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The Protective Effect of Cerebralcare Granule�� on BrainEdema, Cerebral Microcirculatory Disturbance, andNeuron Injury in a Focal Cerebral Ischemia Rat Model

FANG WANG,*,� QIN HU,� CHUN-HUA CHEN,� XIANG-SHUN XU,*,� CHANG-MAN ZHOU,� YA-FANG

ZHAO,*,� BAI-HE HU,*,� XIN CHANG,*,� PING HUANG,*,� LEI YANG,� YU-YING LIU,*,� CHUAN-SHE

WANG,*,�,§ JING-YU FAN,*,� KE ZHANG,– GUO-YU LI,– JING-HUI WANG,– AND JING-YAN HAN,*,�,§

*Tasly Microcirculation Research Center, Peking University Health Science Center, Beijing, China; �Key Laboratory of Microcirculation, State

Administration of Traditional Chinese Medicine, China; �Department of Anatomy, School of Basic Medical Sciences, Peking University, Beijing,

China; §Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China; –School

of Pharmacy, Shihezi University, Xinjiang, China

Address for correspondence: Jing-Yan Han, M.D., Ph.D., Professor and Chairman, Department of Integration of Chinese and Western Medicine,

School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China. E-mail: [email protected]

Received 25 April 2011; accepted 13 December 2011.

ABSTRACT

Objective: The purpose of the present study was to explore the

protective effects of CG on rat cerebral injury after focal cerebral I ⁄ R.

Methods: Male Sprague–Dawley rats were subjected to right

middle cerebral artery occlusion for 60 minutes followed by

reperfusion for 60 minutes or 24 hours. CG (0.4 or 0.8 g ⁄ kg) was

administrated 90 minutes before ischemia. Brian edema was

evaluated by Evan’s blue dye extravasations and brain water

content, leukocyte adhesion, and albumin leakage were

determined with an upright fluorescence microscope, and neuron

damage was assessed by 2,3,5-triphenyltetrazolium chloride

staining, terminal deoxynucleotidyl transferase-mediated dUTP

nick end labeling, and immunohistochemistry of caspase-3, p53,

p53 upregulated modulator of apoptosis.

Results: Focal cerebral I ⁄ R elicited a prominent brain edema, an

increase in leukocyte adhesion, and albumin leakage, as well as

neuron damage. All the insults after focal cerebral I ⁄ R were

significantly attenuated by pretreatment with CG.

Conclusions: Pretreatment with CG significantly reduced focal

cerebral I ⁄ R-induced brain edema, cerebral microcirculatory

disturbance, and neuron damage, suggesting the potential of CG

as a prophylactic strategy for patients in danger of stroke.

Key words: ischemia and reperfusion, middle cerebral artery

occlusion, neuron injury, cerebral microcirculatory disturbance,

brain edema

Abbreviations used: BBB, blood–brain barrier; CG, Cerebralcare

Granule; dUTP, 2¢-deoxyuridine, 5¢-triphosphate; EB, Evan’s blue;

FITC, fluorescein isothiocyanate; I ⁄ R, ischemia and reperfusion;

MCAO, middle cerebral artery occlusion; PBS, phosphate-buffered

saline; PUMA, p53 upregulated modulator of apoptosis;

TTC, 2,3,5-triphenyltetrazolium chloride; TUNEL, terminal

deoxynucleotidyl transferase-mediated dUTP nick end labeling;

UPLC-TOF-MS, ultra performance liquid chromatography-time-

of-flight-mass spectrometer

Please cite this paper as: Wang F, Hu Q, Chen C-H , Xu X-S, Zhou C-M, Zhao Y-F, Hu B-H, Chang X, Huang P, Yang L, Liu Y-Y, Wang C-S, Fan J-Y,

Zhang K, Li G-Y, Wang J-H, Han J-Y. The protective effect of cerebralcare granule� on brain edema, cerebral microcirculatory disturbance and neuron

injury in a focal cerebral ischemia rat model. Microcirculation 19: 260–272, 2012.

INTRODUCTION

Stroke, as a serious neurological disease, inflicts a major

impact on the public health in almost every country. Ische-

mic stroke frequently results in brain edema causing severe

or even fatal outcome. However, several available strategies,

such as decompression craniotomy, hypothermia, and

osmotic diuresis therapy, are applied only as a rescue ther-

apy [6,19,24], while options for preventing brain edema are

limited.

