Experimental animals
Male Sprague Dawley (SD) rats weighing 300 g to 350 g were used. This study was reviewed and approved by China Medical University Institutional Animal Care and Use Committee (Permit Number: 100-215-c), and the committee recognized that the proposed experimental procedures compiled with the Animal Protection Law by the Council of Agriculture, Executive Yuan, Taiwan. All the procedures with animals avoided or minimized discomfort, distress, and pain to the animals.
The MCAo model
The MCAo model was established in the SD rats using an intraluminal suture method as described previously [26]. Briefly, the rats were anesthetized with chloral hydrate (400 mg/kg, intraperitoneally), and the right common carotid artery (CCA) and internal carotid artery (ICA) were exposed by way of an incision in the midline neck prior to ligation of the pterygopalatine artery close to its branch. A 3–0 nylon filament suture, blunted at the tip by a flame and coated with poly-L-lysine (Sigma, USA), was inserted into the right external carotid artery (ECA) through the CCA into the ICA for a distance of 20 mm to 25 mm to block the origin of the middle cerebral artery (MCA). The suture was removed slowly to reestablish the blood flow after 15 min of MCAo. The rectal temperature of the rats was maintained at 37 ± 0.5°C throughout the experimental procedure using an electrical heating pad.
Electrode implantation
Following the completion of the MCAo operation, the rat’s head was fixed to the stereotactic frame and its scalp or costal skin was incised. The electrode consisted of 0.5-mm stainless steel wires used for acupoint (or nonacupoint) stimulation. It was implanted in Baihui (midpoint of the parietal bone, 4-mm depth of insertion forward) and Dazhui (below the spinous process of the seventh cervical vertebra, 5-mm depth of insertion vertically) acupoints, or in bilateral costal regions (nonacupoints). The rat was then returned to the cage.
Assessment of neurological status
The neurological status of each rat was assessed after 1 d and 3 d of reperfusion. Motor, sensory, balance, and reflex functions were determined using the modified neurological severity score as described previously [27]. The neurological function of each rat was graded using a numeric scale from 0 to 18. (normal score, 0; maximal deficit score, 18). Excepting the sham-operation group, rats with neurological deficit scores equal to or greater than 7 after 1 d of reperfusion were included in further analyses, whereas rats with neurological deficit scores less than 7 were excluded from subsequent analyses.
Experiment A
Grouping
Rats were randomly divided into 6 groups (n = 5 or 6): the EA-like stimulation at acupoints (EA group), EA-like stimulation at nonacupoints (non-acup), model, sham-operation (sham), treatment with U0126 in the EA (U0126 + EA) and treatment with vehicle in the EA (vehicle + EA) groups. Rats in the EA group were subjected to 15 min of MCAo. After 1 d of reperfusion, rats received EA at acupoints once daily for 2 consecutive days. Rats were then sacrificed after 3 d of reperfusion. Rats in the non-acup group were subjected to the same procedure as rats in the EA group but received EA at nonacupoints. Rats in the model group were subjected to the same procedure as rats in the EA group but did not receive EA. Rats in the sham group were subjected to the same procedure as rats in the model group but the MCA origin was not occluded. Rats in the U0126 + EA group were subjected to the same procedure as rats in the EA group but also received an intracerebroventricular (ICV) injection of the MEK1/2 inhibitor U0126 30 min prior to the onset of EA at acupoints. Rats in the vehicle + EA group were subjected to the same procedure as rats in the EA group but also received an ICV injection of the vehicle 30 min prior to the onset of EA at acupoints.
Intracerebroventricular injection of U0126 or vehicle
Rats were anesthetized with a 2% isoflurane/oxygen mixture and an ICV injection of a 4 μl solution containing U0126 (4 μg in vehicle, #662005 Calbiochem) or vehicle (DMSO diluted in saline) was administered to the right hemisphere. Injections were performed using a Hamilton syringe with a 26 gauge needle (Hamilton Company, Nevada, USA). The location of each injection was 0.8 mm posterior to the bregma, 1.5 mm lateral to the midline, and 3.5 mm deep into the skull surface.
