Design
A randomized, controlled, animal study.
Time and setting
The experiment was performed at the Department of Pharmacology of Basic Medical School, Wuhan University and Institute of Traditional Chinese Medicine, Chengde Medical College, China from June to December 2011.
Materials
Animals
Forty adult, male Sprague-Dawley rats, aged 3–3.5 months, weighing 350–400 g, were purchased from the Laboratory Animal Center, Hebei Medical University (Clean grade, Certification No. 04057), China. The rats were housed in groups (four or five per cage) in a room controlled at 23 ± 1°C and maintained in an alternating 12-hour light/dark cycle. Food and water were freely available. All animal procedures were performed in accordance with the Regulations of Experimental Animal Administration, issued by the State Committee of Science and Technology of China[22].
Drugs
Flavonoids, isolated from the stems and leaves of Scutellaria baicalensis Georgi (Huangqin), were prepared by the Phytochemistry Laboratory, Institute of Traditional Chinese Medicine, Chengde Medical College, and the Huangqin herb was planted by our laboratory. With regard to preparation, the dried aerial part of Scutellaria baicalensis (1 kg) was boiled with 80% (v/v) alcohol for 1 hour and then filtered. The filtrate was concentrated to the appropriate volume at 60°C under vacuum distillation. The concentrated solution was adjusted to pH 2 with 1 M HCl and the suspension was filtered after still standing for 24 hours at room temperature[23]. The residue was identified as flavonoids from Scutellaria baicalensis Georgi, in which the purity of flavonoids from Scutellaria baicalensis Georgi was not less than 80% and scutellarein was determined to be the major ingredient by high performance liquid chromatography analysis. flavonoids from Scutellaria baicalensis Georgi were dissolved in distilled water prior to administration, and the pH of the solution was adjusted to 7.2–7.4 with saturated sodium bicarbonate.
Methods
Establishment of middle cerebral artery occlusion models
The rats were anesthetized with 240 mg/kg chloral hydrate by intraperitoneal injection. The right middle cerebral artery was occluded according to an animal model of focal cerebral ischemia as previously described[24]. The rats were placed in a supine position and the skin was incised along the midline of the neck. The right common carotid artery, external carotid artery and internal carotid artery were exposed and separated. The common carotid artery and internal carotid artery were occluded using a bulldog clamp. The proximal and distal tips of the external carotid artery were ligated with 0# thread, and were cut at the bevel portal at the middle of the two ligated sites of the external carotid artery. The distal end of the external carotid artery was lifted so that it was in line with the internal carotid artery. A nylon thread (50 mm length and 0.26 mm diameter) was inserted approximately 20 mm from the bevel portal of the external carotid artery distal tip and through the internal carotid artery to the bifurcation of the common carotid artery. At this position, the tip of the nylon thread was inside the middle cerebral artery branches of the Willis circle of the brain, where blood flow was blocked by the nylon thread. The middle cerebral artery occlusion in rats lasted 20 minutes and then the nylon thread was retracted 10 mm to allow for blood reperfusion for 10 minutes. The same procedure was repeated three times. After the final ischemia and reperfusion, the external carotid artery stump was ligated and the surgical incision was sutured. The rats that received the same surgery without ischemia/reperfusion served as the sham-surgery group. The surgical rats were placed on a heating pad until they recovered from anesthesia.
Intragastric administration of flavonoids from Scutellaria baicalensis Georgi
Rats from the three treatment groups received flavonoids from Scutellaria baicalensis Georgi at doses of 35, 70 and 140 mg/kg daily by intragastric administration. The model and sham-surgery groups were subjected to the same volume of distilled water for 13 days from 24 hours after the surgery (2 mL per 100 g body weight). The dosage was based on a previous report[5].
Morris water maze test
Learning ability and memory retention was assessed using the Morris water maze[9]. Assessment of learning began on day 8 and the probe trial was carried out on day 12 after drug administration. During the learning performance test and the probe trial, the drug or its vehicle (distilled water) was administered 60 minutes before the trial. The water maze was a stainless steel circular tank of diameter 120 cm and depth 50 cm and was supplied by the Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College. A circular platform, diameter 10 cm and 30 cm high, was placed in a quadrant of the tank. When the water maze test was carried out, the tank was added with water to a depth of 31.5 cm and the platform was set 1.5 cm under the water surface. The tank water was maintained at 23 ± 1°C and made white/opaque with non-fat milk powder. All spatial marks around the maze were kept invariable during the water maze test. The tank was subdivided into four equal quadrants by imaginary lines. The hidden platform was placed in the third quadrant. Learning ability was assessed over 5 consecutive days with two trials each day. The time taken to find the hidden platform (latency) was recorded and an average of the two trials was used to determine learning ability. On the first training day, each rat was left to swim for 120 seconds in the water maze to become familiar with the water conditions. On the second day, the rats were left to search for the hidden platform for 60 seconds. In cases where rats did not find the platform, the rats were placed on the hidden platform within 60 seconds. Regardless of whether rats discovered the hidden platform or not, the rats were maintained on the platform for 20 seconds and then removed from the tank. Rats were allowed to recuperate for 10 seconds between the two trials. The probe trial was performed immediately after the last learning ability test. The rats were allowed to swim for 60 seconds, in which the platform was taken away from the water. The time spent in the target quadrant (third quadrant), where the platform had been available during the learning ability test, was recorded as memory retention. Swimming activity was monitored using a video camera linked to computer-based graphics analytic software (Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College).