CG was approved in 1996 by the China State Food and

Drug Administration for treatment of headache and dizzi-

ness associated with cerebrovascular diseases. CG is com-

posed of 11 herbs, including Radix angelicae sinensis

(Dang Gui), Rhizoma chuan xiong (Chuan Xiong), Raidix

paeoniae alba (Bai Shao), Ramulus uncariae cum uncis

(Gou Teng), Caulis spatholobi (Ji Xue Teng), Spica prunel-

lae (Xia Ku Cao), Concha margaritifera usta (Zhen Zhu

Mu), Radix rehmanniae preparata (Di Huang), Semen cas-

siae (Jue Ming Zi), Rhizoma corydalis yanhusuo (Yan Hu

DOI:10.1111/j.1549-8719.2011.00155.x

Original Article

260 ª 2012 John Wiley & Sons Ltd

The Official Journal of the Microcirculatory Society, Inc. and the British Microcirculation Society

Suo), and Herba asari (Xi Xin). Each of these herbs has

previously been characterized and commonly used in tra-

ditional Chinese medicine. A number of studies have been

conducted to identify the active constituent contained in

these herbs and the mechanism behind their actions. For

example, ligustrazine (the major constituent of Rhizoma

chuan xiong) was reported to inhibit neutrophil adhesion

to endothelial cells [15,17], and peoniflorin (the major

constituent of Raidix paeoniae alba), ligustrazine, and feru-

lic acid (the major constituent of Radix angelicae sinensis)

were found to decrease the infarct size and brain tissue

damage [12,15,17,18,29].

Our previous studies undertaken in Mongolian gerbils

demonstrated that CG attenuates the bilateral common

carotid artery occlusion-elicited cerebral microcirculatory

disturbance, hippocampal neuron injury, as well as BBB dis-

ruption, indicative of the potential of CG in protecting brain

from edema after ischemia and reperfusion (I ⁄ R) [28,30].

The purpose of the present study was to verify the ability of

CG as a prophylactic management in the development

of brain edema following I ⁄ R, in addition to attenuation of

microcirculation disturbance and neuron injury, using a focal

cerebral I ⁄ R model in which rats were subjected to MCAO.

MATERIALS AND METHODS

AnimalsMale Sprague–Dawley rats weighing 270–300 g were pur-

chased from the Animal Center of Peking University

Health Science Center. The animals were fasted for

12 hours before each experiment and allowed free access to

water. All animals were handled according to the guidelines

of the Peking University Animal Research Committee. The

surgeries and protocols were approved by the Committee

on the Ethics of Animal Experiments of the Health Science

Center of Peking University.

Cerebralcare GranuleCG was produced by Tianjin Tasly Pharmaceutical Co. Ltd

(Tianjin, China). The batch number of the granules used in

this study was Z10960082. No steroid is included in the con-

tent of CG. The processing of the product followed a strict

quality control, and the ingredients were subjected to stan-

dardization. The herbs were manufactured as granules after

dynamic cycle extraction and concentration by evaporating

and spray drying. Then the CG was packed with aluminum

foil composite, 4 g per bag. The compound was dissolved in

water to a concentration of 80 or 160 mg ⁄ mL before use [30].

UPLC–TOF-MS Analysis of CG Components inRat PlasmaRats were fasted overnight before experiment, and adminis-

trated by gavage with 0.8 g ⁄ kg CG dissolved in saline. Ani-

mals that received equivalent volume of saline were used as

control. Blood was harvested from orbit veins one, two,

three, and four hours after drug administration, and anti-

coagulated with heparin. Plasm was separated by centri-

fugation for 10 minutes at 3000 rpm (956 rcf), and pro-

cessed for analysis by UPLC–TOF-MS (UPLC Waters

ACQUITY and LCT Premier XE TOF-MS) [31]. MassLynx

4.1 Software (Waters Corporation, Milford, MA, USA) was

used to acquire data. Data were collected over the mass

range of 50–1500 m ⁄ z.

Animal Model and Drug AdministrationFocal cerebral ischemia was induced by MCAO, as

described elsewhere [3]. Briefly, animals were anesthetized

using 20% urethane. The right common carotid artery,

including its bifurcation, was exposed. The external carotid

artery was divided, leaving a stump of 3–4 mm. The inter-

nal carotid artery was then isolated, and the pterygopalatine

artery was ligated close to its origin. The internal carotid

artery was then clamped with a small vascular clip. The

common carotid artery was also clamped. The stump of

the external carotid artery was reopened, and a 4-0 mono-

filament nylon suture with a slightly enlarged and round

tip was inserted up through the internal carotid artery.

When a small resistance was felt, insertion was stopped.

The distance from bifurcation of the common carotid

artery to the tip of the suture was 18–22 mm. After occlu-

sion for one hour, the suture was withdrawn through the

internal carotid artery into the external artery, allowing

reperfusion. The animals in the sham group underwent the

same surgical procedures, but without arterial occlusion.

