All procedures involving the use of animals were approved by the ethics committee of animal experiment of the Fourth Military Medical University (Xi′an, China), and proceeded in accordance with the guidelines for animal experimentation of the same university. The Sprague Dawley rats were bought from Fourth Military Medical University and acclimatized for one week in a 12 h:12 h of light:dark cycle before interventions.
Experimental design
Experiment 1. To test the time course of sevoflourane preconditioning on the alteration of Trx-1 after MCAO, rats were randomly divided into two groups (n = 15 for each group, 3 for each time point): Sevoflurane MCAO group (Sev MCAO) and Oxygen MCAO group (Oxy MCAO). Rats were subjected to MCAO at 24 h after sevoflurane or its vehicle oxygen preconditioning. Animals were decapitated to extract proteins at 24 h after last exposure of preconditioning without surgery as well as at 2 h, 8 h, 24 h and 72 h after MCAO, respectively. The expression of Trx-1 in brain ischemic penumbra tissue was examined by western blotting. The activity of Trx-1 at penumbra was measured by fluorescent plate with the spectrometer. The nitrotyrosine of protein in brain tissue was determined by chemilumineseence method.
Experiment 2. To assess the effects of sevoflurane preconditioning and MCAO ischemia/reperfusion on the nitration of Trx-1, animals were randomized into four groups (n = 4 for each group): Oxygen group (Oxy), Sevoflurane group (Sev), Oxygen MCAO group (Oxy MCAO) and Sevflurane MCAO group (Sev MCAO). Rats in Oxy and Sev groups underwent the sham operations, which didn’t induce ischemia. Meanwhile, rats in Oxy MCAO group and Sev MCAO group were subjected to MCAO at 24 h after preconditioning. The levels of nitrated Trx-1 were tested at 8 h after MCAO by immunoprecipitation, and the correlation between Trx-1 nitration and its activity was analyzed.
Experiment 3. To elucidate whether the enhancement of Trx-1 nitration could reverse the brain ischemic tolerance induced by sevoflurane, rats were randomly divided into four groups (n = 10 for each group, 7 for neurological scores and infarct volume, 3 for TUNEL staining): Oxy MCAO group, Sev MCAO group, Nitrated human-Trx-1 (3 N-Trx-1) group and human-Trx-1 (hTrx-1) group. Animals in Oxy and Sev MCAO groups were treated in the same way as those in Experiment 2. In 3 N-Trx-1 and hTrx-1 groups, sevoflurane-preconditioned rats received the nitrated hTrx-1 or hTrx-1 injection through tail vein at dose of 0.2 mg/kg, respectively, prior to MCAO. Neurological scores, brain infarct volumes and TUNEL staining were evaluated at 24 h after reperfusion.
Sevoflurane preconditioning
The protocols of sevoflurane preconditioning were based on our previous publications [2]. In brief, rats were placed in a temperature-controlled, transparent and air-tight (30 × 30 × 20 cm3) chamber with a gas inlet port and an outlet port. During sevoflurane preconditioning, the box was flushed with 2% sevoflurane in 100% oxygen; As for oxygen preconditioning, the box was flushed with only 100% oxygen. The expired fraction of sevoflurane, oxygen, and carbon dioxide were monitored continuously (MP-60, Phillips Medical Systems, Best, The Netherlands). Soda-lime (Molecular products limited, Essex, United Kingdom) was placed at the bottom of container for carbon dioxide absorption. The sevoflurane preconditioning was performed for 1 h per day, repeated for five consecutive days.
Focal cerebral ischemia and reperfusion
The animals had free access to water and food before the surgery. MCAO was operated as described previously [2]. SD rats were anesthetized with 60 mg/kg pentobarbital sodium (intraperitoneal injection), breathing spontaneously. Once the right common carotid artery and its branches were separated, a nylon monofilament (RWD, China) was inserted from the external carotid artery to internal carotid artery then to the right middle cerebral artery. Ninety minutes after occlusion, the filament was withdrawn to induce the reperfusion in the right middle cerebral artery territory. During the surgery, cerebral blood flow was monitored by laser-Doppler flowmetry (periflux 5000, perimedAB, Sweden). A reduction in cerebral blood flow of more than 80% baseline (pre-ischemia) was confirmed as the effective occlusion; otherwise, animals were excluded from the further experiments. Throughout the surgery, the temporal temperature was maintained at 37 °C ± 0.5 °C by a thermostatic blanket and a heating lamp. All the animals had a comparable surgical duration of time.
