In previous studies, we have investigated
enhanced thromboxane (TX) biosynthesis, as reflected by the
urinary excretion of a major TXA2 metabolite, 11-dehydro-TXB2,
in patients with acute ischemic stroke. Increased TX production
was found to occur episodically during the first 2 to 3 days
after the onset of ischemic stroke,1,3 a noninvasive index of
platelet activation, was present in the chronic phase after a
transient ischemic attack (TIA) or stroke.1,3.
It has been demonstrated that plasma indexes of
6-Keto-PGF1a/TXB2 disequilibrium or hemorrheological
abnormalities possibly participate in nosogenic patho-physiological
mechanisms of cerebrovascular disease.4,8
For the restriction of clinical research, rat
models with cerebral ischemia has become an indispensable tool
in the study of mechanism and prevention of cerebral disease. It
is important for hemorrheological detection in the onset,
prognosis and prevention of cerebrovascular disease (CVD).9,10
As is known to now, TXB2 is one Vol. 18, No1-2, 2009 / Revista
Ecuatoriana de Neurología 33 of the strongest vaso-excitor
material(VEM) and platelet aggregation agent that can promote
thrombosis. As a platelet functional inhibitor, 6-Keto-PGF1
alpha has protective effect for vasospasm caused by platelet
aggregation. They keep balance under physiological status.4,5
The purpose of the study is to monitor
hemorrheology and plasma contents of TXB2 and 6-Keto-PGF1 alpha
24 hours after cerebral ischemia using middle cerebral artery
occlusion (MCAO) model, bilateral common carotid artery ligation
(BCCA) model, and unilateral common carotid artery ligation (UCCA)
model, to find difference between them and to screen the most
similar clinical feature model.
Materials and methods
Animal protocols were approved by the Stanford University
Administrative Panel on Laboratory Animal Care. Institutional
guidelines were followed in all protocols. All animal
experiments were conducted in accordance with the NIH guide for
the care and use of laboratory animals (NIH publication 80-23).
All efforts were made to minimize animal suffering, and only the
smallest numbers of animals were used to generate reliable
Animals and Experimental groups. Adult
male Sprague-Dawley rats, weighing 200-250 g, were obtained from
the Experimental Animal Center of Beijing University, China.
They were maintained under controlled lighting (lights on
07:00–19:00 h) and temperature (22º C) and given free access to
water and the commercial laboratory rodent diet. Twelve hours
prior to experiment, the rats were fasted, but allowed free
access to water. They were randomly divided into four groups (32
rats per group): MCAO group, BCCA group, UCCA-group and normal
Middle cerebral artery occlusion model.
Male Sprague-Dawley rats weighing between 290 and 320 g (Charles
River, Wilmington, Del) were anesthetized with 3% halothane by
facemask and were subsequently maintained with 1% halothane in
200 ml/min oxygen and 800 ml/min air. Depth of anesthesia was
assessed every 15 min by hind-limb pinch. A thermistor probe was
inserted 50 mm into the rectum and rectal temperature was
maintained between 36.5ºC and 37.5ºC during ischemia. ECG leads
were placed to monitor heart rate and respirations.
Physiological parameters were monitored every 15 min and
maintained in the normal range throughout surgery. The MCA was
occluded using an intraluminal suture pre viously used by our
lab.11,12 In brief, a midline incision was made in the neck to
expose the common carotid (CCA), external carotid (ECA),
internal carotid (ICA), and pterygopalatine (PPA) arteries. The
CCA, ECA, and PPA were ligated with a 6-0 silk suture. Ischemia
was induced by inserting an uncoated, 30-mm long segment of 3-0
nylon monofilament suture (tip rounded by flame) 19-20 mm from
the bifurcation of the CCA to induce ischemia in the arterial
territory supplied by the MCA. After 2 h of ischemia, the suture
was removed and the animal was allowed to recover. After
recovering from anesthesia, the rats were allowed free access to
food and water. Behavioral tests were performed in rats by Zea
longa 5 cent method of preparation, the method for assessing
praxiology of rat, animals with 1-3 cents will be selected.13
Bilateral common carotid artery ligation model.
All procedures were performed under anesthesia with the
intraperitoneal injection of pentobarbital (50mg/kg). For
chronic bilateral occlusion, five days prior to angiography, the
left CCA was exposed through a midline cervical incision
under anesthesia and then it was ligated by 6-0 nylon suture,
and cut by microscissors. The wound was thereafter
closed with a suture.14
After recovering from anesthesia, the rats were
allowed free access to food and water. All rats were observed
at room temperature for 24h.
