Animals and treatment
All experimental procedures were approved by the Institutional Animal Ethical Committee 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 40 male Sprague–Dawley rats weighing 250-280g were used for this experiment. Rats were housed in the same animal care facility during a 12 hour light/dark cycle throughout the protocol. Sprague–Dawley rats were subjected to transient focal cerebral ischemia induced by left transient middle cerebral artery occlusion (MCAO) as mentioned earlier [1, 28]. Briefly, rats were anesthetized with intra-peritoneal injection of 3.5% chloral hydrate (350 mg/kg) and were placed in a supine position. Body temperature of the rats was maintained at 37±0.5°C on a heating pad. The left common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) were surgically exposed. The CCA was ligated distally and the ECA was ligated proximally to the bifurcation of the ICA and the ECA. A 3–0 silk suture was tied loosely around the ICA, and a micro-vascular clip was placed distally across the ICA. A filament (4–0 nylon suture with rounded tip) was inserted into the ICA through the CCA and gently advanced from the common carotid artery bifurcation to block the middle cerebral artery (MCA) at its origin. The suture around the ICA was tightened, and the microvasculature clip was removed. Mean arterial blood pressure, heart rate and arterial blood gases were analyzed during the process of surgery. The suture was pulled back until the tip reached the suture around the ICA to restore blood flow (reperfusion) after 90 min of MCAO. The animals were not allowed to recover from anesthesia until the wound was closed, and they were sent to their cages.
The neurological function was evaluated using the Bederson’s neurological function test, after 6 hour MCAO. The Bederson’s scores are as follow: no deficits score, 0; unable to extend the contra-lateral forelimb score, 1; flexion of contra-lateral forelimb score, 2; mild circling to the contra-lateral side score, 3; severe circling and allying to the contra-lateral side score, 4. The rats with scores 1–3 were then selected and randomly divided into three groups: the physical exercise group (n=15), which was given running exercise everyday at 3 days after transient MCAO, the control group (n=20), and sham-operated group (n=5, filament was not inserted into the artery), which were fed in standard cages with no special exercise training and served as controls. To normalize for handling stress, sedentary animals in control and sham-operated group were placed on nonmoving wheels for time duration equal to exercised treatments. Exercised rats were further randomized into one of three groups with different exercise durations: 7, 14 and 21 days groups after transient MCAO. Correspondingly, sedentary rats were randomly divided into four groups: 3 days group (basal control group) and 7, 14 and 21 days after transient MCAO.
Exercise training and function testing
All animals submitted to the running wheel exercise were placed into a programmable, motorized wheel apparatus (21cm diameter, 40cm long, made in China), which was easy to quantify the exercise intensity. The rats in physical exercise group were put into the wheel to run at 3 days post-ischemia. At the beginning, the running speed was set as 5 rev/min (about 3m/min), for 20 minutes twice a day (morning and afternoon), then gradually increased to 10rev/min (about 6m/min) on the seventh day, 15 rev/min (about 10m/min) on the fourteenth day. The control group and sham-operated group were housed in a standard cage (n=5) with no special exercise training, supplied with enough food and water. Body weight was monitored every 3 days.
All rats in this study were given 1 week pre-conditioning exercise before MCAO and the investigator was blinded to the experimental groups. Neurological function was assessed on a scale of 0–18 (normal score, 0; maximal deficit score, 18) [14, 29, 30]. Neurological severity score is a combination of motor, sensory, reflex and balance tests [14, 31].
Tissue preparation for histochemistry
Rats were sacrificed after the completion of motor functional evaluation at 3, 7, 14 and 21 days after transient MCAO (n=5 per group at each time point) with an overdose of 10% chloral hydrate and perfused transcardially with 0.9% saline at 4°C followed by 4% paraformaldehyde in phosphate buffer (0.1 mol/L, pH 7.4) . The brains were removed, fixed in the above fixative for 8 hours at 4°C, and then immersed sequentially in 20% and 30% sucrose until sinking occurred. Coronal sections (10-μm thick) were cut on a cryostat (CM1900; Leica, Heidelberger, Germany) from bregma +4.0 to −6.0 mm and used for Nissl staining, immunoflourescence staining or TUNEL staining.
