) at a Cryostat. Each set of
tissue sections was immunostained for PCNA, V W F
or VEGF. For EPO analyses, imbedded brain tissue
was used to cut as 5μm se ct ions. For immunohisto-
chemistry, tissue sections were incubated with rabbit
polyclonal antibody against EPO (1:200, santa cruz,
USA), VEGF(1:200, Wuhan Booster Biotech. Co.,
China). For double-fluo rescen ce labeli ng, cr oss se c-
tions were incubated with the vessel m a r k e r a n t i b o d y ,
rabbit polyclonal against VWF (1:200, Zhongshan
Biotech. Co., China), together with the mouse mo-
noclonal antibody against the proliferation marker
PCNA (1:200, Wuhan Booster Biotech. Co., China).
Immunohistochemistry for EPO was done with b i o-
tinylated goat anti-rabbit IgG (1:500; Vector Labora-
t or i es ) a nd p e ro xi da s e-conjugated avidin-biot in co m-
plex (ABC kit; Wuhan Booster Biotech. Co., China),
bound antibodies were visualized by addition of di-
aminobenzidine. V E G F s ec t i o ns then incubated with
Cy3-labeled goat anti-rabbit IgG (Wuhan Boster Bio-
tech. Co., China). PC NA a nd VWF then incubated
with Cy3-labeled goat anti-rabbit IgG (Wuhan Boster
Biotech. Co., China) a n d F I T C -labe led goat an -
ti-m o u s e I g G . After through washing, immunostained
ce lls w ere observed under a Nikon microscope and
were documented with a Nikon digital camera. Im-
munofluorescence staining for PCNA (green) and
VWF (red) or VEGF (red) was visualized and docu-
m e n t e d w i t h a c o n f o c a l m i c r o s c o p e (Le i c a) . According
to Acker [25] (Acker et al., 2001), the number of
double-stained vessels and the intensity of staining
were analyzed with Image Pro Plus Version 6.0.
software for cerebral microvessels at the boundary
zone of ischemia. Five fields of each slice were ran -
domly selected for blinded scoring and analyses. E a c h
experiment was performed three times.
2.9 Electron microscopy observation of brain
vascular endothelial cells
Rats in each group were anesthetized with 3.5%
chloral hydrate (35 mg/mL, i.p) and perfused for 2-3
min through the ascending aorta with 0.9% normal
saline, followed by ice-cold fixative (4% paraformal-
dehyde in 0.1 M PBS for 30 min). The brains were
removed for immunocytochemical electron micro-
scopic studies and fixed in 4% paraformaldehyde in
0 . 1 M P B S p H 7 . 4 f o r 3 4 h a t 4 ° C . T h e b r a i n w a s t h e n
rinsed in PBS for 30 min, treated with 1% OsO
4
for 30
min, dehydrated in sequential ethanol gradients, and
embedded in Epon 618. Ultrathin sections were
processed according to the postembedding proce-
dure. Briefly, ultramicrocut was made by Leica ul-
tramicrotome to collect 60nm-thick sections. The sec -
tions were mounted on formvar-c o at e d c o pp e r g r id s ,
incubated in uranyl acetate- acetate lead double elec -
t r o n s t a i n , a n d o b s e r v e d f o r b r a i n v a s c u l a r e n d o t h e l i a l
ce lls.
2.10 Western blotting
According to K.N. Na m [26] and Cao Hu ang
[27], brain cortex in peri-ischemic were lysed on ice in
lysi s b uf fer [50 m m T r i s -HC l (pH 8.2), 0.5 M saccha-
rose, 10 mMHEPES (pH 7.9), 1.5 mM MgCl2, 10 mM
KCl, 1 mM EDTA, 10% (v/v) glycerine, 1 mM DTT, 1
mM PMSF, 10μg/mL Aprotinin, and g/mL Leu-
peptin]. After centrifugation at 16,000 ×g for 10 mi-
nutes. Protein content in cleared lysate was deter-
mined by Bradford Assay. Lysate samples containing
40μg of p r o te i n were fractionated by SDS-1 0 % p o-
lyacrylamide gel electrophoresis and then electrob-
lotted onto P CV F membranes. The membranes were
probed with primary antibodies as EPO, VEGF (1:250,
1:300, Satcruze Co., USA) , an d β-a c t i n (1:200, Booster
Biotech. Co., Wuhan, China), then incubated with the
hors e ra d ish peroxidase-conjugated goat anti-mous e
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or anti-rabbit IgG (1:2500; Booster Biotech. Co., Wu-
han, China), the PVDF membrane was put into DAB
fluid f o r coloration. Immunoreactivity was di gitall y
scanned b y ScanMaker E6 system a n d quantified us-
ing Q uanti ty one 4. 5.1 ( Bi o-Rad) software. β-ac t i n w a s
used as an internal control for all Western blotting.
