Animals
Adult male Sprague-Dawley rats weighing 250 to 270 g were purchased from the Experimental Animal Center of Soochow University (Suzhou, Jiangsu, China). Animals were housed in a room with temperature of 21 to 23°C, relative humidity at 30% to 70%, and a 12-h light/12-h dark cycle. They had free access to food and water. All experimental procedures in this study were performed in accordance with the guidelines for the care and use of laboratory animals of Soochow University.
Experimental groups
The rats, under deep anesthesia, were exposed to cerebral ischemia (see below) for 2 h followed by 72-h reperfusion to induce I/R injury in the brain. We have previously shown DOR activation induced protection from ischemic injury [5, 12]. Since this study was mainly for the purpose of determining if DOR activation has any effect on major antioxidant enzymes and oxidant free radicals after I/R, we randomly assigned the animals to various groups after the same procedure for cerebral ischemia (see below), and sampled cortical and hippocampal tissues for biochemical and molecular measurements. The animals that died before the timepoint (72 h after MACO) were removed from the experiments, since no brain tissues could be sampled from them. There was no significant change in the death rate among various groups except for the PD98059 group (see below and Results).
In the I/R plus DOR activation group (n = 12), the I/R rats were treated with DADLE, a DOR agonist purchased from Sigma (St Louis, MO, USA). DADLE was applied through intraperitoneal injection (1 mg/kg in saline) [18] within 30 min starting at 30 min after the onset of suture occlusion. The reason we chose to apply DADLE in the early stage of ischemia was because an early application of DADLE displays better neuroprotection against excitotoxic injury, as shown in our previous work [3]. In the I/R control group (n = 12), the procedures were the same except for vehicle solution without DADLE. In the sham control group (n = 6), rats were subjected to sham I/R operation. In addition, in a group of rats (n = 12), PD98059 (Sigma, St Louis, MO, USA; 1 μg in 6 μl of vehicle solution), a specific inhibitor of mitogen-activated protein kinase (MAPK) extracellular signaling-regulated kinase (ERK) kinase, was microinjected into the right lateral ventricle of the ischemic rats with or without DADLE, through a stereotaxically inserted 27-gauge needle (ALCB10, Shanghai Alcott Biotech, Shanghai, China) (1.5 mm lateral and 1 mm posterior to the bregma, 4 mm in depth) [35]. The microinjection was started at 30 min after the onset of cerebral ischemia and was completed within 30 min. After the microinjection, sterile procedures were performed to suture the skin.
Induction of cerebral ischemia
To mimic cerebral I/R injury, the model of stroke was established by performing MCAO in the rat [5, 12, 36]. In brief, rectal temperature was recorded and maintained at 37°C ± 0.5°C throughout the surgical procedure and up to 2 h after reperfusion. The right common carotid artery was carefully separated from around tissues and a 4-0 monofilament nylon suture coated with paraffin was inserted through the external carotid artery stump into the internal carotid artery to occlude the right middle cerebral artery. After 2 h of occlusion, the nylon monofilament suture was withdrawn for blood flow reperfusion. In the sham-operated group, the surgical procedures were the same as in the ischemic animals except for real insertion of the suture.
Sample preparation
After 72-h reperfusion, the rat brains were quickly removed under deep anesthesia and placed into cold ethylenediaminetetraacetic acid (EDTA) buffer (0.02 M, pH = 7.4) for removing tissue debris and blood clots. All visible vascular structures were also removed. Then, the cortex and hippocampus were dissected from the hemispheres, respectively.
For immunohistochemistry, the tissues were fixed in 10% formalin, pH 7.4. After dehydration in graded ethanol and xylene, the tissues were paraffin embedded and cut into coronal sections (4 μm thick). For RNA and other measurements, the samples were frozen in liquid nitrogen and stored at -80°C until use.
For detection of the activities of antioxidant enzymes (SOD and GSH-Px) and the content of MDA and nitric oxide (NO), the tissue samples were weighed and homogenized with a buffer consisting of 10 mM sucrose, 10 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, and 0.1 mM EDTA (pH 7.4), and then centrifuged at 3,000 g for 15 min at 4°C. The supernatant was used for bioassays. Total protein concentration was measured using the method of bicinchoninic acid protein assay. All the data were normalized to protein concentration (a unit value per μg of protein).
Caspase 3 immunohistochemistry
Procedures were processed according to the manufacturer's protocol recommended for the caspase 3 immunohistochemistry kit (Beijing Zhongshan Biotechnology, Beijing, China). Following deparaffinzation and rehydration, brain sections were irradiated in 0.1 mol/l sodium citrate buffer (pH 6.0) in a microwave oven for 12 min. The sections were then exposed to 3% H2O2 for 10 min to bleach endogenous peroxidases, and subsequently subjected to three rinses with phosphate-buffered saline (PBS) for 10 min each. These sections were incubated with an anti-caspase 3 primary antibody (1:100, Beijing Zhongshan Biotechnology) for 1 h at 37°C and then washed in PBS 3 times. Finally, the sections were incubated with a biotinylated goat secondary anti-mouse antibody (Beijing Zhongshan Biotechnology) for 30 min at 37°C. The primary antibody was omitted in the control sections. After washing in PBS, caspase 3 signals were visualized with 3,3'-diaminobenzidine tetrahydrochloride (DAB) and counterstained with hematoxyline. Finally, the sections were dehydrated in graded ethanol, immersed in xylene and covered by cover slips.