Serial sections from bregma +4.0 to −6.0 mm were selected for Nissl staining to measure infarct volume in the ipsilateral hemisphere. Nissl staining was performed with 0.1% cresyl violet (Sigma) on basis of a standard procedure. For quantification of infarct volume, five successive coronal sections at 2.0-mm intervals from bregma +4.0 to −6.0 mm were selected. The infarct areas marked by black stars (see Figure 1C) are referred to as zones of irreversible ischemic damage that have been exposed to the most severe reduction in cerebral blood flow and exhibit severe, consistent, ischemic damage. The area immediately outside this zone is referred to as the ‘peri-infarct’, in which most neurons do not display the histological signs of irreversible damage . The volumes of the ipsilateral and the contra-lateral hemisphere were counted as previously mentioned and relative infarct volume was described as a percentage of the contra-lateral hemisphere [33, 34].
Another set of sections was used for immunofluorescence. Immunofluorescence staining of LC3-II, IGF-1 and Ki67 were performed as following: Sections were pretreated for 5 minutes with hot (85°C) citrate buffer (0.01 mol/L, pH 6.0) for antigen retrieval followed by 5% normal goat serum for 1 hour at room temperature. Next, sections were incubated with mouse anti-LC3 (microtubule-associated protein 1A light chain 3, 1:200; MBL, Naka-Ku Nagoya, Japan), anti-IGF-1 (insulin-like growth factor, 1:100; Millipore, USA) or rabbit anti-Ki67 (rabbit monoclonal to Ki67, 1:400; Abcam, England) overnight at 4°C. After rinsing in phosphate-buffered saline (PBS) 3 times for 5 minutes each, sections were incubated with peroxidase-marked mouse secondary antibody (anti-mouse IgG, 1:1000, Cell Signaling) or rabbit secondary antibody (anti-rabbit IgG, 1:1000, Cell Signaling) for 1 hour at room temperature. Fluorescence signals were detected with a microscope (BX51; Olympus). Negative control sections were incubated with PBS instead of primary antibodies and showed no positive signals.
Double-Immunofluorescence studies were performed for LC3 plus TUNEL (the terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling). Staining steps were the same as those described above: sections were pretreated for 5 minutes with hot (85°C) citrate buffer (0.01 mol/L, pH 6.0) for antigen retrieval followed by 5% normal goat serum for 1 hour at room temperature. Then, sections were incubated with mouse anti-LC3 (microtubule-associated protein 1A light chain 3, 1:200; MBL, Naka-Ku Nagoya, Japan), overnight at 4°C. After rinsing in phosphate-buffered saline (PBS) 3 times for 5 minutes each, sections were incubated with peroxidase-marked mouse secondary antibody (anti-mouse IgG, 1:1000, Cell Signaling) plus Terminal deoxynucleotidyl transferase and digoxigenin-labeled nucleotides (In Situ Cell Death Detection Kit, AP, Roche Corp., Switzerland) for 1 h at 37°C. After rinsing, the co-location of LC3 and TUNEL signals were observed with a microscope (BX51; Olympus).
Image analysis and quantification
All histological images were captured at the same exposure and analyzed with Image-Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD, USA) by one author who was not aware of the assignment of animals’ group assignment. The regions of interest were defined as a zone with 700 μm width and length in the peri-infarct region, which is immediately outside the infarct zone (Figure 1C) . For cell counting of LC3/TUNEL/IGF-1/Ki67-immunopositive cells, eight consecutive sections at 240-μm intervals from bregma 0.20 to −2.20 mm were analyzed. The number of LC3/TUNEL/IGF-1/Ki67-immunopositive cells in the peri-infarct region (Figure 1C, marked by black squares) was counted by Image-Pro Plus image analysis software in 4 non-overlapping fields (425 μm X 320 μm) under X 400 magnification and was presented as the average cell number per field on each section . The final cell number per rat was the average cell number of all the sections .
Numerical data were presented as mean ± SD. Repeated measures ANOVA was used to evaluate MNSS variables. A nonparametric test was used to evaluate MNSS and infarct volume values. Two independent samples t-test was used for 2-group comparisons of the number of LC3/TUNEL/Ki67/IGF-1 positive cells variables. Pearson bivariate correlation was used to run correlation analysis. Statistical analysis was performed using SPSS 16.0 for windows (SPSS Inc, Chicago, IL, USA). *p < 0.05, **p < 0.01, ***p < 0.001 when comparison was made.