2.11 Image and data analysis
After capturing images with a digital camera,
quantification of the results from immunohistoche-
mistry, immunofluorescence, western blotting was
performed with Image Pro Plus Version 6.0. software.
EPO or VEGF-positive cells were counted at five dif-
ferent fields in the inner border of the peri-ischemic
cortex in five sections per rat, the to ta l nu mb er o f EP O
o r V E G F -positive cells per image (cells/cm
2
, o b j e c t i v e
× 20) was calculated by an observer blind to the ex-
perimental treatment. In each section, five pe -
ri-ischemia cortical areas outside labeled neurons
were chosen randomly to obtain an average value for
the subtraction of background by an observer blind to
the experimental treatment.
2.12 Statistical analyses
Data were expressed as mean ± S .E .M. Al l d ata
were analyzed by one-way analysis of variance
(ANOVA) using SPSS 11.0 software. A value of p <
0.05 was co nsider ed sta tistic ally s ignifi cant.
3. Results
3.1 Catalpol improve sensorimotor performance
in stroke rat s
Post-stroke administration of catapol reduced
Bederson's score in rats, indicating improved motor
function re lative to v ehicle c ontrol , catap ol sign ifi-
cantly reduced Bederson's scores in rats at 7 and 15
da ys after s trok e (Fig. 1 A).
The Beam walking test w as us ed to m e as ur e
sensorimotor function. During the course of treat -
ment, beam walking scores were reduced; by day 15
following stroke, score reductions in catapol-treated
versus vehicle-treated animals reached statistical sig-
nificance (P< 0.05) ( Fi g. 1B ).
Figure 1. Effects of intraperitoneal injection with catalpol on sensorimotor performance in post-surgical
rats at days 1, 4, 7 and 15. (A) Post-stroke treatment with catalpol reduced Bederson’s score in stroke rats and (B)
decreased beam working score in stroke rats. T h e d a t a a r e p r e s e n t e d a s m e a n ± SE. * p<0.05 compared with vehicle group,
#
p<0.01 compared with sham operation g ro up.
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3.2 Effect of catalpol on the vascular pattern of
the cerebral cortex surface
The effect of catalpol on the vascular pattern of
the rat cerebral cortex surface was examined follow-
ing pMCAO. In vehicle-treat ed a nimals , li quefa c t i v e
necrosis of the brain was observed 15 days after
p MC AO . B ra in s fr o m vehicle-treated animals exhi-
bited a few deranged microvessels (F ig u re 2A ). B y
contrast, the focus of cerebral ischemia was near
normal in catalpol-treated animals; the brains of thes e
animals exhibited more branched vessels crossing and
gathering radially to the surface of cerebral ischemia.
All these vessels arborized to form a continuous net-
work of small blood vesse l s (F ig ur e 2B ).
Figure 2. T h e vascular pattern in cerebral cortical surface in rats 15 days after pMCAO. (A) In the ve-
hicle-tr e ated group, the pale brain surface had few vessel branch points, infarct areas were characterized by liquefactive
necrosis, cortical surface vessels were scarce and rearranged, several discontinued vascular structures were observed, and
the radial patterns were lost . (B) In the catalpol-treated group, brain surface vessel branch points increased obviously,
vascular structures continued, focus on the infarct area was present, and the vessel radial patterns and brain tissue infarct
area were close to normal. Arrow points to vascular structures around the ischemia area.
3.3 Catalpol enhanced brain angiogenesis in the
peri-infarcted area of the cortex
The effect of catalpol on angiogenesis was then
examined by immunostaining of brain sections for
von Willebrand Factor (vWF), a marker of endothelial
cells, and for proliferating cell nuclear antigen
(PCNA), a marker of cell proliferation. V WF a n d
PCNA co-localization points in the sham o p e ra t i o n
group were scarcly observed (F ig 3A).C o m p a r e d w i t h
the v ehic le-treated g r o u p ( 34 ± 3.25) (Fi g 3B) , t he
number of VWF and PCNA co-localization points in
the catalpol-treated group (Fig 3C an d D ) in c re a se d
significantly (p < 0.05). The number of vWF and
PCNA co-localization points in the catalpol-treated
gr ou p (F ig 3C and D) was 233.67 ± 89.51, nearly 6
times that in the vehicle group (p < 0.01) (Fig 3B an d
D). These results also agreed with results o b ta in e d
from integral optical density (IOD) analyses in the
vWF-P C N A c o -localization area (Fig 3E). These data
demonstrate that catalpol plays an important role in
cerebral ischemia angiogenesis.
3.4 Effects of catalpol on brain capillary endo-
thelial cells (BCECs)
The influence of catapol treatment on BCEC mi-
crostructure following pMCAO were examined by
transmission electron microscopy. Compared to ve-
hi cle co tntrol (F ig 4A), catalpol significantly reduced
BCEC edema (Fig 4B). The number of chondriosomes
in the catalpol-treated group was hi ghe r than that in
the vehicle group and close to normal levels.
