12 J
and chemical-induced lung tumorigenesis [25]. There is each brain slice was defined by the 2,3,5-triphenyltet-
also considerable epidemiological evidence suggesting that razolium chloride TTC staining method. After 48 h
the consumption of green tea lowers the risk of heart reperfusion, the gerbils received an intracardiac perfusion
disease as well as several types of cancer incidences as a of 0.9 buffered saline. The brain was then removed, and
result of these antioxidant mechanisms [1]. cut into 2-mm serial slices starting 1 mm from frontal pole.
In the present study, the protective effect that green tea The coronal slices were then immersed in a 2 phosphate-
extract has on ischemia reperfusion-induced brain injury buffered solution for 50 min at 378C. After TTC staining,
was examined. In particular, the ischemia reperfusion- the slices were fixed in 10 phosphate-buffered formalin
induced production of hydrogen peroxide, lipid peroxida- and the infarction area was then determined by an image
tion and oxidative DNA damage formation of 8-hydroxy- analyzer using the leicaQwin programme Leica Mi-
deoxyguanosine, and cell death as well as locomotor crosystem Image Solution Ltd., Cambridge, UK. The
2
activity on the Mongolian gerbil were focused. infarct area mm from each thicken-brain slice was
3
determined, and the infarct volume mm was calculated from sum of the slice areas 7 slices in all3thickness 2
2. Materials and methods mm.
2.1. Animals 2.4. Determination of hydrogen peroxide level, lipid
peroxidation and 8-hydroxydeoxyguanosine 8-oxodG
The female Mongolian gerbils used were maintained in accordance with the National Institute of Toxicological
The level of hydrogen peroxide in the whole brain tissue Research of the Korea Food and Drug Administration
was measured using the BIOXYTECH H O -560 assay kit
2 2
guideline for the care and use of laboratory animals. The OXIS International Inc., USA. The formation of malon-
female Mongolian gerbils body weight 50–60 g were aldehyde and 4-hydroxynonenal, as lipid peroxidation
housed 4–5 per cage and maintained at 22628C with a products in the whole brain homogenate was also de-
constant humidity for at least 1 week prior to the com- termined using a lipid peroxidation assay kit OXIS
mencement of the experiments. All the animals were International Inc., Ohio, USA according to the methods
allowed free access to food and water before and after described in the manufacturer’s protocol. For the determi-
ischemia surgery. The 2 doses 0.5 or 2 of green tea nation of 8-oxodG, isolated DNA from whole brain
extract were added into the drinking water and were homogenate was purified and digested as described previ-
accessed by animals for 3 weeks ad libitum before the ously [5]. The digested DNA was then analyzed by the
induction of ischemia. The method of green tea extraction ELISA assay kit Japan Institute for the Control of Aging,
and compounds in the extracts are described elsewhere [8]. Nikken Foods Co., Japan to detect 8-oxodG formation.
Briefly, dried green tea leaves were extracted with 70 ethanol at 90–958C for 6 h. The extract was then fraction-
2.5. Detection of apoptotic cells ated by column chromatography using Amberite XAD-7
mean pore diameter, 90 A. The total catechin content of The terminal deoxynucleotidyltransferase TdT-medi-
the extract was determined by UV spectroscopy, and the ated dUTP-biotin nick end-labeling TUNEL method was
composition was determined using HPLC. used for the detection of apoptotic bodies. The brains were
fixed with 4 phosphate-buffered paraformaldehyde pH 2.2. Ischemic surgery
7.4, and stored in 4 phosphate-buffered paraformal- dehyde pH 7.4. After 24 h, they were transferred into a
The acclimatized Mongolian gerbils were anesthetized 20 phosphate-buffered sucrose solution and cut into 10
with a gas mixture of 2 isoflurane, 75 N O and 25 mm cryostat coronal slices starting between 7 and 9 mm
2
O . The bilateral common carotid arteries were occluded from the frontal pole. The tissue sections were fixed with
2
using sugita aneurysm clips for 5 min. During the occlu- 4 phosphate-buffered paraformaldehyde pH 7.4 for 30
sion and postoperative period, the gerbils were kept on min at room temperature, and then incubated with a
thermostat-control warming plates in order to maintain blocking solution 3 H O in water for 5 min at room
2 2
body temperature at 378C. Following the occlusion, the temperature. This was followed with immersion in a
clips were removed to restore the blood flow. The same permeabilization solution 0.1 Triton X-100 in 0.1
surgical operated animals without carotid ligation were sodium citrate for 2 min on ice. 50 ml of the TUNEL
served as sham control animals. mixture Boehringer Mannheim, USA was deposited on
the tissue sections and incubated in a humidified chamber 2.3. Morphometric determination of infarct volume
for 60 min at 378C. A 50 ml Converter-POD solution horse-radish peroxidase, Boehringer Mannheim was then
For detection of the ischemia infarction area of the added and then further incubated for 30 min at 378C.
brain, the cross-sectional infarction area on the surfaces of Subsequently, a 100 ml DAB 3,39-diaminobenzidine
J .T. Hong et al. Brain Research 888 2001 11 –18
13
tetrachloride substrate solution was added onto tissue tein in brain of animals pretreated by 0.5 and 2 green tea
sections for 10 min at room temperature. After each step, for 3 weeks, respectively. Consistent with the hydrogen
the tissue sections were rinsed twice with a phosphate- peroxide level, the level of lipid peroxidation products
buffered saline solution PBS, pH 7.4. The apoptotic malonaldehyde and 4-hydroxynonenal in the animals had
bodies were identified under microscope 3200 as con- also increased as a result of ischemia reperfusion from
taining brown colored nuclei. The quantity of apoptotic 1020660 to 14106210 nmole mg protein. The animals
bodies were expressed as the average number of apoptotic pretreated with green tea had substantially reduced lipid
cells per high power field visible apoptotic cells HPF. peroxidation products especially the ones that were ad-
ministered the 2 extract. The malonaldehyde and 4- 2.6. Measurement of locomotor activity
hydroxynonenal levels were 930640 and 330620 nmole mg protein in brains of animals pretreated with 0.5 and 2
The locomotor activity was determined by the distances green tea for 3 weeks, respectively Fig. 2B. The 8-oxodG
traveled by the animals for 1 h counts cm in the level had also increased in brains of ischemia reperfusion
22
locomotor activity apparatus OPTI-Varimex , Columbus animals 3.960.1 ng mg DNA, 310
compared to the
22
Instrument, Ohio, USA. Spontaneous locomotor activity control animals 2.160.7 ng mg DNA, 310
. These
22
was monitored by a single break defined as an activity in values were reduced to 2.860.3 ng mg DNA, 310
,
22
the photocell light beam emitted from 15 sites in the 1.960.3 ng mg DNA, 310
as a result of pretreatment photocell chamber.
with 0.5 or 2 green tea for 3 weeks, respectively Fig. 2C.
2.7. Statistics 3.3. Formation of apoptotic cells
The data was expressed as the mean6standard error. The data was analyzed with a one-way analysis of the
In order to determine whether green tea ingestion had variance followed by either Dunnett’s or Bonferroni’s
the effect of reducing the level of oxidative damage to method as a post hoc test. Differences were considered
macromolecules and hence prevent neuronal death, the significant at P,0.05.
level of apoptotic cell formation was measured. In the preliminary study, it was found that the number of
surviving neurons was greatly reduced in the striatum and
3. Results cortex as well as the hippocampus [16]. Therefore, neuro-
