After purchasing male Sprague-Dawley rats, weighing 200±10 g (7 weeks old) from a commercial breeder (Orient Co., Seoul, Korea), the experimental procedures were performed in accordance with the animal care guidelines of the National Institutes of Health and the Korean Academy of Medical Sciences. The rats were classified into four groups (n=10 in each group): sham-operation group, brain inflammation-induced group, brain inflammation-induced and treadmill exercise group, brain inflammation-induced and wheel exercise group.

Brain inflammation was induced, as described previously (Kim et al., 2013). Zoletil 50 (10 mg/kg, intraperitoneally; Vibac Laboratories, Carros, France) was used to anesthetize the rats. For the induction of brain inflammation, the rats were placed in a stereotaxic frame. Though a hole drilled in the skull, a 26-gauge needle was implanted into the intraventricle area at the following coordinates: 1.2 mm lateral to the midline, 0.9 mm anterior to the coronal suture, and depth 3.3 mm deep from the surface of brain. Seven μL of solution containing LPS (Sigma Chemical Co., St Louis, MO, USA) was infused over 3 min. The needle remained in place for an additional 3 min after infusion and subsequently was withdrawn slowly. Opposite site was operated at the next day as the same procedure.

Treadmill running applied to the rats in the treadmill exercise group for 30 min per a day during 6 weeks. The running speed was 5 m/min for 5 min, 8 m/min for 5 min, and then 10 m/min for 20 min, without inclination. The daily running distance was 265.00±0.00 m.

The rats in the wheel exercise group was housed in each rat cage containing plastic running wheels (diameter, 15 cm) during 6 weeks. A magnet attached to a running wheel triggered a magnetic reed switch that input to an electrical counter. Revolution number was recorded between 6:00 p.m. and 9:00 a.m., as the rat ran almost exclusively during the dark period. Wheel revolutions were counted irrespective of the direction of the wheel and the number was converted to distance in meters per day. The daily running distance was 258.25±10.40 that was similar with the treadmill running.

Rota-rod test was used to determine balance, as described previously (Jung and Kim, 2017). Rota-rod test was performed at 38 days after induction of brain inflammation. For this test, the rat was placed on the rod and then the rat ran while rod was rolling. At the starting, speed was set at 4.5 rpm, and then speed was increased from 4.5 to 8 rpm within 5 min. The trial ended if the rat fell off from the rung. An arbitrary time limit of 300 sec was set for the testing procedure.

To assess the motor coordination, vertical pole test was performed, as described previously (Yun et al., 2014). The rat was trained in a vertical pole test at 39 days after induction of brain inflammation. The rat was placed on a cloth-tape-covered pole (diameter, 3.0 cm; length, 150 cm), then the pole was gradually lifted to a vertical position and the time a rat stayed on the pole was recorded for a maximum of 180 sec. The rats were habituated to the task in 2 trials per day during 2 days. On test day (third day), 3 measures were taken over 5 trials per rat.

The foot fault score of the foot fault test was determined to evaluate walking ability at 41 days after induction brain inflammation, as described previously (Rodriguez et al., 2016). The rat was placed on an elevated, grid floor (34 mm×29 mm) for 2 min. The openings in the grid were 3 mm². While moving around on the grid, the rat typically placed their paws on the wire frame for foot holds. A foot-fault is counted when there is incorrect placement of an impaired forward limb which leads to a slip (called foot-fault). The number of foot fault for both fore- and hind-limb and the total number of forelimb step were recorded for each animal. A primary measure of interest was the percentage of contralateral forelimb foot faults per forelimb step minus the percentage ipsilateral forelimb foot faults per forelimb step. Foot fault score (%)=contralateral forelimb error/contralateral forelimb steps×100.

The rats were sacrificed one day after determining foot fault test (42 days after inducing brain inflammation). After anesthetizing by Zoletil 50 (10 mg/kg, intraperitoneally; Vibac Laboratories), 50 mM phosphate-buffered saline was transcardially perfused, and fixed with a 4% paraformaldehyde in 100 mM phosphate buffer (pH, 7.4) solution. After dissecting, the brains were postfixed in the same fixative solution during overnight and then transferred into a 30% sucrose solution. Coronal sections with 40-μm thickness were made by a freezing microtome (Leica, Nussloch, Germany). Ten slice sections on average in the motor cortex (M1) were collected from each rat. The sections of 2.70 mm to 1.00 mm posterior from the bregma were used for immunohistochemistry.

Bax and Bcl-2 expressions were detected by western blot analysis, as described previously (Shin et al., 2016). The tissues were homogenized with lysis buffer with 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl·6H₂O, 1 mM EGTA, 1 mM PMSF, 1 mM Na₂VO₄, and 100 mM NaF, and then centrifuged at 14,000 rpm during 30 min. Bio-Rad colorimetric protein assay kit (Bio-Rad, Hercules, CA, USA) was used to measure protein content. Protein with 30 μg was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane. As primary antibodies, mouse beta-actin antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse Bcl-2 antibody (1:1,000; Santa Cruz Biotechnology), and rabbit Bax antibody (1:1,000; Santa Cruz Biotechnology) were used. Horseradish peroxidase-conjugated anti-mouse antibody for beta-actin and Bcl-2 (1:3,000; Vector Laboratories, Burlingame, CA, USA), and horseradish peroxidase-conjugated anti-rabbit antibody for Bax (1:3,000; Vector Laboratories) were used as secondary antibodies. Experiment was performed in normal lab conditions and at room temperature except membrane transfer. Membrane was transferred at 4°C with the cold pack and prechilled buffer. Enhanced chemiluminescence detection kit (Santa Cruz Biotechnology) was used for band detection.