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Figure 3. Angiogenesis surrounding ischemic cortical area as demonstrated by immunocytochemistry and
laser scanning confocal microscopy. The effects of Catalpol on angiogenesis were indicated by double-stai ning for
VWF, a marker of endothelial cells and for proliferating cell nuclear antigen (PCNA), a marker of cell proliferation.
Co-labeling of PCNA (green) and VWF (red) demonstrates angiogenesis, i.e., endothelial proliferation in the capillaries, in
the peri-i n f a r c t e d a r e a a t 1 5 d a y s a f t e r p M C A O . C o -localization of P C N A a n d V W F i s y e l l o w . ( A ) S h a m o p e r a t i o n g r o u p , ( B )
Vehicle-treated group, (C) Catalpol-t r e a t e d g r o u p . B a r s = 1 5 0 μm i n A , B , a n d C . T h i s a n a l y s i s d e m o n s t r a t e d that few vessels
were double-s t a i n e d b y V W F a n d P C N A i n s h a m -operated rats (A), but significa n t r e m o d e l i n g o f t h e m i c r o v e s s e l n e t w o r k
o c c u r r e d i n t h e i n f a r c t e d h e m i s p h e r e a n d t h e n u m b e r o f v e s s e l s w i t h s m a l l d i a m e t e r a n d s h o r t s e g m e n t i n c r e a s e d a t 1 5 d a y s
after the stroke. More vessels were double-s t a i n e d f o r V W F a n d P C N A i n c a t a l p o l -treated group (C). Statistical analyses are
s h o w n i n t h e g r a p h o f t h e n u m b e r o f v e s s e l s c o - l a b e l e d f o r P C N A a n d V W F ( D ) . T h e r e s u l t s a b o v e a g r e e d w i t h t h e r e s u l t s
of IOD analyses in the co-localization area (E). (
*
P < 0.01).
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449
Figure 4 Ultrastructural observations of brain capillary endothelial cells (BCECs). In the vehicle-t re at ed gr ou p
( A ) , B C E C e d e m a , c h r o m a t i n r a r e f a c t i o n ( s h o r t a r r o w ) , a n d c h o n d r i o s o m e s w e l l i n g ( l o n g a r r o w ) w e r e o b s e r v e d . ( B ) I n t h e
catalpol group, BCECs, pykno-chromatin (short arrow), and chondriosome number and shape (long arrow) were close to
normal or near normal. Bars = 1μm.
3.5 Catalpol upregulated EPO and VEGF ex-
pression in rat brain following pMCAO
To monitor the influence of catapol on EPO and
VEGF expression, we performed immunohistochem-
ical analyses of brain sections derived from rats fol-
lowing pMCAO. EPO and VEGF- positive cells w er e
detected in the cell membrane and cyt o p l a sm . Fe w
EPO positive cells were observed in brain sections
derived from sham-treated animals ( Fi g 5A). Com-
pared to vehicle-treated animals (Fig 5B), brain sec-
tions derived from catapol-treated animals exhibited
significantly increased EPO expression (Fig 5C). Sta-
tistical analyses revealed 7.2 ± 1.40 positive cell/cm
2
vs 15.3 ± 2 positive cell/cm
2
in vehicle-treated versus
catapol-treated animals (p < 0.05). Similar results were
obtained by western blot analyses (Fi g 5G and I); A s
for V EG F, immunofluorescence analyses demon-
strated that catalpol at the dose of 5mg/ kg signifi-
cantly upregulated VEGF expression compared with
the vehicle group; the number of positive cell/cm
2
vehicle-treated versus catapol-treated animals was 8±
1.6 vs 17 ± 2.5; (p < 0 .01) (F ig . 5E , F, H) . Once again,
similar results were obtained by western blot analyses
(Fig 5G a n d I ) . Statistical analyses were shown in Fig
5I.
4. Discussion
There are three principal findings emerged from
the present study. Firstly, catalpol treatment im -
proved neurofunction after stroke, as evidenced by
enhanced scores in the beam walking test designed to
evaluate sensorimotor reflexes, motor strength and
coordination. Secondl y, o ur resu lts show that catapol
enhances brain angiogenesis following stroke without
wo rs en i ng stroke brain ed e ma . Fina lly, we demon-
strate that t h e ameliorative effects of catalpol on
stroke brain are mediated by enhanced expression of
EPO and VEGF. Our findings thus provide new in -
sights into the likely regulatory mechanisms of cata-
pol.
Ischemic stroke is a serious dis e as e caused by a
thrombus (blood clot), w h i c h c a n r e s u lt i n p e r m an e n t
neurological damage, complications, and even d ea th
[26].Th e standard m e th o d of treatment is t o dissolve
the clot a n d r e s t o r e blood flow in the b l o c k e d vessel.