Unilateral common carotid artery ligation
model. All procedures were performed under anesthesia with
the intraperitoneal injection of pentobarbital (50mg/kg). For
acute unilateral occlusion, the right ICA was carefully exposed
through a midline cervical incision, and a microsurgical clip (Zen
temporary clip, Oowa-Tsusho, Tokyo, Japan) was applied to the
proximal part of the ICA to acquire the images of immediately
after occlusion. After recovering from anesthesia, the rats were
allowed free access to food and water.14
Statistical analysis. Statistical analyses
for continuous data were performed using a one-way analysis of
variance followed by a multiple comparison procedure (Bonferroni
post-hoc test). All data are expressed as mean±S.E.M. P﹤<0.05
was considered significant.
Main instruments and reagent. Heparin (Jiangsu
wanbang biochemistry medicine limited company, Batch No:
0507110,12500u ),red cell deformity reagent (Peking sidi
scientific instrument company , Batch No: 060628, 100ml/bot).
The LG- R-80 type blood viscosity tester (Peking sidi scientific
instrument company ), the LG- B-190 type cytomorphosis/
aggregation tester (Peking sidi scientific instrument company ),
GL-20 type completely automated high speed refrigerated
centrifuge (xiangxi instrument gauge total factory),
thromboxanB2 (TXB2) and 6-keto-prostacyclinF1a radio-immunity
kit (Peking kemeidongya biotechnology limited company).
Hemorheological detection. Blood samples
were collected from abdominal aorta 24h after cerebral ischemia.
The index of hemorheolgy included the whole blood viscosity,
reduced viscosity, plasma viscosity, hematocrit, the index of
red cell deformity.
1: Comparison of hemorheological index after
cerebral ischemia 24h between 3 model groups with
normal group. Mean±SEM. n=8. *P<0.05, **P<0.01 vs
All rats abstained from food but could not
refrain from water 24h before taking blood sample. They were
anesthetized with 10% Chloral Hydrate intraperitoneal injection
the next day, taking blood sample from abdominal aorta. 6ml
blood sample was kept in an anticoagulation cuvette with heparin
100μl inside, with LG- R-80 type blood viscosity tester and LG-
B-190 type cytomorphosis/aggregation tester , at 37 ºC ± 0.1.
Whole blood hypsi-tomy coefficient of viscosity (shear rate
150s-1),hypo-tomy coefficient of viscosity (shear rate 5s-1),
plasma viscosity, index of red blood cell deformation and
aggregation were detected. The HCT determination used decigram
method centrifugation in common temperature with rotary speed
3000 r/min for 30 min.
Determination plasma contents of TXB2, 6-Keto-PGF1a.
24h after cerebral ischemia, blood samples from abdominal aorta
were taken from all rats. Each group of rats abstained from food
but could not refrain from water 24h before taking blood sample.
Next day, 10%Chloral Hydrate was used to anesthetize. Through
abdominal aorta blood sample was taken: 3ml in anticoagulation
cuvette with 0.2 ml 10%EDTA. After misce bene, GL-20 type
completely automated high speed refrigerated
centrifuge was used , 4°C, 3500 r/min, for 15 min, and conserved
at -20°C for use. Determination for plasma contents of TXB2 and
6-Keto-PGF1a with radio-immunity proceeded according to
description of kit.
1.Comparison of hemorheological index after 24 hours of cerebral
ischemia between 3 model groups and normal group.
There were significant differences for indexes of
rats between the three cerebral ischemia groups and the normal
group(P<0.05). The whole blood viscosity value, plasma viscosity
value and hematocrit of rats in MCAO- roup were the highest
among the three model groups, their values were (19.29±2.996,mPa•s/5s-1,
(1.812±0.348) mPa.s and (47.5±5.28)%. Meanwhile the deformity
index of RBC 0.47±0.017, of rats in MCAO-group was significant
lower than that of rats in normal-group, (P<0.05).
The results showed that the expression of
Erythrocyte aggregation in 3 model groups was enhanced but there
was no difference between 3 model groups. (P>0.05). There were
no significant differences for other index of hemorheological
change in rats among the three cerebral ischemia groups
(P>0.05). Results are listed in Table1.