For evaluating the CG effect, animals in the CG + I ⁄ Rgroups received the drug orally 90 minutes before MCAO

at either 0.4 g ⁄ kg (CG 0.4 + I ⁄ R group) or 0.8 g ⁄ kg (CG

0.8 + I ⁄ R group).

Evan’s Blue Dye Extravasation and Brain WaterContentDisruption of the BBB was analyzed 24 hours after the

MCAO using EB dye. Briefly, rats were anesthetized with

pentobarbital sodium (0.1 g ⁄ kg body weight, i.p.), and EB

dye (Sigma-Aldrich, St. Louis, MO, USA; 4%, 3 mL ⁄ kg) in

saline was injected within two minutes into the left femoral

vein and allowed to circulate for 120 minutes. Rats were

transcardially perfused with PBS until colorless perfusion

fluid drained from the right atrium. The amount of extrava-

sated EB in the brain was determined by spectrofluropho-

tometry at an excitation wavelength of 620 nm. The brain

was removed and weighed immediately for determination of

wet weight, and weighed after drying in an oven at 105�C for

24 hours for determination of dry weight. The brain water

content was presented as [(wet weight ) dry weight) ⁄ wet

weight] · 100% [13].

Chinese Medicine and Cerebral Reperfusion Injury

ª 2012 John Wiley & Sons Ltd 261

Observation of MicrocirculationAfter being anesthetized with 20% urethane (2.0 g ⁄ kg

body weight, i.m.), the rats were tracheotomized and

mechanically ventilated with a breathing machine designed

for small animals (ALC-V8). With a hand-held drill, a

skull window of 4 · 6 mm was created 1 mm behind the

coronal suture, and 1 mm on the right side of the sagittal

suture. The dura was superfused contiguously with 37�C

warm physiological saline. A single unbranched venule was

selected for determination, which was without obvious

bend with diameter ranging between 30 and 50 lm and

length of about 200 lm. The dynamic of cerebral microcir-

culation was continuously observed for 60 minutes using a

biological microscope (DM-LFS, Leica, Germany) equipped

with a color monitor (J2118A, TCL, Huizhou, China), a

video timer (VTG-55B, For-A, Tokyo, Japan), and a DVD

recorder (DVR-R25, Malata, China). The microvessel

images were recorded through a high speed video camera

system at a rate of 1000 frames ⁄ sec (FASTCAM-ultima

APX, Photron, Tokyo, Japan), and the recordings were

replayed at a rate of 25 frames ⁄ sec from the stored images.

The RBC velocity in venule was measured with Image Pro

Plus software (IPP, Media Cybernetic, Bethesda, MD,

USA) before ischemia and 1, 20, 40, and 60 minutes after

reperfusion, respectively. Using the same software, the

inner diameter of cerebral venules was measured and pre-

sented as the mean of three measurements at one location

[30]. To assess leukocyte adhesion in venules, the fluores-

cence tracer Rhodamine 6G (Sigma, St Louis, MO, USA)

was administrated (5 mg ⁄ kg body weight) to the animal

via the femoral vein 10 minutes before ischemia. After

craniotomy, the cerebral microvessels were observed under

an upright fluorescence microscope (DM-LFS, Leica,

Germany) in combination with a CCD camera (USS-301,

Uniq, Santa Clara, CA, USA) using a helium-neon laser

beam for illumination. The adherent leukocytes were identi-

fied as those that attached to the venular walls for more than

30 seconds. The number of adherent leukocytes was scored

under basal condition and 1, 20, 40, and 60 minutes after

reperfusion. For evaluation of albumin leakage from venular

wall, FITC-albumin (Sigma-Aldrich) was infused (50 mg ⁄ kg

body weight) slowly through femoral vein 10 minutes before

ischemia, and the upright fluorescence microscopy was

employed. Using IPP software, the fluorescence intensities of

FITC-albumin inside the lumen of selected venules (Iv) and

in the surrounding interstitial area (Ii) were measured. The

ratio Ii ⁄ Iv was calculated and compared with the baseline as

an indication of albumin leakage.

Infarct VolumeTwenty-four hours after reperfusion, the rat was anesthe-

tized with 20% urethane (2.0 g ⁄ kg body weight, i.m.) and

the brain was rapidly excised and sliced coronally into five

sections (2 mm thick) beginning from optic chiasma. The

slices of the brain were incubated for 30 minutes at 37�C

in a 2% solution of TTC in PBS and then photographed as

digital images (Digital Sight DS-5M-U1; Nikon, Tokyo,

Japan). The infarct volume was calculated based on the

ratio of the infarct area of the ipsilateral hemisphere to

total non-infarct area from both the ipsilateral and contra-

lateral hemispheres to avoid the influence of tissue edema

[3].