The drug TP A is approved for this use; however, TPA
is used onl y 3-6 hours after stroke, a n d the more ra-
pidly blood flow is restored to the brain, the fewer
brain cells die [27]. Recent research has suggested that
an alternative approach to restore blood flow is to
promote angiogenesis in regions surrounding the
ischemic brain.
As one of the most potentangiogenic factors,
VEGF is up-regulated by focal cerebral ischemia not
only in animal models but also in human patients
[10-11] as an angiogenic, neurotrophic, and neuro-
protective factor [28-30]. VEGF also plays a vital role
d u r i ng n e u r a l [29,31] and vascular remodeling [32 -33]
after stroke. Our results showed that ischemia in -
duced VEGF expression, which was not enough for
vascular remodeling, but catalpol treatment increased
VEGF expression together with increased microvessel
f or m at i on , he al ed t h e ischemic brain, and improved
neurobehavioral score. These data suggested that cat-
alpol stimulated brain angiogenesis after stroke by
increasing the secretion of endogenous VEGF.
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Figure 5 Catalpol upregulated EPO and VEGF expression in pMCAO rat brains. Immunohistochemistry results
showing neurons with EPO (A, B, C, 200×) and VEGF (D, E, F, 20) in the peri-infarcted area of a pMCAO rat, a
sham-operated rat (A & D), a vehicle-treated rat (B & E), and a catalpol-treated rat (C & F). EPO and VEGF expression
detected by western blot showed in (G). The internal control was β-actin. Vs vehicle group
*
p < 0.05 . The experiments
repeated three times and 6 rats used in eac h group. Statistical bars shown as H and I respectively.
Int. J. Biol. Sci. 2010, 6
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451
Moreover, VEGF, known as vascular permeabil-
ity factor, is associated with angiogenesis, vascular
permeability and neuroprotection. VEGF enhances
angiogenesis, but worsens rather than improves ce -
rebral haemodynamics after stroke [34-35]. Lo w-dose
VEGF aggravates hemorrhagic transformation [36].
Inhibition of endogenous VEGF by topical application
of anti-VEGF antibody in the ischemic cortex de-
creased the blood-brain barrier (BBB) disruption [37].
VEGF is in part responsible for the BBB disruption
during the early stage of focal cerebral ischemia [37] .
These adverse effects suggested that caution should
be heeded when considering the use of only VEGF for
stroke patients [38].
EPO is a pleiotropic factor [39] . EP O and EPO
receptor (EP O -R) are expressed in neurons, astrocytes,
and endothelial cells after focal permanent ischemia
in mice. EPO is a neuroprotective factor [40-41], which
improves functional recovery and reduces neuronal
apoptosis [42] and inflammation [43]. EPO has a mi-
togenic and positive chemotactic effect on endothelial
cells and endothelial progenitor cells [44-45]. It sti-
mulates angiogenesis in vitro and i n v i v o [18,40].
Therefore, the EPO/EP O -R system is implicated in
the process of neuroprotection [4 6] and restructuring
(such as angiogenesis) after ischemia [18,46]. In this
study, catalpol increased EPO expression in neurons
and endothelial-like cells surrounding the vessels.
Therefore, catalpol may improve neurofunction and
neural and vascular remodeling after stroke by acti-
vating EPO .
In this study, we observed that catalpol in-
creased VEGF expression but did not increase vascu-
lar permeability (Fig 3b), which may be related to the
simultaneous increase of EPO expression in the brain.
EPO reduces the side effects of VEGF, which protects
the BBB against VEGF-induced permeability in vitro
[19]. EPO and VEGF promote neural outgrowth
(Bocker-Meffert et al., 2002) and exhibit equal angi-
ogenic potential [18]. Furthermore VEGF modulates
erythropoiesis through regulation of adult hepatic
erythropoietin synthesis [ 47], a n d EPO -R promotes
V EG F e xp re ssion and angiogenesis in peripheral
ischemia in mice [48]. Therefore, the advantageous
reciprocal interactions between EPO and VEGF on
angiogenesis, which can be induced by catapol may
be an effective way to treat stroke patients. Brain in -
j u r y h e l p s E PO to cross the BBB [49-50], w h i c h m a y
then produce a synergistic effect with VEGF. Catalpol
is the effective component of Rehmannia glutinosa,
which can increase blood level o f EPO (data not
shown). Moreover, catapol can cross the BBB, as de-
tected by HPLC even in normal rats [51].
In conclusion, our data suggested that catalpol
modul ated angiogenesis through increased EPO and
VEGF after stroke, which may be the mechanism by
which catalpol reduced ischemic neuronal damage
and enhanced functional recovery. Taken to ge t he r ,
these data suggested that catalpol may improve col-
lateral circulation and pr o vi d e impact on stroke pa-
tients through new blood vessel formation. Future
research may elucidate the specific signaling path-
ways through which catalpol increases angioge n es i s.
