Design
This is a randomized controlled animal study.
Time and setting
Experiments were performed at the Experimental Center, Hebei United University, China from March 2010 to October 2011.
Materials
A total of 66 clean male Sprague-Dawley rats, aged 2–4 months and weighing 200–250 g, were supplied by the Laboratory Animal Institute, Chinese Academy of Medical Sciences (license No. SCXK (Jing) 2010-0013). The rats were housed in the Animal Laboratory of Environmental Barriers, Hebei United University, China. They were kept in a temperature controlled environment (23 ± 2°C) with natural illumination, and were acclimatized for 2 weeks before experiments. The protocols were conducted in strict accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China[17].
Methods
Establishment of cerebral ischemia/reperfusion models
In accordance with a modified Longa’s method[18], the rat model of middle cerebral artery occlusion was established. The rats were fasted for 12 hours with no water for 4 hours prior to surgery. The rats were intraperitoneally anesthetized with 10% chloral hydrate (0.3 mL/kg), and laid in the supine position. A longitudinal incision (approximately 25 mm) was made on the neck to expose the right common carotid artery. The proximal part of the common carotid artery and bifurcation of the external carotid artery were ligated, and the external carotid artery was reversely straightened. A cut was made about 5 mm from the end of the common carotid artery. A 0.26-mm diameter fishing line was inserted along the internal carotid artery, and withdrawn 0.5 mm after meeting resistance. The depth of penetration was approximately 18.5 ± 0.5 mm. The artery was ligated at the proximal part. The incision was sutured using a full-layer suture. After 2 hours, the fishing line was pulled out to enable reperfusion. The surgery was performed at 22 ± 2°C. Inclusion criteria[18] were as follows: rats turned left after consciousness; and no spreading of the left forelimb. In the DKK1 group, 20 μL of recombinant human DKK1 (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), at a concentration of 1.0 mg/mL, was injected into the lateral ventricle[19]. One hour after drug administration, rat models were created according to the above method. The depth of penetration was less than 9 mm in the sham surgery group with no middle cerebral artery occlusion.
Preparation of tissue specimens
Six rats from each group were collected at corresponding time points. Three rats were sacrificed and perfusion fixed in paraformaldehyde. The right cerebral hemisphere was dissected. Tissue sections (2 mm) were obtained from the region 2 mm posterior to the optic chiasma, and fixed, dehydrated in alcohol, permeabilized with xylene, immersed in wax, embedded and serially sliced into 5 μm coronal sections. These sections were used for TUNEL staining and immunohistochemistry. The remaining three rats from each group, were deeply anesthetized with 10% chloral hydrate (0.3 mL/100 g) and then decapitated. The brain was removed on ice and washed with cold PBS. Fresh hippocampi were dissociated[20], lysed and homogenized. The supernatant was stored at 4°C for western blot analysis.
TUNEL staining to determine cell apoptosis in the rat hippocampal CA1 region
TUNEL staining was performed in accordance with a kit (Beijing Zhong Shan-Golden Bridge Biological Technology Co., Ltd., Beijing, China), with sections developed using 3,3’-diaminobenzidine (Maixin-Bio, Fuzhou, Fujian Province, China), and counterstained in hematoxylin. Cell apoptosis in the rat hippocampal CA1 region was observed under the light microscope (Olympus, Tokyo, Japan). Positive cells were quantified using a CMIAS real-color automatic image analyzer system (Air Force General Hospital-Biomedical Engineering Institute, Beihang University, Beijing, China). Six fields from each section were observed under × 200 magnification, and the number of positive cells in each field counted, obtaining an average value. Nuclei with brown particles represented TUNEL-positive cells.
β-catenin and glycogen synthase kinase-3β immunohistochemistry in the rat hippocampal CA1 region
Immunohistochemistry was performed in accordance with a kit (Maixin-Bio). Sections were blocked in normal goat serum, incubated with rabbit anti-β-catenin or GSK-3β polyclonal antibodies (both 1:175; Beijing Biosynthesis Biotechnology Co., Ltd.) at 4°C overnight, followed by incubation in biotinylated goat anti-rabbit IgG (1:200; Maixin-Bio) and horseradish peroxidase-labeled streptavidin (Maixin-Bio). Antibody signals were detected by 3,3’-diaminobenzidine coloration. Sections were examined using a light microscope. Positive cells were quantified using the CMIAS real-color automatic image analyzer system. Cells with brown cytoplasm represented positive cells. Six non-overlapping fields of the hippocampal CA1 region from each rat were quantified under × 200 magnification, with the average value calculated.
Western blot analysis of β-catenin and glycogen synthase kinase-3β protein expression in the rat hippocampal CA1 region
Hippocampal tissue homogenates were collected for protein quantification. Protein was isolated by SDS-PAGE. The specimens were incubated with rabbit anti-GSK-3β (1:175) and β-catenin (1:175) polyclonal antibodies at 4°C overnight, then incubated in biotinylated goat anti-rabbit IgG (1:300) at room temperature for 0.5 hours, followed by streptavidin- horseradish peroxidase complex (Maixin-Bio) at room temperature for 0.5 hours, and then 3,3’- diaminobenzidine coloration. Absorbance values were measured using the CMIAS real-color automatic image analyzer system, under the same conditions for each antibody. The experiment was repeated in triplicate, with β-actin used as an internal reference.
Statistical analysis
Data were managed using Excel and analyzed using SPSS 17.0 (SPSS, Chicago, IL, USA). All values were expressed as mean ± SD. The mean values at various time points from the model and DKK1 groups were compared using t-tests. A value of P < 0.05 was considered statistically significant.
.jpg)
