Animals
Adult male Sprague Dawley (SD) rats weighing 220-250g were supplied by Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). This study was performed under supervision of the Animal Care and Use Committee of Qilu Hospital of Shandong University. To verify roles of EA in cerebral I/R injury and specific signaling pathways involved in EA, SD rats were randomly divided into four groups: Sham, I/R, EA and VX-765 (a specific inhibitor of Caspase-1).
Cerebral Ischemia-reperfusion Model
Middle cerebral artery occlusion (MCAO) followed by reperfusion was used to simulate process of cerebral I/R. Briefly, after sodium pentobarbital (40mg/kg, i.p.) anesthesia, central skin of SD rat neck was cut open, and right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were fully exposed by blunt separation. Then, after ligating ECA, a monofilament (2636-A4, Shadong Co. Ltd., China) was inserted slowly into ICA to block blood flow to MCA. After 2h of occlusion, monofilament was withdrawn to achieve 7d of reperfusion. All operations performed in Sham group were consistent with those performed in I/R group except that MCA was not blocked. Cerebral blood flow (CBF) was determined in area of MCA by laser Doppler flowmetry (Model Moor VMS-LDF2, Moor Instruments Ltd, UK). The Garcia JH neurological function rating system was used to evaluate the motor and sensory function of the rats. Body temperature was maintained in rats at 37 ℃ throughout process.
Electroacupuncture (Ea) And Drug Treatment
In EA group, after fixation and application of 75% alcohol disinfectant at acupoints, needles (0.3 mm×40 mm, Hwato, China) were inserted into rats at a depth of 5mm at “Baihui” (GV20, midline of head, approximately midway along line connecting apices of auricles) and left “Zusanli” (ST36, 2mm lateral to anterior tubercle of tibia and 5mm below capitulum fibulae under knee joint). Electrodes were fixed to these needles, and 20Hz electrical stimulation with compressional waves was performed for 30min using an EA device (KWD-808 II, Great Wall Brand, China). In VX-765 group, VX-765 (50mg/kg) was dissolved in saline and injected intraperitoneally. Rats in EA group and VX-765 group received first treatment at onset of reperfusion, and were then treated once a day for a total of 7 days.
Cerebral Infarct Size Measurement
Fresh brains were frozen at -20℃ for 10min and then cut into 2mm-thick coronal slices. These slices were immersed in 2% TTC (G3005, Solarbio, China) at 37℃ without light for 30min and then fixed in 4% paraformaldehyde for 1h. The stained slices were photographed, and infarct size was calculated with ImageJ software as follows: right infarct size (%) = (total size of left brain slices - red size of right brain slices)/total size of left brain slices × 100%.
Histological Study
First, the rats were perfused into the heart with 250mL of PBS followed by 400mL of 4% paraformaldehyde in PBS. Then, brain tissues were fixed again in 4% paraformaldehyde for 12h and dehydrated with 15 and 30% sucrose. Finally, brain tissues were embedded in optimal cutting temperature (OCT) compound (4583, SAKURA, USA) or paraffin and cut into 10–20µm-thick slices. H&E staining was stained using hematoxylin and eosin. For Nissl staining, paraffin-embedded slices were covered with 0.2% Nissl staining solution (G1432, Solarbio, China) for 10min. Then, slices were sealed and observed by light microscopy (CI-S, Nikon, Japan). The percentages of surviving neurons in right hemisphere/left hemisphere, in cortex/mm2 and in cornu ammonis 1 (CA1)/mm were quantified using ImageJ software.
In electron microscopic observation, after fixed with 3% glutaraldehyde, brain tissues were incubated with 2% OsO4 and dehydrated in ethanol. Brain tissues were then dried, uranium lead double staining, and observed using transmission electron microscopy (hitachi, HT7700).
Western Blot Analysis
Total proteins were extracted from right brain hippocampus in ischemic area and quantitated using BCA kit (23250, Thermo Fisher Scientific, USA). Equal amounts of protein were separated with 8–12% SDS-PAGE gels and electrically transferred to NC membranes. The membranes were incubated at 4℃ overnight with claudin-5 (sc-17667, SANTA CRUZ), zonula occluden 1 (ZO-1, (61-7300, Invitrogen), NLRP3 (MA5-32255, Invitrogen), ASC (ab180799, Abcam), Caspase-1 (MA5-32909, Invitrogen), GSDMD (PA5-116815, Invitrogen), IL-1β (PA5-95455, Invitrogen) and β-actin (A5441, Sigma) antibodies. They were then incubated in secondary antibodies for 1h at room temperature. After chemiluminescence detection was performed using ECL reagents (32109, Thermo Fisher Scientific, USA) and Bio-Rad ChemiDoc™ XRS + System, the relative optical densities of protein bands were analyzed using ImageJ software.
Measurement Of Various Nitro/oxidative Stress Indexes
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) concentrations were measured using a rat NOX ELISA Kit (KE1649, ImmunoWay, USA). The activity of superoxide dismutase (SOD) was analyzed using a Total SOD Assay Kit with WAT-8 (S0101, Beyotime, China). Malondialdehyde (MDA) levels were tested using a Lipid peroxidation MDA Assay Kit (S0131, Beyotime, China). The level of 3-nitrotyrosine (3-NT) was measured with a 3-nitrotyrosine ELISA Kit (ab116691, Abcam, USA).
Immunofluorescence
For immunofluorescence, frozen slices were incubated with inducible nitric oxide synthase (iNOS, ab15323, Abcam) antibodies at 4℃ overnight and then with fluorescent dye-conjugated secondary antibodies for 2h at room temperature. After nuclear staining was performed with DAPI for 10min at room temperature, the slices were observed using a Zeiss 780 reverse laser-scanning confocal microscope (Zeiss, LSM 780, Germany).
Tunel Assay
One Step TUNEL Assay Kit (C1088, Beyotime, China) is a sensitive, rapid and simple method for DNA fragmentation detection. The positive cells presenting green fluorescence can be detected by fluorescence microscope after one-step staining. The experimental process was carried out according to instructions.
Statistical Analysis
All data are presented as mean ± SEM. Statistical analysis was performed with SPSS13.0. Comparisons between groups were made using two-way ANOVA followed by Bonferroni post hoc test. Values of P < 0.05 were considered statistically significant.