2. hr light and dark cycle), 60% relative humidity

 

2. Materials and Methods

2.1. Animals

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The protocols of the present study were done based on the acknowledged
standards of animals care and use that approved by the Institutional Care and
Use of Animals Committee of Baqiyatallah University of Medical Sciences. To
perform the current study, male Wistar rats, weighing approximately 280-320 g, were
purchased from the animal house facility center of the University of Baqiyatallah
Medical Sciences. The rats were kept in a standard situation with controlled
light period (12 hr light and dark cycle), 60% relative humidity and
temperature (22-24°C), and also ad
libitum access to the rat chow and water.

2.2. Preparation of hydroalcoholic extract of Dorema aucheri leaves

Dorema aucheri leaves were collected around the Zagros mountain
ranges. After drying the leaves under the shade and ground, an electrical
grinder was used to powder the Dorema aucheri leaves. Then, the maceration
method was applied to prepare the extraction. Macerating the powder was
performed by using 70% ethanol and 30% water for 72 hr at room temperature. The
Whatman filter paper (No 1) was used to filter the mixture. After centrifuging
the filter (3000 rpm, 20 min), the supernatant was evaporated at ambient
temperature. Then, the extract was dried at room temperature and kept at 4?C
until used. The obtained semisolid mass was freshly used for the daily
treatment of rats.

2.3. Development of brain ischemia-reperfusion

In the present study, we used the intraluminal filament method to
achieve brain ischemia-reperfusion by the middle cerebral artery occlusion
(MCAO) in the right hemispheres of the ischemic rats, which previously described in detail by Longa et al,. 21. First, the
rats were anesthetized with 2.5% isoflurane (Forane, UK). Surgery was performed
in the dorsal recumbent position. Core temperature of animals was continuously
recorded and maintained at 37±1ºC by using a heating pad and lamp during the
surgery. After exposing the right common carotid artery and also the right external
and internal carotid arteries through a midline incision in the neck area, a
4-cm Poly-L-Lysine-coated nylon thread (3-0) was inserted into the internal
carotid artery via the external carotid artery. The prepared filament smoothly advanced
up until feeling a resistance was met and seeing a sharp decline in the blood
flow trace, which was recorded using a laser Doppler flowmeter (AD Instrument,
Model: ML191, Australia). There was a 75-85 % reduction in regional
cerebral blood flow (rCBF) of ischemic zones during the MCAO (results not
shown). After 90 min MCAO, reperfusion phase was initiated by gently taking out
of  the filament to reestablish the blood
flow to the ischemic areas 22. All incisions were ultimately sutured and the rats were recovered
from anesthesia.

2.4. Experimental protocols and grouping

The surgery was
performed in the rats of sham group (n=12) at the neck area without induction
of the middle cerebral artery (MCA) occlusion. The rats of control ischemic group
(IR, n=12) underwent the surgery at the neck region same as sham group
to achieve MCAO. Then, brain ischemia-reperfusion was developed by 90 min occlusion
of the MCA followed by twenty-four hr reperfusion. The rats of pretreated
ischemic group (IR+D. aucheri, n=12) received the Dorema aucheri
extract orally for 14 days (200 mg/kg/day in distilled water), and then
brain ischemia-reperfusion was done. Other procedures were followed in the same
way of control ischemic rats. After termination of the surgical
procedures, the rats returned to a warm cage for recuperation during the reperfusion
period. The rats that survived during twenty-four hr reperfusion period are the
number of animals presented for each group.

2.5.
Evaluation of brain infarct volume

Based on the method of 2, 3,
5-triphenyltetrazolium chloride (TTC, Sigma) staining, we evaluated the brain
infarct volume. “In brief, the brains were separated under deep anesthesia,
cleaned, and solidified by immersing in pre-cooled normal saline (4oC).
Then, six slices were prepared from each brain using brain matrix. The slices
were stained with TTC solution (2%) and fixed in 10% buffered formalin
solution. After staining, the color of the non-ischemic areas was red and of
ischemic areas was white. The slice images were digitized by using a Cannon
camera. Images of the stained sections were taken. Grossly visible infarction
zones were quantified using image analysis software (NIH Image Analyzer).
Cerebral infarct volume for each hemisphere was calculated by the sum of
infarct sizes for six slices and multiplying by 2 (thickness of each slice)”
23. The following formula was used to calculate the corrected infarct
volume for edema. Finally, the values of brain infarction were expressed as mm3.

Corrected infarct
volume= Left hemisphere volume – (Right hemisphere volume – Measured
infarct volume)

2.6.
Determination of tissue swelling for ischemic hemispheres

The
percent of tissue swelling for ischemic (right) hemisphere was performed as
follow; first, the total volume of each
hemisphere (right and left) was determined by the sum of hemisphere sizes for
six slices and multiplying by 2 (thickness of each slice). Then, the percent of tissue swelling was calculated using the
following formula:

Tissue swelling (%)= (VRight Hemisphere-VLeft Hemisphere)/VLeft
Hemisphere × 100

2.7. Evaluation of
brain antioxidant capacity and oxidative damage

Ischemic (right) hemispheres were quickly
removed under deep anesthesia for evaluation of the antioxidant factors and
oxidative damages. First, the ischemic hemispheres were weighed and washed in
an ice-cold phosphate buffer saline (PBS). After homogenization of the
hemispheres in ice-cold PBS (1:10), the homogenized solutions were centrifuged
at 14000×g at 4?C for 15 min. Then, supernatants were used to
determine the brain contents of glutathione (GSH), malondialdehyde (MDA) and
nitrate as well as the activities of superoxide dismutase (SOD) and catalase. Ultimately,
the method of Bradford was used to quantify the protein content of each
hemisphere for calculation of data 24.