The number of caspase 3-positive cells was blindly counted by a researcher blinded to the assignment of experimental groups. In brief, images were taken with an Olympus BX-51 microscope (Olympus, Tokyo, Japan). Cells with a blue color were counted as neurons. Cells showing both blue and brown color were counted as caspase 3-positive neurons. At least three view areas were randomly selected for a given region on each section. The ratio of caspase 3-positive cells per neurons in each view area was calculated and then three or more values from the randomly selected view areas were averaged to represent the value of the region.
Assay of SOD activity
SOD activity was measured using an assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's protocol. It uses a xanthine-xanthine oxidase system to determine the inhibition of nitroblue tetrazolium (NBT) reduction due to superoxide anion generation. Briefly, 500 μl of supernatant was mixed with 50 μM xanthine and 2.5 μM xanthine oxidase in 50 μM potassium phosphate buffer and incubated at 37°C for 40 min. Then, NBT is added. Total nitrite (nitrite + nitrate) produced by the oxidation of oxyamine was measured by detecting the absorbance at 550 nm. A unit of SOD was defined as the enzyme amount causing 50% inhibition of the NBT reduction rate. The SOD activity was expressed as units of nitrite per mg protein.
Assay of GSH-Px activity
The GSH-Px activity was determined using an assay kit from Nanjing Jiancheng Bioengineering Institute according to the manufacturer's protocol. In brief, 200 μl of the supernatant was mixed with 2.68 ml of 0.05 M phosphate buffer (pH 7.0) containing 5 mM of EDTA, followed by the addition of 0.1 ml of 8.4 mM nicotinamide adenine dinucleotide phosphate (NADPH), 0.01 ml of glutathione reductase, 0.01 ml of 1.125 M NaN2, and 0.1 ml of 0.15 M glutathione. The enzymatic reaction was initiated by the addition of 0.1 ml of 2.2-mM H2O2. The changes in absorbance at 340 nm were continuously recorded between 2 and 4 min after initiation of the reaction (converting NADPH to NADP). A GSH-Px unit was defined as the enzyme activity required to convert 1 mmol of NADPH to NADP per mg tissue protein. The GSH-Px activity was expressed as units per mg protein.
MDA assay
The MDA content was determined by the thiobarbituric acid method using an assay kit from Nanjing Jiancheng Bioengineering Institute according to the manufacturer's recommendations. It was assayed in the form of thiobarbituric acid reacting substances (TBARS). A total of 100 μl of the supernatant was mixed with 1 ml of 20% trichloroacetic acid and 1.0 ml of 0.1% TBARS reagent and incubated at 95°C for 80 min. After cooling on ice, the mixture was centrifuged at 1,000 g for 20 min. The absorbance of the supernatant was measured at 532 nm. The amount of TBARS was determined using tetraethoxypropane as a standard. The content of TBARS, as an index of MDA, was expressed as nmol per mg protein.
Nitric oxide assay
The concentration of nitrite was measured to reflect the production of NO using a Griess reagent system kit (Jiancheng Institute of Biotechnology), according to the manufacturer's recommendations. In brief, the supernatant was mixed with the Griess reagent (1% sulfanilamide, 0.1% N-l-naphathyletylenediamine dihydrochloride and 2.5% phosphoric acid) at room temperature for 10 min. Nitrite products in the supernatants were determined by measuring absorbance at 550 nm with NaNO2 being used for a standard curve. The results were expressed as μmol/g protein.
Quantitative real-time polymerase chain reaction (PCR)
Total RNA was extracted from the brain tissues with Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and then reverse transcribed using a RevertAid First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania).
The cDNA sequences for IL1β, TNFα, and β-actin were obtained from NCBI GenBank. Specific PCR primers and TaqMan fluorogenic probes were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA) and commercially synthesized by Invitrogen. 6-Carboxyfluorescin (6-FAM) was used as the reporter dye and 6-carboxytetramethylrhodamine (TAMRA) as the quencher dye.
Real-time reverse transcriptase (RT)-PCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems) on a Lightcycle (Roche, Basel, Switzerland). Briefly, the reaction mixture contained cDNA 2 μl, 10 × buffer 2.5 μl, 25 mM MgCl2 1.5 μl, 10 mM dNTP 0.5 μl, Taq 0.5 μl, 0.1 μl each of the 100 μM sense and antisense primers and 0.1 μl of 100 μM probe. All samples were tested in duplicate. The conditions of amplification for TNFα were 95°C for 2.5 min, 95°C for 5 s and 56°C for 45 s for 40 cycles. The conditions of amplification for IL1β were 95°C for 2.5 min, 95°C for 15 s and 60°C for 45 s for 40 cycles. The amplification for β-actin was performed according to the manufacturer's instructions (Invitrogen).
The relative levels of these mRNAs were calculated by 2-ΔΔCT method and the levels of TNFα and IL1β mRNAs were normalized to that of β-actin.
Statistical analysis
All data were subjected to statistical analysis using Graph Prism v. 5.0 software (GraphPad Software, San Diego, CA, USA). Statistical significances among groups were determined by one-way analysis of variance (ANOVA) or unpaired t test. P < 0.05 was considered as statistically significant.