Using In Situ Cell Death Detection Kit (Roche, Mannheim, Germany), TUNEL staining was performed, as described previously (Park et al., 2016). The sections were postfixed in ethanol-acetic acid (2:1) and rinsed. Then, the sections were treated with proteinase K (100 μg/mL), rinsed, incubated in 3% H₂O₂, permeabilized with 0.5% Triton X-100, rinsed again, and treated in TUNEL reaction mixture. The sections were rinsed and visualized using Converter-POD with 0.03% 3,3′-diaminobenzidine (DAB). Nissls (DAKO, Glostrup, Denmark) was used for counter-staining, and the sections were finally mounted onto gelatin-coated slides. After the slides were dried under the room conditions, Permount (Fisher Scientific, Fair Lawn, NJ, USA) was used for the coverslips mounting.

Caspase-3 immunohistochemistry was performed, as described previously (Park et al., 2016). The sections were drawn from each brain and treated with mouse anti-caspase-3 antibody (1:500; Santa Cruz Biotechnology) during overnight, and then treated with biotinylated mouse secondary antibody (1:200; Vector Laboratories) for another 1 hr. Bound secondary antibody was then amplified with Vector Elite ABC kit (1:100; Vector Laboratories). The antibody-biotin-avidin-peroxidase complexes were visualized using 0.03% DAB and the sections were finally mounted onto gelatin-coated slides. After the slides were dried under the room conditions, Permount (Fisher Scientific) was used for the coverslips mounting.

To compare relative expressions of Bcl-2 and Bax, detected bands were calculated densitometrically using Molecular Analyst version 1.4.1 (Bio-Rad). The cross-section area of muscle tissue from each slice was measured using Image-Pro Plus computer-assisted image analysis system (Media Cyberbetics Inc., Silver Spring, MD, USA) attached to a light microscope (Olympus, Tokyo, Japan). The numbers of TUNEL-positive and caspase-3-positive cells in the motor cortex were counted.

Statistical analysis was performed using one-way analysis of variance followed by Duncan post hoc test, and the results are expressed as the mean±standard error of the mean. Significance was set as P<0.05.

The latency of the rota-rod test is presented in Fig. 1. The present results showed that latency in the rota-rod test was decreased in the brain inflammation-induced rats (P<0.05), whereas, treadmill exercise and wheel exercise enhanced the latency in the brain inflammation-induced rats (P<0.05). Treadmill exercise and wheel exercise exerted similar effect on latency in the rota-rod test.

The descending time of the vertical pole test is presented in Fig. 2. The present results showed that descending time in the vertical pole test was decreased in the brain inflammation-induced rats (P<0.05), whereas, treadmill exercise and wheel exercise increased descending time in the brain inflammation-induced rats (P<0.05). Treadmill exercise and wheel exercise exerted similar effect on descending time in the vertical pole test.

The foot fault score of the foot fault test are presented in Fig. 3. The present results showed that foot fault score in the foot fault test was increased in the brain inflammation-induced rats (P<0.05), whereas, treadmill exercise and wheel exercise suppressed latency in the brain inflammation-induced rats (P<0.05). Treadmill exercise and wheel exercise exerted similar effect on descending time in the foot fault test.

To verify the apoptosis induced by brain inflammation, we ascertained relative expressions of Bax and Bcl-2 (Fig. 4). The present results showed that brain inflammation enhanced the expression of Bax (P<0.05) and enfeebled the expression of Bcl-2 (P<0.05). Therefore, brain inflammation enhanced the ratio of Bax to Bcl-2. Treadmill exercise and wheel exercise suppressed the expression of Bax in the brain inflammation-induced rats (P<0.05), resulting in decrease of Bax to Bcl-2 ratio (P<0.05). Treadmill exercise and wheel exercise exerted similar inhibitory effect on Bax expression.

Photomicrographs of TUNEL-positive cells in the motor cortex are presented in Fig. 5. The present results showed that brain inflammation enhanced the number of the TUNEL-positive cells (P<0.05), whereas, treadmill exercise and wheel exercise suppressed this number of the TUNEL-positive cells in the brain inflammation-induced rats (P<0.05). Treadmill exercise and wheel exercise exerted similar inhibitory effect on the number of the TUNEL-positive cells.

Photomicrographs of caspase-3-positive cells in the motor cortex are presented in Fig. 6. The present results showed that brain inflammation enhanced caspase-3 expression (P<0.05), whereas, treadmill exercise and wheel exercise suppressed this caspase-3 expression in the brain inflammation-induced rats (P<0.05). Treadmill exercise and wheel exercise exerted similar inhibitory effect on the caspase-3 expression.