Neurological ScoresTwenty-four hours after reperfusion, the neurological

scores were performed in a blinded fashion, as previously

reported with some modifications [13]. Each rat was sub-

jected to 6 tests (spontaneous activity, symmetry in the

movement of four limbs, forepaw outstretching, climbing,

body proprioception, and response to vibrissae touch) with

the minimum neurological score being 3 and the maximum

18. The score given to each rat at the completion of the

evaluation was the summation of the 6 individual test

scores [13].

Nissl and Immunohistochemistry StainingFor this propose, the brain samples were collected 24 hours

after reperfusion. After transcardiac perfusion with 250 mL

of 4% paraformaldehyde under anesthesia, rat brain was

removed and postfixed in the same fixative for 48 hours.

The brain tissue located in the middle between the optic

chiasma and the cerebral caudal end was cut into blocks,

embedded in paraffin, and sectioned at 10 lm. The

sections were deparaffinized and rehydrated, sequentially,

and processed for either Nissl staining or immunohisto-

chemistry. For immunohistochemistry, the sections were

incubated with the following primary antibodies: mouse

anti-caspase-3, goat anti-p53 (1:1000, Santa Cruz Biotech-

nology, Santa Cruz, CA, USA), and rabbit anti-PUMA

(1:1000, Cell Signaling, Boston, MA, USA). After being

washed, the samples were incubated with a biotinylated

secondary antibody followed by avidin–biotin–peroxidase

complex, and visualized with diaminobenzidine. As control,

a consecutive section was treated similarly except that the

primary antibody was omitted. IPP software was used to

assess the number of positive cells of caspase-3, p53, and

PUMA in the cortex penumbral region of the ischemic side

of brain, as described previously [3,13].

TUNEL AssayThe animals under anesthesia were infused via the left ven-

tricle with 4% paraformaldehyde in 0.01 M PBS (pH 7.4)

24 hours after reperfusion. Brain was removed and post-

fixed with the same fixative for six hours, and cryoprotect-

ed in 30% sucrose in PBS for at least 48 hours at 4�C. The

coronal brain sections (10 lm thick) were cut from the

F. Wang et al.


middle between the optic chiasma and the cerebral caudal

end on a cryostat (Leica CM1800, Bensheim, Germany). To

evaluate the apoptotic neuron cells in the cortex penumbral

region, TUNEL was conducted using an in situ cell death

detection kit (Fluorescein dUTP Kit; Roche Inc., Indiana-

polis, IN, USA), according to the instruction of manufac-

turer. The nuclei were counterstained with hoechst33342

(2 lg ⁄ mL) for five minutes. The slides were rinsed with

PBS, coverslipped with mounting medium and observed

under a laser confocal microscope (Axiovert 2000, Zeiss,

Germany) with a 63· objective at an excitation wavelength

of 480 nm and emission wavelength of 530 nm. Five fields

were randomly selected for each rat. The number of TUN-

EL-positive nuclei and the total number of nuclei in each

field were scored, and the ratio of the two values was auto-

matically calculated with IPP 5.0 software. A similarly trea-

ted consecutive section without addition of TdT was used

as control [31].

Ultrastructure of Cerebral CortexAnother set of rats were anesthetized 24 hours after reper-

fusion, and perfused with a fixative composed of 4% form-

aldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer

at a speed of 3 mL ⁄ min through the left ventricle. The ani-

mals were decapitated and a cerebral slice of 1 mm thick

was taken from the middle between the optic chiasma and

the cerebral caudal end, and tissue samples smaller than

1 mm3 in the cortex penumbral region were prepared as

routine for transmission electron microscopy. Ultrathin

sections of cortex were examined by an electron micro-

scope (JEM 1230, Jeol, Japan).

Statistical AnalysisAll parameters were expressed as mean ± SE. Statistical

analysis was performed using ANOVA followed by Tukey

test for multiple comparisons. A p-value <0.05 was consid-

ered statistically significant.

RESULTS

Identification and Kinetics of CG Components inRat PlasmaUPLC–TOF-MS system is characterized by high sensitivity

and specificity. By this approach, two components, coryda-

line and cassialactone, were detected in the plasma of CG

treated rats, which are obviously distinct from that in

plasma of control animals (Figures 1 and 2). These two

components appeared one hour after CG administration,

and maintained detectable over the next three hours.