Glutathione
levels of the ischemic hemispheres: According
to the method of Tietz, the glutathione levels of ischemic hemispheres were assessed
25. First, by adding sulfosalicylic acid (5%), the cellular protein
was precipitated. After centrifugation of solution at 2000 g for 10 min the
supernatant was removed and glutathione level was assayed  as follows: “100 µL of the protein-free
supernatant of the cell lysate, 100 µL of 0.04% 5,5?-dithiobis-(2-nitrobenzoic acid) (DTNB) in 0.1% sodium citrate and 800 µL of 0.3 mM Na2HPO4.
After five min, the DTNB absorbance was recorded at 412 nm. Based on the
measurement sensitivity, the standard curve for glutathione was done between 1
and 100 µM” 26. Ultimately,
the glutathione levels of ischemic hemispheres were calculated as nmol/mg
protein.

Superoxide
dismutase (SOD) activity of the ischemic hemispheres: According to the capability of superoxide dismutase (SOD) to
inhibit the reduction of nitroblue tetrazolium (NBT), the enzyme activity was measured
using the method of Winterbourn 27. “First, potassium phosphate buffer (0.067 M and pH 7.8) was
added to 0.1 M EDTA containing 0.3 mM sodium cyanide, 1.5 mM NBT and 0.1 mL of
sample. Then to initiate the reaction, riboflavin (0.12 mM) was added to each
sample. After 12 min incubation, the absorbance of samples was recorded by a spectrophotometer
(UV 7500, Spectro Lab, England) at excitation of 610 nm for five min. The
amount of enzyme needed to induce 50% inhibition was taken as 1 U” 26. Ultimately,
the activity of the SOD enzyme in the ischemic hemispheres was calculated as
U/mg protein.

Catalase
activity of the ischemic hemispheres: The
method of Aebi was used to determine the activity of catalase in tissue
homogenate 28. First, the homogenate was incubated in the reaction mixture that
contained 0.1 mL homogenate and 0.85 mL potassium phosphate buffer (50 mM and pH
7.0) at room temperature for 10 min. Then, the reaction was begun by adding 0.05
mL H2O2 (30 mM prepared in potassium phosphate buffer 50
mM and pH 7.0). Ultimately, a decrease in the absorbance was recorded by a
spectrophotometer at excitation of 240 nm for 3 min. Specific activity of
catalase was calculated as 1µmol H2O2 decomposed U/mg
protein.

Malondialdehyde
(MDA) assay in the ischemic hemispheres: In
the current study, the Satoh method was utilized for malondialdehyde (MDA)
assay 29. First, 1.5 mL of trichloroacetic acid (TCA, 10%), was added to
0.5 mL of tissue homogenate. The mixture was vortexed and incubated at room
temperature for 10 min. Then, 2 mL thiobarbituric acid (0.67%) were added to 1.5
mL supernatant and incubated in a boiling water bath for 30 min in sealed tubes.
After cooling the samples to room temperature, 1.25 mL n-butanol was added and vortexed.
The samples were finally centrifuged at 2000 g for 5 min and the supernatants were
separated. The absorbance of the solutions was recorded by a spectrophotometer
at excitation of 532 nm. 1,1,3,3-tetraethoxypropane was used as standard to
determine the levels of MDA 30. The MDA levels of the ischemic hemispheres were ultimately calculated
as nmol/mg protein.

NOx (nitrate
and nitrite contents) assay in the ischemic hemispheres: To determine the nitrosative damage of ischemic hemispheres, the colorimetric
reaction method (Griess reagent) was used. First, the homogenate (0.1 mL) was
deproteinized by adding zinc sulfate (0.2 mL). The mixture was centrifuged at
4000 g and 4 ºC for 20 min to separate supernatant. Then, 0.1 mL vanadium III
chloride was added to the 0.1 mL of supernatant (as a sample) or pure water (as
blank) or sodium nitrite (as standard) to reduce nitrite to nitrate. After
adding the 0.05 mL sulfanilamide (0.01 %) and 0.05 mL N-1-naphthyl ethylenediamine
dihydrochloride (NED, 0.01 %), the mixture was incubated at 37 ºC for 30 min in
dark place. Ultimately, the absorbance of the solutions was recorded by a
spectrophotometer at excitation of 540 nm. To determine a standard curve for
calculation of the nitrate concentration, the sodium nitrate solution was used
in the different concentrations 30. The nitrate levels of the ischemic hemispheres were ultimately expressed
as nmol/mg protein.

2.8. Statistical analysis

All
data in the current study were expressed as mean±SEM. Data were analyzed with
analysis of variance (ANOVA) followed by Tukey post-hoc test. All states,
differences were considered significant if P

x

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