Sample preparation of the hippocampus and cerebral cortex
Under ether anesthesia, all rats were sacrificed 60 minutes after the final administration of flavonoids from Scutellaria baicalensis Georgi or distilled water on day 14 of the operation. The hippocampus and cerebral cortex were separated according to a previously described method[25], and were placed on the ice for determining the neural morphology and biochemistry.
Measurement of neuronal morphology in the hippocampus and cerebral cortex
The brain was rapidly placed on ice, and the hippocampus and cerebral cortex were gently dissected. The separated cerebral cortex of the right hemisphere in four rats from each group was fixed with 4% (v/v) formalin and embedded in paraffin. Coronal sections were cut at 6 μm and stained with hematoxylin and eosin as previously described[8]. Stained cells were observed and pictures at 400 × magnification were captured with an Olympus VANOX microscope (Olympus, Tokyo, Japan). In addition, another cerebral cortex was fixed with 2.5% (v/v) glutaraldehyde and 1% (v/v) osmic acid, and then cut at 50 nm using an ultra-microtome. The slices were put on a copper grid with 200-mesh, and stained with uranyl acetate and lead nitrate–sodium citrate according to a previously described method[25]. The neuron ultra-status was viewed using a JELO100 CX-II-transmission electron microscope (JEOL, Japan) and images were captured at a magnification of 6 700–20 000 ×.
Detection of lactate dehydrogenase, Na+-K+-ATPase and Ca2+-ATPase activity in the hippocampus and cerebral cortex
The hippocampus and cerebral cortex were homogenized with cold saline to obtain 2% homogenates for measuring lactate dehydrogenase, Na+-K+-ATPase and Ca2+-ATPase activity. Lactate dehydrogenase activity was determined according to the manufacturers’ protocol (Beikong Bio-technology & Science Inc, Beijing, China) and using an ultraviolet spectrophotometer[26] (Shanghai Analytic Factory, Shanghai, China). ATPase activity was determined according to the manufactures protocol (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China). ATPase activity was determined by measuring the formation of phosphoric acid from ATP[27]. The phosphoric acid combines with ammonium molybdate to produce phosphato-molybdic acid salt. The latter was reduced into molybdenum blue, and the maximum absorptive wavelength of molybdenum blue was measured at 660 nm. ATPase activity was defined as the change in absorbance of molybdenum blue.
Detection of malonaldehyde production and superoxide dismutase activity in the hippocampus and cerebral cortex
The hippocampus and cerebral cortex were separated and homogenized with cold saline to obtain 10 % (w/v) and 1% (w/v) homogenates for assaying malonaldehyde production and superoxide dismutase activity, respectively. The principle of determining malonaldehyde was based on the methods of thiobarbituric acid[28]. The malonaldehyde production was determined by a change in absorbance. The measurement of superoxide dismutase activity was based on the xanthine-xanthine oxidase method[29]. Superoxide dismutase activity was determined by measuring the absorbance at 550 nm. Both assays were performed in an ice-cold environment according to the manufacturer’s instructions (Nanjing Jiancheng Institute of Biological Engineering).
Statistical analysis
The Statpark software package (Oracle Corporation, Redwood Shores, CA, USA) was used to analyze the experimental results. Data are expressed as mean ± SEM and the latency from the Morris water maze training trial was evaluated by two-way analysis of variance with repeated measures for between group comparisons. One-way analysis of variance followed by the Duncan’s multiple-range test was used to analyze group differences for the probe trial and biochemistry data. The level of significance was set at P < 0.05.
Funding: The study was supported by the State Administration of Traditional Chinese Medicine of China, No. 02-03-ZP18; Hebei Provincial Education Department, No. 20015; and Hebei Provincial Hundred Outstanding Innovated Talents, First Batch.
Author contributions: Hui Wang was in charge of funds, designed the study, provided technical support and revised the manuscript. Yazhen Shang was also in charge of funds, performed the experiments with Hong Zhang, and wrote the manuscript. Jianjun Cheng and Hong Miao provided other technical help. All authors approved the final version of the paper.
Conflicts of interest: None declared.
Ethical approval: The study received full approval by the Animals Ethics Committee of Chengde Medical College, China.
Author statements: The manuscript is original, has not been submitted to or is not under consideration by another publication, has not been previously published in any language or any form, including electronic, and contains no disclosure of confidential information or authorship/patent application/funding source disputations.
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