EB Dye Extravasation and Brain Water ContentAfter 24 hours of reperfusion, EB was clearly observed in

the ipsilateral hemisphere of the coronal sections (Fig-

ure 3A). The EB content of the brain tissue increased sig-

nificantly in the I ⁄ R group compared with the sham group,

while CG treatment mitigated the I ⁄ R-caused increase in

EB content of the brain tissue (Figure 3B). Likewise, a sig-

nificant increase in brain water content was revealed in the

ipsilateral hemisphere 24 hours after the MCAO, when

compared with the sham group, which was alleviated sig-

nificantly by CG treatment at 0.8 g ⁄ kg (Figure 3C). No sig-

nificant differences in brain water content were observed

among the groups in the contralateral hemisphere.

Albumin LeakageCG pretreatment prevented FITC-labeled albumin leakage

from cerebral venules evoked by I ⁄ R, and the representative

images are presented in Figure 4A. Transvascular efflux of

FITC-labeled albumin was quantified for different groups

(Figure 4B). There was no detectable leakage under the

basal condition in all groups. In the sham group, only a

small amount of albumin was found to have leaked from

the venular wall during reperfusion. By contrast, albumin

leakage in the I ⁄ R group increased markedly after one

minute of reperfusion and kept increasing with time. The

I ⁄ R-induced albumin leakage was diminished significantly

in the animals receiving CG at both doses tested.

Leukocyte AdhesionLeukocytes in the microcirculation are easily visualized and

quantified after rhodamine injection (Figure 5A). The

changes in the number of leukocytes adherent to the venu-

lar wall in the four groups are shown in Figure 5B. In the

sham group, the number of adherent leukocytes increased

slightly after reperfusion but showed no significance when

compared with baseline. The number of adherent leuko-

cytes in the I ⁄ R group significantly increased immediately

after reperfusion and remained increasing until the end of

observation. Pretreatment with CG attenuated I ⁄ R-elicited

enhancement of leukocyte adhesion with the higher dose

being more significant than the lower dose.

RBC Velocity and Venular DiameterWith the use of the microscope equipped with a high-speed

video camera, cerebral microvessels and RBC moving inside

the venules were clearly visible in the cerebral cortex (Fig-

ure 6A). Impressively, the number of open capillaries after

I ⁄ R reduced considerably (a2) in comparison with the

sham group (a1), indicating a decreased blood supply in

this area. CG pretreatment, especially at 0.8 g ⁄ kg, obviously

attenuated this alteration by I ⁄ R (a4).

No significant difference in venular diameter was

observed among the sham, I ⁄ R, and CG-treated groups at

baseline, nor over the 60 minutes reperfusion (Figure 6B).

The time courses of RBC velocity in cerebral venules in dif-

ferent groups are depicted in Figure 6C. The mean RBC



of strategy for blunting brain edema is considered to be of

crucial importance in improving outcome of the patients

after stroke. With the focal cerebral I ⁄ R model, the present

study demonstrated that pretreatment with CG significantly

attenuated I ⁄ R-induced brain edema, as indicated by the

decline in EB dye extravasation and brain water content.

Furthermore, the I ⁄ R-imposed insults on cerebral microcir-

culation and neuron were alleviated by pretreatment with

A a1 a2 a3 a4

Sham I/R CG 0.4 + I/RB

C

2.5sue)

0.82 ††

CG 0.8 + I/R

#1.5

2.0 †

of b

rain

tis

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er c

onte

nt

0.79

0.82 †

#†

0.5

1.0#†

s blu

e (μ

g/g

Bra

in w

at

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Figure 3. EB extravasation and brain water content. (A) Representatives of EB-stained brain sections from rats 24 hours after MCAO. No dye

leakage was observed in the sham group (a1), while intensive staining was seen in ischemic area in the I ⁄ R group (a2). In contrast, the brains from

the CG treatment groups showed weak EB staining in infarct areas (a3 for CG0.4 + I ⁄ R and a4 for CG0.8 + I ⁄ R, respectively). (B) Statistical analysis

of EB extravasation. (C) The brain water content of the ipsilateral ischemic hemisphere (right) and the contralateral hemisphere (left). The ipsilateral

hemisphere of CG 0.8 + I ⁄ R group had a significantly decreased water content compared with the I ⁄ R group. Data are means ± SE from six animals.�p < 0.05 vs. sham group, #p < 0.05 vs. I ⁄ R group.

ba

c

V

d

500

ShamI/RCG 0.4 + I/RCG 0.8 + I/R

400

300 **

**

†

†

††

200 **

* *

* *#

# #

#

#

#

Alb

umin

leak

age

(%)