Electroacupuncture-like stimulation at Baihui and Dazhui acupoints or nonacupoints
An EA apparatus (Trio 300, ITO Co., Germany) was used to generate EA at acupoints or nonacupoints for 25 min once daily for 2 consecutive days. The stimulation parameters were 5 Hz amplitude-modulated wave, 2.7 mA to 3.0 mA intensity, and 150 μs pulse width. The rats were awake and moving freely in the cage during EA at acupoints or nonacupoints.
Measurement of cerebral infarct area
Following their neurological status evaluations after 3 d of reperfusion, the rats were sacrificed under deep anesthesia. The brains were removed immediately and cut into 2-mm sections using a brain matrix. The sections were then stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Merck, Germany) for 15 min at 37°C. Brain tissue was differentiated according to staining: white for infarct area and red for noninfarct area. The cerebral infarct areas of the first 6 sections from the frontal lobe were measured using image analysis software (ImageJ, Java). The ratio of infarct area to total brain area was also calculated.
Experiment B
Rats were randomly divided into 5 groups: EA, non-acup, model, sham and U0126 + EA groups. They were then subjected to the experimental procedure described in Experiment A.
Immunohistochemical (IHC) analysis
After 3 d of reperfusion and 15 min of cerebral ischemia, rats were sacrificed under deep anesthesia (n = 5 or 6). Rats were transcardially perfused with 200 ml 0.9% saline and 200 ml 4% paraformalaldehyde (PFA; pH 7.4). Rat brains were removed quickly and postfixed in 4% PFA followed by 30% sucrose (weight/volume) for 3 d, after which they were cut into 15-μm sections using a cryostat. Brain sections were rinsed with Dulbecco’s phosphate buffered saline (DPBS; Sigma-Aldrich) containing 0.01% Tween-20 and immersed in 3% hydrogen peroxide (H2O2)/methanol for 15 min to inhibit endogenous peroxidase activity. They were then incubated with a 10% normal animal serum (ScyTek, Logan, Utah, USA) for 20 min at room temperature (RT) before incubation in moist chambers with a rabbit anti-BDNF (1:500 dilution, AB1779 Millipore), rabbit anti-phospho-Raf-1 (pRaf-1) (1:100 dilution, sc-28005-R Santa Cruz), rabbit anti-phospho-MEK1/2 (pMEK1/2) (1:200 dilution, #2338 Cell Signaling Technology), rabbit anti-phospho-ERK1/2 (pERK1/2) (1:200 dilution, #4376 Cell Signaling Technology), or rabbit anti-phospho-p90RSK (pp90RSK) (90 kD, 1:250 dilution, #9344 Cell Signaling Technology) antibody overnight at 4°C. Following incubation with the appropriate secondary antibody and avidin-biotin peroxidase complexes (ABC kit, ScyTek, Logan, Utah, USA), sections were colored using a 3,3′-diaminobenzidine (DAB) kit (ScyTek, Logan, Utah, USA), and counterstained with hematoxylin. The stained sections were mounted in mounting media (Assistant-Histokitt, Germany) and immunopositive cells were detected using microscopic analysis (Axioskop 40, Zeiss). Negative controls for BDNF, pRaf-1, pMEK1/2, pERK1/2, and pp90RSK staining were prepared using adjacent serial sections from the EA group incubated without primary antibodies.
Immunohistochemical costaining
Brain sections were immersed in 3% H2O2/methanol for 15 min and then incubated with a diluted normal blocking serum (Vector Laboratories, CA, USA) at RT for 25 min. Sections were then incubated with a mouse antineuronal nuclei (NeuN) antibody (1:200 dilution, MAB 377 Chemicon) 1.5 h at 37°C and washed with DPBS. Following their incubation with the diluted biotinylated secondary antibody and an ABC-AP reagent (AK-5002, Vectastain), the sections were stained with an alkaline phosphatase substrate solution (SK-5300, Vector Blue). They were then incubated with a rabbit anti-active caspase-3 antibody (17 kD, 1:100 dilution, AB3623 Chemicon) for 1.5 h at 37°C and washed with DPBS. Following their incubation with the diluted biotinylated secondary antibody and an ABC-AP reagent (AK-5001, Vectastain), the sections were stained with an alkaline phosphatase substrate solution (SK-5100, Vector Red), dried, and mounted in mounting media (Assistant-Histokitt, Germany). Finally, the immunopositive cells were detected using microscopic analysis (Axioskop 40, Zeiss).