Acknowledgements
This work was supported by grants from the
Fundamental Research Funds for the Central Univer-
sities (No.XDJK2009C081) and Southwest University
Dr. Foundation (No.104290-20710906) and N SF C
General Projects (No.81073084).
Conflict of Interests
The authors have declared that no conflict of in-
terest exists.
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... administration, to either DEPs or saline. The dose of catalpol used in the present work has been selected from previous studies that demonstrated its effectiveness, including in reducing oxidative stress and exerting cardioprotective effects against an ischemia/reperfusion insult in rats [11], increasing brain angiogenesis and ameliorating the edema of the brain capillary endothelial cells, following permanent middle cerebral artery occlusion in rats [21], ameliorating atherosclerotic lesions in hypercholesterolemic rabbits [22] and improving behavioral impairment and cerebral blood flow in rats after cerebral ischemia [23]. The animals were separated at random into four groups and were treated as follows: ...
... The limitations of the present work include the fact that we have assessed the protective effect of only one selected dose of catalpol [11,[21][22][23] on the acute cardiovascular effects of pulmonary exposure to DEPs. Additional studies are required to assess the impact of various doses of catalpol following subchronic and chronic lung exposure to DEPs, and to include inflammatory cell invasion quantification in the heart and lung by histology. ...
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Inhaled particulate air pollution exerts pulmonary inflammation and cardiovascular toxicity through secondary systemic effects due to oxidative stress and inflammation. Catalpol, an iridiod glucoside, extracted from the roots of Rehmannia glutinosa Libosch, has been reported to possess anti-inflammatory and antioxidant properties. Yet, the potential ameliorative effects of catalpol on particulate air pollution—induced cardiovascular toxicity, has not been studied so far. Hence, we evaluated the possible mitigating mechanism of catalpol (5 mg/kg) which was administered to mice by intraperitoneal injection one hour before the intratracheal (i.t.) administration of a relevant type of pollutant particle, viz. diesel exhaust particles (DEPs, 30 µg/mouse). Twenty-four hours after the lung deposition of DEPs, several cardiovascular endpoints were evaluated. DEPs caused a significant shortening of the thrombotic occlusion time in pial microvessels in vivo, induced platelet aggregation in vitro, and reduced the prothrombin time and the activated partial thromboplastin time. All these actions were effectively mitigated by catalpol pretreatment. Likewise, catalpol inhibited the increase of the plasma concentration of C-reactive proteins, fibrinogen, plasminogen activator inhibitor-1 and P- and E-selectins, induced by DEPs. Moreover, in heart tissue, catalpol inhibited the increase of markers of oxidative (lipid peroxidation and superoxide dismutase) and nitrosative (nitric oxide) stress, and inflammation (tumor necrosis factor α, interleukin (IL)-6 and IL-1β) triggered by lung exposure to DEPs. Exposure to DEPs also caused heart DNA damage and increased the levels of cytochrome C and cleaved caspase, and these effects were significantly diminished by the catalpol pretreatment. Moreover, catalpol significantly reduced the DEPs-induced increase of the nuclear factor κB (NFκB) in the heart. In conclusion, catalpol significantly ameliorated DEPs–induced procoagulant events and heart oxidative and nitrosative stress, inflammation, DNA damage and apoptosis, at least partly, through the inhibition of NFκB activation.
... Catalpol ( Figure 4D) is the main active component of the radix from Rehmannia glutinosa Libosch, and it belongs to the iridoid monosaccharide glycoside family (Ismailoglu et al., 2002;Zhang et al., 2008), which has pleiotropic protective effects on many diseases, including neurodegenerative diseases (Xia et al., 2012), ischemic stroke (Zhu et al., 2010), metabolic disorders (Zhu et al., 2010) and others. It's reported that the efficacy of catalpol pretreatment on cerebral I/R injury may be attributed to reduction of free radicals and inhibition of lipid peroxidation and endothelin-1 (ET-1) production . ...
... Catalpol ( Figure 4D) is the main active component of the radix from Rehmannia glutinosa Libosch, and it belongs to the iridoid monosaccharide glycoside family (Ismailoglu et al., 2002;Zhang et al., 2008), which has pleiotropic protective effects on many diseases, including neurodegenerative diseases (Xia et al., 2012), ischemic stroke (Zhu et al., 2010), metabolic disorders (Zhu et al., 2010) and others. It's reported that the efficacy of catalpol pretreatment on cerebral I/R injury may be attributed to reduction of free radicals and inhibition of lipid peroxidation and endothelin-1 (ET-1) production . ...