#

Baseline 1 20 40 60 (minutes)100 * * *

I R

e f

A B

Figure 4. The effect of pretreatment with CG on the albumin leakage from the venule. (A) Representative images of the sham group (a and b), I ⁄ Rgroup (c and d), and CG 0.8 g ⁄ kg + I ⁄ R group (e and f). Before I ⁄ R injury (baseline), no obvious albumin leakage was detected in the three groups

(a, c, e). This situation persisted to the end of observation in the sham group (b). The albumin leakage from venular wall was observed at 60 minutes

after I ⁄ R injury (d). The I ⁄ R injury-induced albumin leakage from cerebral venule was apparently suppressed by the pretreatment with 0.8 g ⁄ kg CG

(f). Bar = 50 lm. (B) Time course of albumin leakage in the sham, I ⁄ R, CG0.4 + I ⁄ R and CG0.8 + I ⁄ R groups. Data are mean ± SE from six animals.

*p < 0.05 vs. baseline, �p < 0.05 vs. sham group, #p < 0.05 vs. I ⁄ R group.

F. Wang et al.


CG as well, which is in accordance with our previous find-

ings using gerbil as a global I ⁄ R model [28,30]. Finally, the

result of neurological score proved the prospective benefi-

cial action of CG on the outcome. The results by UPLC–

TOF-MS analysis showed that the protocol used can ensure

the appearance of the CG components in the plasma when

ShamI/R

8CG 0.4 + I/RCG 0.8 + I/R

6

*

**

*†

†

2

4

* * * *

* * * *

# #

# # #

††† #

Baseline 201 40 60 (minutes)

Num

ber

of a

dher

ent l

euko

cyte

s(p

er 2

00 μ

m v

enul

e)

0

I R

ba

dc

V

e fe f

A B

Figure 5. The effect of pretreatment with CG on the number of leukocytes adhering to venular wall. (A) Representative images of the sham group (a

and b), I ⁄ R group (c and d), and CG 0.8 g ⁄ kg + I ⁄ R group (e and f) acquired at baseline (a, c, e) and 60 minutes after I ⁄ R injury (b, d, f). Dotted line

arrows: adherent leukocytes; Actual line arrow: rolling leukocyte. Bar = 50 lm. (B) The time course of the number of adherent leucocytes in different

conditions. Compared with the I ⁄ R group, the number of adherent leukocytes in the CG administration groups decreased significantly starting from

20 minutes after reperfusion. Data are mean ± SE from six animals. I ⁄ R, ischemia and reperfusion; V, venule. *p < 0.05 vs. baseline, �p < 0.05 vs. sham

group, #p < 0.05 vs. I ⁄ R group.

A

a31 2 4a3a1 a2 a4

50 μm

Sham

CB

45

50

μm)

/s)

1.6

I/R CG 0.4 + I/R CG 0.8 + I/R

40

diam

eter

(

eloc

ity (m

m/

0.8

1.2

*

†

†

30

35

Venu

lar

d

RB

C v

e

0.4*

††

Baseline 1 20 40 60 (minutes)Baseline 1 20 40 60 (minutes)0

Figure 6. The effect of pretreatment with CG on rat cerebral cortical venular diameter and RBC velocity in venules. (A) The representative images of

venules observed by the high-speed video camera system. Arrows show the venules in the sham group (a1), I ⁄ R group (a2), CG 0.4 + I ⁄ R group (a3),

and CG 0.8 + I ⁄ R (a4). (B) The time course of the cerebral venular diameter. (C): The time course of the velocity of RBC in cerebral venule. Data are

mean ± SE from six animals. *p < 0.05 vs. baseline, �p < 0.05 vs. sham group.



A

CG 0.8 + I/R

I/R CG 0.4 + I/R

Sham

B C

volu

me

(%)

30

40

#

†

†15

20

l sco

res

#†

††

of i

nfar

ct v

10

20 #†

5

10

Neu

rolo

gica

l † †

Rat

io

0 0

N

CG 0.8 + I/R

I/R CG0.4 + I/R

Sham CG CG 0.8 + I/R

I/R0.4 + I/R

Sham

Figure 7. The influence of CG pretreatment on neuronal injury of rats subjected to focal cerebral I ⁄ R. (A) Representatives of TTC-stained brain

sections 24 hours after cerebral I ⁄ R. The white areas represent the infarct regions. (B) A quantitative evaluation of the influence of CG on infarct

volume. (C) Neurological scores 24 hours after MCAO. Grades of 3–18 were used. Administration of CG at 0.8 g ⁄ kg significantly improved

neurological function compared with the I ⁄ R group. Data are mean ± SE from six animals. �p < 0.05 vs. sham group, #p < 0.05 vs. I ⁄ R group.