Western blotting
Brain ischemic penumbra was dissected, and then homogenized in the RIPA lysis buffer (Beyotime, Nantong, China) containing whole proteinase inhibitor cocktail. A BCA protein assay kit (Beyotime, Nantong, China) was used to determine the protein concentration. Equivalent amount of protein (30 μg per lane) was loaded and separated by a 12% SDS-PAGE gel. After electrophoresis, the protein was transferred to a polyvinylidene difuoride (PVDF) membrane, which was then blocked with 2% bovine serum albumin (BSA) in TBST. The sample membrane was incubated overnight at 4 °C with the anti-Trx-1 (1:1000, Abcam, Cambridge, UK) and β-actin primary antibodies. At last, the membrane was incubated with horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (1:5000, Pierce, Rockford, IL) for 1 h at room temperature. Analysis software program Image J was used to quantify the optical density of each band.
Immunoprecipitation
The tissue was homogenized in the RIPA lysis buffer (Beyotime, Nantong, China) on ice. After centrifugation at 13000 rpm for 10 min, the supernatant was collected and protein concentration was measured by BCA protein assay kit (Beyotime, Nantong, China). Equivalent amount of protein was incubated with 2μg of mouse monoclonal anti-3-nitrotyrosine (Abcam, ab61392, UK) for 12 h with gentle rotation at 4 °C, and then added 50 μl of protein G/A beads for 3 h at 4 °C. Beads were then washed with cell lysis buffer for three times and the bound proteins were eluted with 2 × loading sample buffer and subjected to SDS-PAGE in 12% polyacrylamide gels, followed by blotting onto nitrocellulose filter membrane with the anti-Trx-1 antibody (Abcam, ab26320, UK). Immunoreactive bands were detected using Chemidoc MP imaging system (Bio-rad). Analysis software program Image J was used to quantify the optical density of each band.
Nitrotyroysine assay
A nitrotyroysine assay kit (Millipore, lot 2,113,321, USA) was used to test the nitrotyrosine of the proteins. The procedure was performed in strict accordance with the instructions. In brief, the sample was homogenized in the RIPA lysis buffer (Beyotime, Nantong, China) on ice, and 50 μl of test sample or standard was incubated with 50 μl of 2 × anti-nitrotyrosine in the plate at 37 °C for 60 min. After washing, the plate was then incubated with 1 × anti-Rabbit lgG, HRP-conjugate (100 μl per well) at 37 °C for 60 min. Lately, the freshly prepared chemiluminescent substrate was added into each well and incubated at room temperature for 10 min. Chemidoc MP imaging system was used to quantify the optical density of the each well.
The activity of Trx-1
Assay kits (IMCO, USA) were used to measure the activity of Trx-1. Specifically, Human-Trx-1 at different concentrations used to draw the standard curve by reacting with 10 μl TrxR and followed by incubation with 5 μl β-NADPH for 30 min at 37 °C in an incubator plate. Then 20 μl of the fluorescent substrate was added into each well for incubation for another 60 min. The emission at 545 nm after 520 nm excitation was recorded in a fluorescent plate reader at room temperature. The activity of samples was calculated by the fluorescence concentration and the standard curve.
In vitro nitration of Trx-1
The hTrx-1 (Sigma, USA) was subjected to in vitro nitration with a modified procedure as described previously [11]. The purified hTrx-1 was dissolved in 0.1 μm phosphate buffer (pH = 7.4), of the final concentration at 50 μm. HTrx-1 was then incubated with Sin-1 (final concentration of 100 μM, Cayman Chemical, USA) at 37 °C for 30 min. The unreacted Sin-1 was removed by ultrafiltration through a 10-kDa cut-off membrane.
Neurobehavioral evaluation and infarct assessment
According to the method of Garcia et al [12], neurological behaviors were assessed at 24 h after MCAO by an observer who was blinded to the animal grouping. Infarct volumes were measured immediately after Neurobehavioral evaluation. Rat brains were sliced into 2-mm coronal sections and stained by 2,3,5-triphenyltetrazolium chloride (1%; TTC, Sigma, USA).
TUNEL staining
The cellular apoptosis was evaluated at 24 h post reperfusion by an in situ terminal deoxynucleotidyl-transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling assay (TUNEL, Roche Diagnostics, Germany). Rat brains were fixed with paraformaldehyde and frozen with embedding agent, and then cut into 14 μm coronal sections from approximately 2 mm rostral to Bregma point. The slices were stained as the manufacture’s instruction. The positive cells were acquired by using a 40× objective lens in the ischemic penumbra, and the counting of positive cells was expressed as number per 100μm2. The ischemic penumbra area was defined according to the previous publications [3].
Statistical analysis
The software SPSS 12.0 for Windows (SPSS Inc., Chicago, IL) was used to analyze the data. All values were presented as mean ± SD, except for neurologic scores as median [interquartile range]. Values in Fig. 1 were analyzed by Two-way analysis of variance (ANOVA), and multiple comparisons within groups were performed by Bonferroni’s test. Values in other figures were analyzed by One-way ANOVA, and between-group differences were detected with Tukey post hoc test. The neurologic scores were analyzed by Kruskal-Wallis test followed by Mann-Whitney U test. Two-tailed values of P < 0.05 were considered statistically significant.
