Original Paper 817
We have reported that pretreatment with isosteviol relieved is-
chemia-reperfusion (IR) injury in Langendorf perfused guinea
pig heart in vitro, and reduced infarct volume of myocardium in-
duced by occluding and re-opening the coronary artery in rat in
vivo [12], [13]. However, whether isosteviol possesses similar ef-
fects on the brain has not been studied to date. This study was
designed to demonstrate the effects of isosteviol on ischemia-re-
perfusion (IR) injury in rat brain induced by MCA occlusion–re-
perfusion.
the filament blocked the origin of the MCA and occluded blood
flow from the internal carotid artery. Two hours after the induc-
tion of ischemia, the filament was slowly withdrawn by 10 mm,
causing a re-opening of the MCA and starting the reperfusion
process. The incision was closed and the rat was then returned
to the cage and monitored until fully recovered from the anes-
thesia. The reperfusion was maintained for 22 h. The rat was al-
lowed free access to food and water. In sham–operated rats, the
carotid arteries were surgically exposed, but no filament was in-
serted. During surgery, the body temperature of the rat was
monitored using a rectal thermometer and maintained at
37 ± 0.5 °C using a lamp.
Materials and Methods
!
Chemicals
Assessment of neurological deficits
Stevioside was purchased from Stevioside Sweetener Factory,
Nankai University (Tianjin, China). Isosteviol was obtained by
the acidic hydrolysis of stevioside according to procedures previ-
ously described in the literature [5]. The structure of the yielded
isosteviol was confirmed by nuclear magnetic resonance spec-
troscopy, infrared spectroscopy and determination of melting
point (212 °C). The purity of the isosteviol (99%) was determined
by high-performance liquid chromatography. Isosteviol was dis-
solved in normal saline for administration to the rats. Nimodi-
pine (purity 99.5%) was provided by Shanghai Institute of Mate-
ria Medica, Chinese Academy of Sciences (Shanghai, China) and
also dissolved in normal saline for administration. Triphenylte-
trasodium chloride (TTC) was purchased from Sigma.
Neurological deficits in the rats were determined after 22-hour
reperfusion. Neurological findings were scored on a 5-point
scale in which 0 is for no neurological deficits, 1 for failure to ex-
tend left paw fully (mild neurological deficit), 2 for circling to the
left (moderate neurological deficit), 3 for falling to left (severe
neurological deficit), and 4 for failure to walk spontaneously
and/or unconsciousness [15]. Except for the SO rats, any rat
without neurological deficits after 22-h `reperfusion' was con-
sidered as an unsuccessful model and excluded from the study.
Determination of infarct volume in the brain
Infarct volume in the brain was evaluated using the TTC staining
method [6]. Briefly, after 22-h reperfusion the rat was anesthe-
tized by pentobarbital (60 mg/kg, i.p.) and killed by decapita-
tion. The brain was quickly removed and placed in ice-cold saline
for 5 min, and then cut coronally into 5 consecutive 2-mm thick
sections. The brain slices were then immersed in 2% TTC solu-
tion at 37 °C for 15 min where normal (non-infarct) tissue was
stained red and necrotic or apoptotic (infract) tissue remained
white. The stained brain sections were fixed in 10% formalin
overnight. Infarct area was measured using Medical Image
Analysis System (BI-2000; Taimeng Science & Technology Pty
Ltd). Infarct volume was calculated as the product of the infarct
area and the thickness of the section and data were expressed
by percentage of the infarct volume to the total volume of the
brain.
Animals
Adult male Sprague-Dawley rats (250 to 300 g, 10 weeks old)
were purchased from the Experimental Animal Center of the
Nanjing Medical University. The rats were kept at a constant
room temperature of 22 °C under 12 h light-dark cycle and free
access to tap water and standard food. Rats were randomly
divided into six groups (n = 30): a sham-operation (SO) control
group and 5 IR groups. The IR groups included an IR vehicle con-
–1
trol, isosteviol low (Iso L + IR, 5 mg·kg ), middle (Iso M + IR,
–
1
–1
1
0 mg·kg ) and high (Iso H + IR, 20 mg·kg ) dose group respec-
–1
tively, and nimodipine (nimodipine + IR, 5 mg·kg ] as a positive
control group. The vehicle, isosteviol or nimodipine was pre-ad-
ministrated intravenously through the left femoral vein. Of the
3
0 rats in each group, 24 rats were used for assessment of neuro-
Determination of contents of malondialdehyde (MDA)
and superoxide dismutase (SOD) activity in brain tissue
After the reperfusion, the right hemisphere of the brain was
weighed and homogenized with buffer (pH 7.4) consisting of
10 mM sucrose,10 mM Tris-HCl and 0.1 mM EDTA, and then cen-
trifuged at 3000×g for 15 min at 4 °C. The contents of SOD and
MDA in the supernatant of cerebral tissues were determined by
the xanthine oxidase method [17] and thiobarbituric acid meth-
od [18].
behavioral deficit and infarct volume (n = 8), determination of
malondialdehyde and superoxide dismutase in brain tissue
(n = 8), and general histological examination (n = 8); the remain-
ing 6 rats were used for immunohistochemical testing for Bcl-2
and NF-κB.
Preparation of cerebral ischemia models
All experimental protocols were approved by our institutional
Committee on Animal Research. The rats were anesthetized
with chloral hydrate (300 mg/kg) injected intraperitoneally
Histological examination of brain tissue
(
i.p.). Cerebral ischemia models of rats were prepared using the
Twenty-two hours after reperfusion of the brain, the rats were
anesthetized with pentobarbital and killed by opening the chest.
The brain was perfused with 200 mL of cold normal saline (4 °C),
followed by 200 mL of 4% paraformaldehyde solution through
ascending aorta. The brain was removed and immersed in 10%
formaldehyde for 7 days and then embedded with paraffin. The
tissue in paraffin was cut into 5 μm thick coronal sections and
stained with hematoxylin and eosin. The morphological changes
in brain tissue (in particular in cortical area and hippocampus
CA1 region) were examined by microscopy.
intraluminal filament technique, as described in detail previous-
ly [14]. Briefly, a midline longitudinal incision of approximately
2
mon carotid artery (CCA), external and internal carotid arteries
were exposed. Twenty minutes after the injection of test com-
pounds or vehicle, a nylon monofilament (diameter: 0.28 mm)
coated with silicone resin/hardener mixture was inserted into
the internal carotid artery for a length of 17–20 mm from the
CCA bifurcation until a slight resistance was felt. At this point,
.0 cm was made in the cervical area of the rat. The right com-
Xu Det al. The Neuroprotective Effects… Planta Med 2008; 74: 816–821