D1A1 B1 C1

A2 B2 C2 D2

A3 B3 C3 D3

20 μm

10 μm

Figure 8. Nissl staining of cerebral cortex in penumbral region of rats. The arrows indicate Nissl-positive neurons. The double arrows indicate nuclear

pyknosis with karyorrhexis. a1, a2, a3: sham group; b1, b2, b3: I ⁄ R group; c1, c2, c3: CG 0.4 + I ⁄ R group; d1, d2, d3: CG 0.8 + I ⁄ R group. Data are

mean ± SE from six animals.

F. Wang et al.


I ⁄ R is initiated, and the CG components do exist in the rat

plasma over the observation of microcirculation when

given 90 minutes before ischemia. Brain edema following

I ⁄ R is the consequence of BBB disruption. Various mecha-

nisms are implicated in maintaining the integrity of BBB,

among which endothelium, basal lamina, and glial cell pro-

cesses are thought to be the prominent contributors

[1,11,25,27]. We did not undertake an experiment in the

present study to discriminate at which target CG exerted

action to attenuate brain edema. However, increasing evi-

dence demonstrates the close link between leukocyte

recruitment and breakdown of BBB [8,14]. The accumu-

lated leukocytes in postcapillary venules not only increase

the hydrostatic pressure within the vessels but also release

active oxygen species, which, in turn, impair the vessel wall

[2,9,10,20,26]. Thus, the preventing effect of CG on I ⁄ R-

induced leukocyte adhesion in venules found in the present

study, as well as in previous work [30], may account, at

least in part, for its favorite role in mitigating brain edema.

The CG is originally developed for treatment of cerebro-

vascular disturbance, such as headache and dizziness, which

is mostly associated with global cerebral ischemia. In an

animal model of global cerebral ischemia, we recently

reported the potential of CG pretreatment on I ⁄ R-induced

cerebral cortex microcirculatory disturbance [30]. Microcir-

culatory disturbance after I ⁄ R triggers the disruption of

BBB, and is exaggerated by the subsequent brain edema. In

the present study, we showed the protective effect of CG

on the cerebral vasculature disorder in a focal cerebral I ⁄ Rmodel that is more relevant to ischemic stroke in clinic,

suggesting a potential use of CG for improving the out-

come of the patients suffering from stroke.

The intravital microscopy in the present study revealed

that I ⁄ R caused only a transient decrease in blood velocity

while it had no effect on the venular diameter. On the

other hand, however, the number of nonperfused capillaries

in the area under investigation increased considerably in

animals subjected to I ⁄ R, in comparison with control,

implying a reduced blood perfusion in the affected terri-

tory. The rationale for the occurrence of nonperfused capil-

laries in the reperfusion phase remains unclear. One

possible mechanism is the activated leukocytes built up in

AShamI/R60B

a1 a2 a3 a4

NE

L po

sitiv

e ds

(%)

I/RCG 0.4 + I/RCG 0.8 + I/R40

†

†#

Num

ber

of T

UN

cells

/5 fi

eld

20#

†#

†

m2

N

0C

b4b3b2b1400

D

†

sitiv

e ce

lls/m

m

c3 c4c1 c2200

300

†

†

†#

umbe

r of

pos

d2 d3 d4d1

0

100†

†

†# #

#

#

#†

†N

Caspase-3 P53 Puma

Figure 9. The effects of CG pretreatment on the number of TUNEL-positive cells and expression of apoptosis-related proteins. (A) Representative

photomicrographs of TUNEL-positive cells in the sham group (a1), I ⁄ R (a2), CG 0.4 + I ⁄ R group (a3), and CG 0.8 + I ⁄ R group (a4). (B) The

quantitative analysis of TUNEL-positive cells. (C) Representative images of immunohistochemistry staining for caspase-3 (b), p53 (c), and PUMA (d) in

the sham group (1), I ⁄ R group (2), CG 0.4 + I ⁄ R group (3), and CG 0.8 + I ⁄ R group (4). The arrows indicate positive immunohistochemically stained

cells. (D) A quantification of immunohistochemistry staining positive cells for caspase-3, p53, and PUMA in different groups. Bar = 50 lm. Data are

mean ± SE from six animals. �p < 0.05 vs. sham group, #p < 0.05 vs. I ⁄ R group.



the capillaries, obstructing the blood flux. Nonetheless,

these nonperfused capillaries will exaggerate ischemia, and,

along with the adherent leukocytes in venules and BBB dis-

ruption, lead to neuron damage. Interestingly, CG pretreat-

ment attenuated I ⁄ R-evoked capillary nonperfusion as well,

particularly at the dose of 0.8 g ⁄ kg.