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The current prevalence of neurodevelopmental, neurodegenerative diseases, stroke and brain injury stimulates studies aimed to identify new molecular targets, to select the drug candidates, to complete the whole set of preclinical and clinical trials, and to implement new drugs into routine neurological practice. Establishment of protocols based on microfluidics, blood-brain barrier- or neurovascular unit-on-chip, and microphysiological systems allowed improving the barrier characteristics and analyzing the regulation of local microcirculation, angiogenesis, and neurogenesis. Reconstruction of key mechanisms of brain development and even some aspects of experience-driven brain plasticity would be helpful in the establishment of brain in vitro models with the highest degree of reliability. Activity, metabolic status and expression pattern of cells within the models can be effectively assessed with the protocols of system biology, cell imaging, and functional cell analysis. The next generation of in vitro models should demonstrate high scalability, 3D or 4D complexity, possibility to be combined with other tissues or cell types within the microphysiological systems, compatibility with bio-inks or extracellular matrix-like materials, achievement of adequate vascularization, patient-specific characteristics, and opportunity to provide high-content screening. In this review, we will focus on currently available and prospective brain tissue in vitro models suitable for experimental and preclinical studies with the special focus on models enabling 4D reconstruction of brain tissue for the assessment of brain development, brain plasticity, and drug kinetics.
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Neurovascular unit (NVU) is organized multi-cellular and multi-component networks that are essential for brain health and brain homeostasis maintaining. Neurovascular unit dysfunction is the central pathogenesis process of ischemic stroke. Thus integrated protection of NVU holds great therapeutic potential for ischemic stroke. Catalpol, classified into the iridoid monosaccharide glycoside, is the main active ingredient of the radix from traditional Chinese medicine, Rehmannia glutinosa Libosch, that exhibits protective effects in several brain-related diseases. In the present study, we investigated whether catalpol exerted protective effects for NVU in ischemic stroke and the underlying mechanisms. MCAO rats were administered catalpol (2.5, 5.0, 10.0 mg·kg⁻¹·d⁻¹, i.v.) for 14 days. We showed that catalpol treatment dose-dependently reduced the infarction volume and significantly attenuated neurological deficits score in MCAO rats. Furthermore, catalpol treatment significantly ameliorated impaired NVU in ischemic region by protecting vessel-neuron-astrocyte structures and morphology, and promoting angiogenesis and neurogenesis to replenish lost vessels and neurons. Moreover, catalpol treatment significantly increased the expression of vascular endothelial growth factor (VEGF) through up-regulating PI3K/AKT signaling, followed by increasing FAK and Paxillin and activating PI3K/AKT and MEK1/2/ERK1/2 pathways. The protective mechanisms of catalpol were confirmed in an in vitro three-dimensional NVU model subjected to oxygen-glucose deprivation. In conclusion, catalpol protects NVU in ischemic region via activation of PI3K/AKT signaling and increased VEGF production; VEGF further enhances PI3K/AKT and MEK1/2/ERK1/2 signaling, which may trigger a partly feed-forward loop to protect NVU from ischemic stroke.
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Stroke is an acute cerebrovascular disease caused by sudden rupture of blood vessels in the brain or blockage of blood vessels, which has now become one of the main causes of adult death. During stroke, hypoxia-inducible factor-1 (HIF-1), as an important regulator under hypoxia conditions, is involved in the pathological process of stroke by regulating multi-pathways, such as glucose metabolism, angiogenesis, erythropoiesis, cell survival. However, the roles of HIF-1 in stroke are still controversial, which are related with ischemic time and degree of ischemia. The regulatory mechanisms of HIF-1 in stroke include inflammation, autophagy, oxidative stress, apoptosis and energy metabolism. The potential drugs targeting HIF-1 have attracted more attention, such as HIF-1 inhibitors, HIF-1 stabilizers and natural products. Based on the role of HIF-1 in stroke, HIF-1 is expected to be a potential target for stroke treatment. Resolving when and what interventions for HIF-1 to take during stroke will provide novel strategies for stroke treatment.
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Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.
