All experimental procedures were approved by the Institutional Animal Ethical Committee of Sun Yat-sen University and were conducted according to the Guide for the Care and Use of Laboratory Animal of the National Institute of Health (Publication No. 80–23, revised 1996). A total of 60 male Sprague–Dawley rats weighing 250–300 g were purchased from the Center for Experimental Animals of Sun Yat-Sen University. Rats were randomly assigned into four groups: sham-operated control group (cont, n = 10), MCAO group (maco, n = 30), MCAO +3-methyladenine (3-MA) group (3-MA, n = 10), and MCAO + normal saline (NS) group (NS, n = 10).
Rats were anesthetized with intraperitoneal (IP) injection of 10% chloral hydrate (3 ml/kg body weight) and subjected to MCAO as described previously, with minor modifications . In brief, a midline neck incision was made, and the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were isolated. The ECA was tied. A 4–0 monofilament nylon suture (Beijing Sunbio Biotech Co. Ltd, Beijing, China) with a rounded tip was aseptically inserted from the right CCA to the ICA through the stump of the ECA and gently advanced to occlude the MCA. Recirculation/reperfusion of cerebral blood flow was allowed by gently removing the monofilament after 1-hour ischemia, followed by different time intervals of reperfusion. In sham-operated animals, all procedures except occlusion of the MCA were performed. Core body temperatures were monitored with a rectal probe and maintained at 37°C during the whole procedure. Following surgery, rats were allowed to recover spontaneous breathing and were kept in their cages with free access to food and water. To evaluate impairment of neuronal function after stroke, neurologic examinations were performed 2,4, and 8 hours after the onset of occlusion and then daily until sacrifice by a blinded examiner who used a modified scoring system based on that developed by Longa et al. . The scoring system used is as follows: 0, no deficits; 1, difficulty in fully extending the contralateral forelimb; 2, unable to extend the contralateral forelimb; 3, circling to the contralateral side; 4, falling to the contralateral side; 5, did not walk spontaneously and displayed a depressed level of consciousness.
One hour before MCAO, rats in the 3-methyladenine (3-MA) group were anaesthetized as above and mounted on a stereotaxic apparatus. 3-Methyladenine (Sigma, St Louis, MO, USA), an autophagy inhibitor, was dissolved in 0.9% saline and injected into the left lateral ventricle at a volume of 10 μL (600 nmol), as previously described [2, 10]. Animals in the vehicle group were anaesthetized and injected with the same volume of normal saline. Animals in the sham-operated and MCAO groups did not receive any intraventricular injections.
At 4, 24 and 72 hours after reperfusion, five rats from each group were sacrificed under deep anesthesia with 10% chloral hydrate (5 ml/kg body weight, IP) and then transcardially perfused with 0.9% sodium chloride at 4°C followed by 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4). Brains were then removed, kept in the same fixative for 48 hours at 4°C and cryoprotected in serial PBS isopropanol sucrose solutions (20% and 30%) at 4°C until brains sank. Coronal sections (10 μm) were cut on a cryostat (CM1900; Leica, Heidelberger, Germany) and used for immunofluorescent staining.