We have previously reported that administration of CG to

gerbils ameliorated hippocampal CA neuron damage caused

by global I ⁄ R [28]. Likewise, CG was found to protect cortex

neurons from focal cerebral I ⁄ R injury in the current study,

as documented by the results of TTC staining, Nissl staining,

and electron microscopy. In the acute phase of ischemia, cell

death in the ischemic core is commonly considered necrotic,

whereas after a short period of cerebral ischemia followed by

reperfusion, the neurons in the penumbral regions undergo

another wave of delayed cell death, which is dominated by

apoptosis [4,32]. Consistent with others, we observed a dras-

tic increase in apoptosis 24 hours after reperfusion in the

penumbral regions of cortex in response to I ⁄ R challenge, as

evidenced by the increase in the number of both TUNEL-

positive and caspase-3-positive cells. Noticeably, the

enhanced apoptosis in the cerebral cortex evoked by I ⁄ R was

ameliorated significantly by pretreatment with CG. Neuron

death is the consequence of a cascade of reactions in

response to I ⁄ R challenge, which take place sequentially and

interplay with each other. The complexity of neuron death

process is superimposed by the multiple components of CG,

which renders it extremely difficult to elucidate the mecha-

nism underlying the effect of CG on I ⁄ R-induced neuron

injury. However, some clues emerge that may plausibly shed

light on this issue. In view of the critical importance of oxi-

dative stress in the initiation of neuron death process, the

antioxidant potential of CG may contribute most to the

observed attenuation effect on neuron death. To this end,

we have previously reported that CG is able to ameliorate

I ⁄ R-induced hydrogen peroxide production [30]. Consistent

with this finding, available evidence from in vitro studies

revealed that at least six chemicals derived from the com-

posed herbs exhibit antioxidant potential; they are: paeoni-

florin from Radix paeoniae alba [22], ligustrazine from

Rhizoma chuan xiong [7], ferulic acid from Radix angelicae

sinensis [23], rehmannioside from Radix rehmanniae prepara-

A1 B1 C1

A2 B2 C2

A3 B3 C3

E

,

E

S

N

N

Figure 10. The effect of CG pretreatment on the ultrastructure of cerebral cortex in penumbral region of rats subjected to focal cerebral I ⁄ R. The

brains were processed for electron microscopy 24 hours after reperfusion. a1, a2, a3: sham group; b1, b2, b3: I ⁄ R group; c1, c2, c3: CG 0.8 + I ⁄ Rgroup. The electron micrographs of low magnification for different conditions are shown in Panel 1, where the capillaries and neurons enclosed in

rectangles are further enlarged in Panels 2 and 3. N, nuclei of neurons; S, swelling perivascular astroglials; E, swelling endothelial cells.

F. Wang et al.


ta [21], genistein from Caulis spatholobi [16], and alaternin

from Semen cassiae [5]. In the present circumstance, these

antioxidants most likely work coordinately, yielding an addi-

tive effect. Secondary, the CG-caused attenuations in cere-

bral microcirculation disturbance, BBB disruption and the

resultant brain edema, as observed in present study, may

lend themselves to blunt the development of neuron death,

as obstruction of these insults improves the cerebral oxygen

supply, on the one hand, and alleviates the inflammatory

reaction, on the other hand. Finally, the fact that CG attenu-

ated neuron apoptosis after I ⁄ R and, meanwhile, depressed

the I ⁄ R-enhanced expression of p53 and PUMA suggests

that the pathway of p53 through PUMA to caspases is at

least one of the targets at which CG acts to protect neuron

from apoptosis after I ⁄ R. We admit that much more works

are required to elucidate the mechanism. The speculations

above are to point to the likelihood for future exploration of

the rationale behind the CG action observed in the present

study.

In conclusion, CG is able to alleviate the brain edema

induced by focal cerebral I ⁄ R in addition to attenuation of

microcirculatory disturbance and neuron damage, provid-

ing a potentially alternative prophylactic management for

the patients in imminent danger of stoke.

PERSPECTIVE

Cerebral microcirculatory disturbance, neuron injury, and

brain edema following I ⁄ R is a complicated pathological

process involving multiple insults. To interfere with this

process, the efforts so far made are targeting a single insult,

and commonly with a poor outcome in clinic. The fact that

CG, a compound medicine consisting of numerous ingredi-

ents, is able to ameliorate I ⁄ R-induced cerebral microcircu-

latory disturbance, neuron injury, and brain edema

suggests a possibility of using a medicine containing multi-

ple components as a protective strategy for the patients in

imminent danger of stoke in clinical practice.

ACKNOWLEDGMENTS

This study was supported financially by the Production of

New Medicine Program of Ministry of Science and Tech-

nology of the People’s Republic of China (2008ZX09401).

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