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Context Tissue plasminogen activator is the only thrombolytic agent approved in the United States for treatment of acute ischemic stroke, and has limitations. Aptiganel hydrochloride is a novel and selective ligand for the ion-channel site of the N-methyl-D-aspartate receptor-channel complex and a promising neuroprotective agent in animal models of focal brain ischemia.Objective To determine whether aptiganel improves the clinical outcome for acute ischemic stroke patients.Design Nested phase 2/phase 3 randomized controlled trial conducted between July 1996 and September 1997.Setting One hundred fifty-six medical centers in the United States, Canada, Australia, South Africa, England, and Scotland.Participants A total of 628 patients with hemispheric ischemic stroke (50.3% male; mean age, 71.5 years).Interventions Patients were randomly assigned within 6 hours of stroke to receive 1 of 3 treatment regimens: high-dose aptiganel (5-mg bolus followed by 0.75 mg/h for 12 hours; n = 214); low-dose aptiganel (3-mg bolus followed by 0.5 mg/h for 12 hours; n = 200); or placebo (n = 214).Main Outcome Measures The primary efficacy end point was the Modified Rankin Scale score at 90 days after stroke onset. Secondary end points included mortality and change in National Institutes of Health (NIH) Stroke Scale score at 7 days after stroke.Results The trial was suspended by the sponsor and the independent data and safety monitoring board because of both a lack of efficacy and a potential imbalance in mortality. There was no improvement in outcome for either aptiganel (low-dose or high-dose) group compared with the placebo group at 90 days (median Modified Rankin Scale score for all 3 treatment groups = 3; P = .31). At 7 days, placebo-treated patients exhibited slightly greater neurological improvement on the NIH Stroke Scale than high-dose aptiganel patients (mean improvement for placebo group, −0.8 points vs for high-dose aptiganel, 0.9 points; P = .04). The mortality rate at 120 days in patients treated with high-dose aptiganel was higher than that in patients who received placebo (26.3% vs 19.2%; P = .06). Mortality in the low-dose aptiganel group was 22.5% (P = .39 vs placebo).Conclusions Aptiganel was not efficacious in patients with acute ischemic stroke at either of the tested doses, and m ay be harmful. The larger proportion of patients with favorable outcomes and lower mortality rate in the placebo group suggest that glutamate blockade with aptiganel may have detrimental effects in an undifferentiated population of stroke patients. Figures in this Article Ischemic stroke is the third leading cause of death in the United States. Epidemiological studies indicate that as many as 730 000 strokes occur annually in the United States,1 of which between 73% and 86% are ischemic.2 Up to half of all stroke survivors are disabled, a third of them seriously enough to require assistance in daily activities.3 However, to date, the thrombolytic agent tissue plasminogen activator is the only therapy approved in the United States and Canada for the treatment of acute ischemic stroke. The limitations of tissue plasminogen activator are well-known. It must be administered within 3 hours of symptom onset, it increases the risk of brain hemorrhage, and only patients in whom cerebral hemorrhage has been definitively excluded are eligible for treatment.4 The approval of tissue plasminogen activator for use in North America has provided impetus for a change in how stroke is perceived both by the general public and among health care professionals. Acute stroke is now widely viewed as a medical emergency. There is a theoretic window of opportunity for minimizing the disability associated with stroke by means other than acute thrombolysis. Injury caused by the initial brain ischemia is compounded by the release of excitatory neurotransmitters, such as glutamate, which precipitate the influx of sodium and calcium into neurons, contributing to secondary damage.5 These observations have led to the concept of improving the stroke outcome by inhibiting these secondary processes. Substances that block glutamate receptors have neuroprotective properties in animal stroke models.6- 8 However, none of the putative neuroprotective agents has yet proven efficacious in humans. Aptiganel hydrochloride (N-[1-naphtyl]-N-methyl-guanidine hydrochloride) is a selective ligand for the ion-channel site of the N-methyl-D-aspartate subtype of glutamate receptor. Aptiganel, when given up to 1 hour after permanent or temporary occlusion of the middle cerebral artery in rat models of ischemic stroke, reduced the amount of brain damage between 40% and 70%.9 Aptiganel produced a significant improvement in neurological outcome after hypothermic circulatory arrest,10 and decreased contusion volume and hemispheric swelling after traumatic brain jury in animal models.11 Aptiganel was efficacious in animal studies at a minimum plasma concentration of approximately 10 ng/mL under steady state and nonsteady-state conditions.12 The half life of aptiganel in humans is 4 hours, and the mean clearance is 18 mL/min per kilogram. It is 88%-protein bound and is metabolized by the liver with primary excretion via the feces. Safety studies indicate that aptiganel is relatively well tolerated in healthy human volunteers and acute stroke patients (who received study medication within 24 hours of stroke onset). Adverse effects include increases in heart rate and blood pressure and neurological disturbances, such as blurred vision, nystagmus, numbness, dizziness, and sedation with increasing doses.13 In preliminary studies, it was determined that the maximum tolerable dose in humans was a 5-mg bolus infused over 3 to 5 minutes followed by a 12-hour, constant-rate, maintenance infusion of 0.75 mg/h.12 The present study was designed to compare the efficacy and safety of 2 doses of aptiganel with placebo in patients with acute ischemic stroke. The 2 dosing regimens were designed to produce steady-state plasma concentrations of aptiganel of approximately 5 and 10 ng/mL.