2. Comparison of plasma contents of TXB 2, 6-Keto-PGF1a
after 24 hours of cerebral ischemia, between 3 model groups and
The results showed that the expression of TXB2 in
3 model groups was enhanced, but plasma contents of 6-Keto-PGF1
was lower. There was significant differece between 3 model
groups and normal group (P<0.05). The content of TXB2 of MCAO-group
was the highest in the three cerebral ischemia groups. There was
significant difference for plasma contents of TXB2 between MCAO-group
and UCCA group (P<0.05), but no significant difference for
plasma contents of TXB2 between MCAO-group and BCCA group
There was no significant difference for plasma
contents of 6-Keto-PGF1a between three cerebral ischemia groups
(P>0.05). Results are listed in Figure 1.
Figure1: Comparison of plasma contents of TXB2, 6-Keto-PGF1a
after cerebral ischemia 24h between 3 model groups
with normal group. Mean ± SEM. n=8. *P<0.05,
**P<0.01 vs normal group.
MCAO-group vs UCCA-group, ˆP<0.05.
3. Comparison of plasma indexes of 6-Keto-PGF1a/TXB2
after 24 hs of cerebral ischemia between 3 model groups with
Based on results of plasma contents of TXB2,6-Keto-PGF1a 24hs
after cerebral ischemia, ratio of 6-Keto-PGF1a and TXB2 was
figured out. Results showed that plasma indexes of 6-Keto-PGF1a/TXB2
of three cerebral ischemia groups were lower than that of normal
group, but there was significant difference of plasma indexes of
6-Keto-PGF1a/TXB2 between MCAO-group and normal group (P<0.05).
There was no significant difference among the three cerebral
ischemia groups (P>0.05). Result are shown in Figure 2.
Figure 2: Comparison of plasma indexes of 6-Keto-PGF1a/TXB2
24h after cerebral ischemia between 3 model groups
with normal group.
Mean±SEM. n=8. *P<0.05, **P<0.01 vs normal group.
Hemorheological detection is important in the course of disease,
including nosogenesis, development, prognosis, turnover, as well
as curative effect assessment, especially for prevention of
cardiocerebrovascular disease. Brain microcirculation slow down
or stagnation is the main pathological change at the beginning
of cerebral ischemia4 TXB2 is one of the strongest vaso-excitor
material (VEM) and platelet aggregation agent at present. 6-Keto-PGF1a
is the platelet function inhibitor, have protective effect for
vasospasm caused by platelet aggregation, they keep balance
under physiological status.4,5,15 Disequilibrium of TXA2 and
PGI2 caused by cerebral ischemia is one of the reasons of
platelet aggregation, vasospasm and thrombogenesis.
There were significant differences between the
three cerebral ischemia groups and normal group for indexes of
whole blood viscosity value, plasma viscosity value, hematocrit
and plasma contents of TXB2, 6-Keto-PGF 1a, plasma indexes of 6-Keto-PGF1a/TXB2(P<0.05).
Results showed that disequilibrium of TXA2 and PGI2 is included
in the of cerebrovascular disease, which is similar with that of
abroad6,8 The whole blood viscosity value, plasma viscosity
value, hematocrit and plasma contents of TXB2 of rats in MCAO-group
were the highest among the three model groups. Index of red
blood cell deformation is the lowest in MCAO group. There is
significant difference of rats between MCAO-group and normal
group with plasma indexes of 6-Keto-PGF1a/TXB2 (P<0.05). This
may happen because model of MCAO bring damage to the brain most,
but there were no significant difference for indexes of
hemorrheology and plasma contents of TXB2,6-Keto-PGF1a between
the three cerebral ischemia groups (p>0.05). Although different
models bring out different degree of injury to the brain, all 3
models have effect on hemorrheology, plasma of TXB2 and 6-the
Keto-PGF1a. It has been demonstrated that hemorrheology
abnormality or plasma indexes of 6-Keto-PGF1a/TXB2
disequilibrium are important in cerebrovascular disease,4,5,15
to display the abnormal hemorrheological changes: TXB2 obviously
to heighten, but 6-Keto-PGF1a to reduce or change slightly.
Results showed that abnormal change of
hemorrheology – increased TXB2 and reduced plasma indexes of 6-Keto-PGF1a/TXB2-
after cerebral ischemia, is similar to domestic and abroad
result,6,7,16,17 Cerebrovascular diseases often affect
latero-vascular territory. MCAO-model (middle cerebral artery
occlusion model) has been accepted by international clinics,
therefore being the most suitable cerebral ischemia model, which
shows obvious changes in hemorheology and plasma contents of
TXB2, 6-Keto-PGF1a and has characteristic features of
We are thankful to Mr. Congshun Song and Mr. Pengwen Wan for
their help in experiment and manuscript preparation. This work
was supported partially by the National Point Foundation
Research Item of People’s Republic of China (Program Grant #
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