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assay
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling analysis was used to identify cells with nuclear DNA fragmentation in the ischemic cortex. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling staining was performed according to the manufacturer’s instructions (QIA33 Calbiochem, USA). Briefly, brain sections adjacent to those used in IHC analysis were incubated with 20 μg/ml proteinase K for 20 min at RT, rinsed with a Tris-buffered saline and incubated with a 1 × TdT equilibration buffer for 30 min at RT. They were then incubated with a TdT labeling reaction mixture for 1.5 h at 37°C. After addition of the stop solution and blocking buffer, sections were incubated with 1 × conjugate solution for 30 min at RT, and the TUNEL-positive cells were visualized using a DAB kit (Calbiochem). Finally, sections were counterstained with methyl green (Calbiochem).
Western blot analysis
Three days after reperfusion, the rats were anesthetized with choral hydrate (n = 4). The rat brains were then removed and sectioned coronally from -4.3 mm to +1.7 mm bregma. The brain was separated into the right cortex, right striatum, left cortex, and left striatum, and the right cortex was weighed and homogenized in an ice cold phosphate buffered saline (PBS) (0.5 ml). Lysates were centrifuged at 500 × g for 10 min at 4°C, and the supernatant was removed. After addition of 200 μl cytosol extraction buffer A (#K266-25 BioVision, USA) and 11 μl cytosol extraction buffer B (#K266-25 BioVision, USA), the suspension was centrifuged at 16000 × g for 30 min at 4°C. The supernatant was collected and saved as the cytosolic fraction. The protein concentration of the cytosolic fraction was determined using a Bio-Rad assay. The samples were boiled at 100°C in a sodium dodecyl sulfate (SDS) gel loading buffer for 10 min and loaded onto a 10% SDS polyacrylamide gel. After electrophoresis, the separated proteins were electrotransferred to a nitrocellulose membrane (Hybond-c Extra, Amersham Biosciences, UK) in transfer buffer. The membranes were incubated in 5% skim milk containing 0.1% Tween 20 for 60 min at RT to block nonspecific binding. They were then incubated with a rabbit anti-pMEK1/2 (1:1000 dilution, #2338 Cell Signaling Technology), rabbit anti-pERK1/2 (1:1000 dilution, #4376 Cell Signaling Technology), rabbit anti-pp90RSK (1:1000 dilution, #9344 Cell Signaling Technology), or rabbit anti-phospho-Bad (pBad) (1:1000 dilution, #9291 Cell Signaling Technology) antibody overnight at 4°C. The transferred membranes were also probed with a monoclonal antibody specific for actin (1:5000 dilution, MAB1501 Chemicon) as an internal control for the cytosolic fraction. After washing, membranes were incubated with an anti-rabbit horseradish peroxidase (HRP)-linked IgG (1:5000 dilution, Jackson ImmunoResearch), an anti-mouse HRP-linked IgG (1:5000 dilution, Santa Cruz Biotechnology), or a HRP-conjugated anti-biotin (1:5000 dilution, Cell Signaling Technology) antibody in a PBS for 1 h at RT. Proteins were detected using an enhanced chemiluminesence reagent kit (#34080 Thermo Scientific, USA) according to the manufacturer’s instructions. Densitometric analysis was performed using Alpha Innotech Analyzer software. The optical density was calculated and the levels of proteins were expressed as the densitometric ratio of proteins to actin.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). All variables showed approximately normal distribution and parametric testing, such as analysis of variance (ANOVA), was appropriate. Data from all experimental groups were compared using one-way ANOVA followed by post-hoc analysis using the Scheffe’s test. A P-value < 0.05 was considered statistically significant.