Article
Background: Therapeutic angiogenesis with vascular endothelial growth factor (VEGF) is a clinically promising strategy in ischemic disease. The pathophysiological consequences of enhanced vessel formation, however, are poorly understood. Methods: We here established mice overexpressing human VEGF165 under a neuron-specific NSE promoter, which exhibited an increased density of brain vessels under physiological conditions. We submitted these mice to transient focal ischemias, as induced by 90 and 30 minutes episodes of intraluminal middle cerebral artery (MCA) occlusions and examined the effects of brain-selective VEGF overexpression on ischemic injury, blood-brain barrier dysfunction, angiogenesis and regional cerebral blood flow (CBF). Results: Following 90 and 30 minutes episodes of MCA occlusions, VEGF overexpression significantly reduced neurological deficits, infarct volume, disseminate neuronal injury and caspase-3 activity. Brain swelling, on the other hand, was not influenced in VEGF-overexpressing animals, while sodium fluorescein extravasation was moderately increased, suggesting VEGF induces mild blood brain barrier leakage. In order to find out whether enhanced angiogenesis improves regional CBF in the ischemic brain, 14C-iodoantipyrine autoradiographies were performed. Autoradiographies revealed that VEGF overexpression reduces regional CBF in ischemic areas and increases blood flow values only outside the MCA territory, suggesting that enhanced angiogenesis facilitates hemodynamic steal phenomena. Conclusions: Our data suggest that brain-selective VEGF overexpression is neuroprotective via inhibition of executive cell death pathways, but worsens cerebral hemodynamics.
Article
Aim: To investigate the effects of catalpol from Radix Rehmanniae on behavior outcome of rats with focal cerebral ischemia and the expression patterns of growth-associated protein-43 (GAP-43) in the cerebral cortex surrounding the ischemic core. Methods: 42 adult male Sprague-Dawley rats were randomized into 7 groups, including sham operation group, model group, normal saline control group, low dose, middle dose and high dose of catalpol treatment group (1, 5 and 10 mg·kg-1, ip, respectively) and citicoline treatment control group (0.4 mg·kg-1, ip). A small craniectomy was performed from the posterior zygoma and along the temporal ridge of the cranium extending ventrally to expose the right middle cerebral artery (MCA) and olfactory tract. The base of MCA was electrico-coagulated, and the permanent focal cerebral ischemia animal model was established. Animals were assigned to receive intraperitoneally either different does of catalpol treatment or citicoline treatment, once a day for 7 successive days, and the first dose was given at the time point of 6h post-operation. Attitudinal reflexes and muscle strength were assessed just before the first dose was given and 1, 4, 7 d and 15 d time points after operation. After 15 days of behavioral testing, animais were deeply anesthetized and then sacrificed. Brain tissue was removed as quickly as possible, and brain sections and brain tissue homogenate were prepared for studying the expression patterns of growth-associated protein-43 in the cerebral cortex surrounding the ischemic core using immunofluorescence histochemistry and Western blot. Results: Compared with the model group, in the normal saline control group and the citicoline control group, attitudinal reflexes and muscle strength in high dose and middle dose of catalpol treatment groups(10 and 5 mg·kg-1, respectively) were ameliorated significantly, and there was no obvious dysfunction in the sham group; the protein expression of GAP-43 in the model rats of the catalpol treatment group(1, 5 and 10 mg·kg-1) increased significantly, higher than those in any other experimental groups (P < 0.05). Conclusion: Catalpol can up-regulate the protein expression of GAP-43 in the cerebral cortex surrounding the ischemic core, and enhance axonal regeneration, Which would be an underlying substrate for catalpol improved the functional recovery of rats with focal cerebral ischemia.
Article
We have examined the incidence and size of infarction after occlusion of different portions of the rat middle cerebral artery (MCA) in order to define the reliability and predictability of this model of brain ischemia. We developed a neurologic examination and have correlated changes in neurologic status with the size and location of areas of infarction. The MCA was surgically occluded at different sites in six groups of normal rats. After 24 hr, rats were evaluated for the extent of neurologic deficits and graded as having severe, moderate, or no deficit using a new examination developed for this model. After rats were sacrificed the incidence of infarction was determined at histologic examination. In a subset of rats, the size of the area of infarction was measured as a percent of the area of a standard coronal section. Focal (1-2 mm) occlusion of the MCA at its origin, at the olfactory tract, or lateral to the inferior cerebral vein produced infarction in 13%, 67%, and 0% of rats, respectively (N = 38) and produced variable neurologic deficits. However, more extensive (3 or 6 mm) occlusion of the MCA beginning proximal to the olfactory tract--thus isolating lenticulostriate end-arteries from the proximal and distal supply--produced infarctions of uniform size, location, and with severe neurologic deficit (Grade 2) in 100% of rats (N = 17). Neurologic deficit correlated significantly with the size of the infarcted area (Grade 2, N = 17, 28 +/- 5% infarction; Grade 1, N = 5, 19 +/- 5%; Grade 0, N = 3, 10 +/- 2%; p less than 0.05). We have characterized precise anatomical sites of the MCA that when surgically occluded reliably produce uniform cerebral infarction in rats, and have developed a neurologic grading system that can be used to evaluate the effects of cerebral ischemia rapidly and accurately. The model will be useful for experimental assessment of new therapies for irreversible cerebral ischemia.
Article
Stroke most commonly results from occlusion of a major artery in the brain and typically leads to the death of all cells within the affected tissue. Mitochondria are centrally involved in the development of this tissue injury due to modifications of their major role in supplying ATP and to changes in their properties that can contribute to the development of apoptotic and necrotic cell death