For immunofluorescent assays, frozen sections (10 μm) were prepared using a cryostat (Leica, CM1900) according to standard procedures. The following antibodies were used: rabbit anti-Mcl1 (1:1000; Abcam, Cambridge, UK), mouse anti- Mcl1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-NeuN (1:400; Chemicon, Temecula, CA), mouse anti-rat GFAP (1:800; Cell Signaling Technology, Beverly, MA), mouse anti-rat OX-42 (1:300; Millipore, Billerica, MA, USA), mouse anti-rat microtubule-associated protein1 light chain 3 (LC3; 1:100; MBL, Japan), rabbit anti- LC3B (1:2000; Novus Biologicals, USA), and mouse anti-caspase-3 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA). Immunofluorescence was performed as described previously [11, 12]. Briefly, sections were pre-incubated with 0.3% Triton X-100 (v/v) in 0.01 M PBS (pH 7.4) for 10 minutes, followed by blocking in 10% normal goat serum (KPL, USA) or 1% bovine serum albumin (MPBIO) for 1 hour at room temperature. Sections were then incubated overnight at 4°C with primary antibodies diluted in primary antibody diluents (Dako, Denmark). After rinsing in 0.01 M PBS (3 × 5 minutes), sections were incubated with FITC-goat anti-rabbit IgG antibodies (1:250; KPL, USA) or Alexa Fluor® 555 conjugated goat anti-rabbit IgG (H + L), F(ab’)2 Fragment (1:1000; Cell Signaling Technology) or Alexa Fluor® 555 conjugated goat anti-mouse IgG (H + L), F(ab’)2 Fragment (1:1000; Cell Signaling Technology) in 0.01M PBS for 1 hour at room temperature. Finally, sections were thoroughly washed (3 × 5 minutes). If necessary, sections were counterstained for nuclei with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1:1000; Roche, Mannheim, Germany), and then mounted in ProLong® Gold antifade reagent (P36930, Invitrogen) prior to imaging. Fluorescence signal was detected with a microscope (BX51; Olympus). Negative control sections were incubated with PBS instead of primary antibodies and showed no positive staining.
Western blot experiments
The remaining rats in each group were sacrificed at 4, 24 and 72 hours after reperfusion (five rats in each group). Rats were sacrificed under deep anesthesia with 10% chloral hydrate (5 ml/kg body weight, IP) and then transcardially perfused with 0.9% sodium chloride at 4°C. Brains were then removed, the ipsilateral ischemic cortex and striatum around the infarct area was rapidly dissected from the brain tissue and then homogenized in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) with complete protease inhibitor cocktail (Roche). Protein (50 μg) extract from each sample was separated by SDS-PAGE gel electrophoresis (Bio-Rad) and then transferred onto polyvinylidene fluoride membrane (Millipore). Nonspecific binding was blocked with Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% nonfat milk (MERBCON, BCR685). The membranes were then incubated with primary and secondary antibodies. Primary antibodies used were as follows: rabbit anti-Mcl1 (1:2000; Abcam), rabbit anti-beclin-1 (1:1000; Abcam), rabbit anti-LC3B (1:2000; Novus Biologicals), and mouse monoclonal anti-β-actin (1:3000; Proteintech Group Inc.). Secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse (1:6000; EarthOx, USA) or goat anti-rabbit IgG antibodies (1:6000; EarthOx). Immunoreactivity was detected with Chemiluminescent HRP Substrate (Millipore) for 5 minutes and then exposed to Kodak X-OMAT films. The exposed X-ray films were scanned. Relative changes in protein expression were estimated from mean pixel density using Image J 1.42q, normalized to β-actin, and calculated as target protein expression/β-actin expression ratios.
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL)
Cell apoptosis was assessed using TUNEL staining. This staining was performed using an in situ cell death detection kit (Roche Applied Science, Nonnenwald, Germany) in accordance with the manufacturer’s instructions. Briefly, brain sections were rinsed three times in PBS, and then were incubated in 0.3% Triton X-100 (v/v) in 0.01 M PBS (pH 7.4) for 20 minutes at room temperature. Subsequently, the TUNEL reaction mixture was then applied for 60 minutes at 37°C. Fluorescence signal was detected using a fluorescence microscope (Olympus BX51) at excitation/emission wavelengths of 492/520 nm (FITC, green).
Image analysis and quantification
All histological images were analyzed with Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA) by one blinded assessor. The number of immunostaining positive cells was counted using Image-Pro Plus image analysis software in nine comparable, nonoverlapping fields (425 μm × 320 μm; 3 fields per section × 3 sections per rat) under × 400 magnification and was presented as the average cell number per field on each section [11, 12].
Data are presented as means ± standard deviation. Statistical analysis was performed by one-way analysis of variance followed by Student’s t-test for post hoc analysis. Statistical analysis was performed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA).