Design
A randomized, controlled animal experiment.
Time and setting
The experiment was performed at the School of Biological Science and Technology, Central South University, China from September 2011 to March 2012.
Materials
Thirty adult male Sprague-Dawley rats were provided by the Department of Experimental Animals, Xiangya School of Medicine, Central South University, China (license No. SYXK (Hunan) 2011-0001). They were aged 8 weeks and weighed 219.2 ± 19.5 g. They were raised separately, allowed free access to food and water, and were maintained at 20–24°C. The use of animals for experimental procedures was in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[29].
Methods
Exercise patterns
According to the Berdford model[30-31], treadmill exercise (Zhejiang Hangzhou Thai Science and Technology, Zhejiang, China) was performed for the exercise groups. Before experimentation, preparation involved 3-day-long exercise to adapt (velocity: 10 m/min, gradient: 0°) and then a 3-day-long rest period. The experiment started on the 7th day according to the following method:
(1) In the medium-intensity exercise group, the gradient was 10 at an initial velocity of 10 m/min, accelerated to 19.3 m/min (equal to 76% VO2max) within 12 minutes.
(2) In the high-intensity exercise group, the gradient was 10 at an initial velocity of 10 m/min, and then accelerated to 26.8 m/min (equal to 92.3% VO2max) within 12 minutes.
Exhaustion was observed when the rat could not maintain the anticipated velocity and lagged during the last one third of the treadmill exercise over 3 attempts. Rats would appear breathless, showed lassitude and lethargy, and would lie down.
Sample preparation
Rats were sacrificed immediately after acute exhaustion exercise using Zoletil 50 (10 mg/kg, intraperitoneal injection)[32]. The rats were transcardially perfused with 0.05 M PBS, and fixed with a freshly prepared solution consisting of 4% (w/v) paraformaldehyde in 0.5 M PBS (pH 7.4). Brains were dissected, post-fixed in the same fixative overnight, and transferred to 30% (w/v) sucrose for cryoprotection. Coronal sections (40 μm thick) were made using a freezing microtome (Leica, Nussloch, Germany). Ten sections on average in the hippocampus were collected from each rat. The sections of 2.5 mm to 2.7 mm posterior from the bregma were used for immunohistochemical staining.
TUNEL staining
Apoptosis was detected by TUNEL[33] using the in situ cell death detection kit (Shenzhen Jinmei BioEngineering Co. Ltd., Shenzhen, China). Shortly after deparaffinization and rehydration, hippocampal sections were rinsed briefly in 0.1 M PBS twice, digested in Proteinase K working solution (10 μg/mL in 10 mM Tris/HCl, pH 7.4–8.0), and incubated at 37°C for 15 minutes. Sections were next treated with TdT enzyme solution, label solution, and converter-peroxidase. The sections were developed with diaminobenzidine substrate for 10 minutes. Negative controls were produced using 0.1 M PBS rather than TUNEL reaction mixture. Apoptotic cells that were TUNEL-positive had brown yellow particles in the nucleus. At a magnification of × 400, 10 microscopic fields in the hippocampal CA1 region were taken randomly, and all cells including TUNEL-positive cells were quantified. The ratio of the number of TUNEL-positive cells to total cells was expressed as a percentage. The sections were observed under a light microscope (Nikon, Tokyo, Japan).
Immunohistochemical staining
Paraffin embedded sections were placed in xylene for 5 minutes × 2 times, and treated with a series of graded ethanol solutions. Sections were incubated in 3% (v/v) H2O2 for 10 minutes, rinsed with distilled water for 5 minutes, washed with PBS, retrieved in citric acid salt antigen retrieval liquid (pH 6.0) using a microwave oven, cooled, and blocked in normal goat serum at room temperature for 10 minutes. The sections were incubated with Bcl-2 or Bax antibody at a dilution of 1:50 (rabbit polyclonal Bcl-2 antibody and mouse anti-rat Bax monoclonal antibody from Shenzhen Jinmei Biological Engineering Co., Ltd., Shenzhen, China) at 4°C in a wet box overnight. The next day, sections were washed with PBS and incubated with horseradish peroxidase-labeled sheep anti-rabbit/mouse (10 μg/mL) in 10 mM Tris/HCl, pH 7.4–8.0 (Shenzhen Jinmei Biological Engineering Co., Ltd.) at 37°C for 30 minutes. Sections were washed, incubated with diaminobenzidine, and counterstained with hematoxylin for 4 minutes. Tissue was examined using optical microscopy (× 400; Nikon), and six fields of view from each slice were analyzed using a Nikon microscope camera system and MIAS medical image analysis system.
Statistical analysis
All data were expressed as mean ± SD and analyzed by SPSS 12.0 statistical software (SPSS, Chicago, IL, USA). One-way analysis of variance followed by Tukey’s method was used for multiple comparisons. A P level less than 0.05 was considered statistically significant.
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