Male, 3-week-old Sprague-Dawley rats (n=30, Samtako Bio. Korea Co. Ltd., Seoul, Korea) were adapted to the laboratory environment (temperature, 22°C±1°C; relative humidity, 55%±3%; 12-hr light/12-hr dark photoperiod) for 2 weeks. All rats were housed in pairs, given free access to water and fed a standard chow diet. Studies were performed in accordance with Korea National Sport University standards for the Care and Use of Laboratory Animals (publication no. KNSU-IACUC-2017-05). Rats were allocated to the following groups: saline control group (SC; n=10), kainic acid control group (KA; n=10), and previous exercise and kainic acid group (KE; n=10).

After 2 weeks adaptation for environment, rats in the KE group were subjected to treadmill exercise 5 days a week for 4 weeks. The treadmill exercise was adapted low-intensity exercise that was increased gradually 5 m/min for first 5 min, 8 m/min next 5 min, and 11 m/min last 20 min.

After 48 hr of exercise period, rats in the KA and KE groups were injected kainic acid (10 mg/kg/mL, intraperitoneally). After injection of kainic acid, the animals were put in cages and observed for 8 hr to evaluate involuntary seizure and response by contact stimulus.

After 24 hr of injection, the animals were sacrificed. For the immunohistochemistry, 5 rats of each group were anesthetized by an intraperitoneal (intraperitoneally) injection with xylazine (8 mg/kg) and ketamine (40 mg/kg). Rats were transcardially perfused with 50 mM phosphate-buffered saline (PBS), and fixed with a freshly prepared solution of 4% paraformaldehyde in 100 mM phosphate buffer (PB; pH, 7.4). The brains were dissected and post-fixed in the same fixative for 2 days, and then transferred into a 30% sucrose solution for cryoprotection. Coronal sections of 40-μm thickness were made using a freezing microtome (Leica, Nussloch, Germany). For the analysis of protein levels, brains were quickly extracted, and the hippocampus was dissected and stored at −70°C until analysis.

To detect nNOS-positive cells in the dentate gyrus, brain sections were transferred in 6-well plates loaded with 0.1 M PBS. Rinse sections twice, 10 min each rinse, with 0.1 M PBS on a shaker. After rinsing, sections were incubated with fresh 0.3% H₂O₂ in 0.1 M PBS for 30 min and then blocking solution (BSA 0.1 g; goat serum 1 mL; 0.1 M PBS 9 mL) for 60 min at room temperature. The sections were incubated 3 days with primary antibody (1:600, mouse anti-nNOS) diluted in blocking solution at 4°C for reducing background staining. The sections were then washed 3 times with PBS and incubated for 1 hr with a biotinylated anti-mouse secondary antibody. For staining, the sections were incubated in a reaction mixture consisting of 0.03% DAB and 0.03% H₂O₂ for 5 min.

Subsequently, the slides were air-dried overnight at room temperature and coverslides were mounted using Permount. The number of nNOS-positive cells in the dentate gyrus of hippocampus was counted hemilaterally in every eighth section throughout the dentate gyrus at 400×magnification. The area of the dentate gyrus was traced using the Image Pro Plus image analyzer (Media Cybernetics Inc., Silver Springs, MD, USA) at 40×magnification. For the number of cells, volume of the dentate gyrus was calculated by means of the Cavalieri method as described (Llorens-Martín et al., 2006). The data of the number of cells are obtained by multiplying the mean cell density by the mean dentate gyrus volume for each individual separately and then obtaining the group mean. The mean cell density of each individual is calculated by using a standard optical dissector protocol as published previously (Trejo et al., 2001).

To prepare protein for western blotting, each hippocampus was crushed in a solution containing 150 mM NaCl, 5 mM EDTA, 50 mM Tris-HCl (pH 8.0), 1% NP-40, 1 mM aprotinin, 0.1 mM leupeptin and 1 mM pepstatin, and centrifuged at 15,294×g for 15 min at 4°C. Proteins were quantified by a Bradford assay and 30 μg was loaded onto a 10% gel, subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked in TBS containing 0.001% Tween-20 (TBS-T) and 5% bovine serum albumin (Bovogen Biologicals Ltd., Victoria, Australia) at 4°C for 90 min. After washing, the membrane was incubated overnight at 4°C with the following primary antibodies: Rabbit anti-GAPDH (1:1,000; EMD Millipore, rabbit anti-SOD-1 (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-SOD-2 (1:1,000; Santa Cruz Biotechnology), and mouse anti-catalase (1: 1,000; Santa Cruz Biotechnology). Subsequently, membranes were washed 3 times with TBS-T for 10 min and incubated with goat anti-rabbit IgG (1:2,000; Santa Cruz Biotechnology) and goat anti-mouse IgG (1:2,000; Santa Cruz Biotechnology.) secondary antibody conjugated to alkaline phosphatase for 1 hr at room temperature. The membrane was washed 3 times with TBS-T for 10 min. Membranes were exposed to Luminata (EMD Millipore) and protein bands were imaged using a Kodak Image Station 440CF (Kodak, Rochester, NY, USA) and were quantified using Kodak ID version 3.5 densitometry software (Kodak).

The accumulation of nitrite, and indicator of the production of NO, was determined using a colorimetric assay with a Griess reagent. Hippocampal nitrite was assayed using a nitric oxide assay kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. The nitrite concentration was obtained according to the standard curve generated after measuring absorbance at a wavelength of 540 nm using microplate reader (Hidex, Turku, Finland).

All data were analyzed using IBM SPSS ver. 18.0 (IBM Co., Armonk, NY, USA) by one-way analysis of variance followed by Tukey post hoc test to compare among the experimental groups. Results are presented as the mean±standard deviation. P<0.05 was considered to indicate a statistically significant difference.

In the present results, KA group showed the number of nNOS-positive cells was increased compared with SC group (Figs. 1, 2). By contrast, in the KE group, decreased the number of nNOS-positive cells was observed in dentate gyrus compared with the KA group (Figs. 1, 2).

In the present results, KA group showed increased nitrite level in hippocampus compared with SC group (Fig. 2). By contrast, in the KE group, decreased nitrite level was observed in hippocampus compared with the KA group (Fig. 2).

In the present results, KA group showed reduced SOD-1, SOD-2, and CAT expressions in hippocampus compared with SC group (Fig. 3). By contrast, in the KE group, increased SOD-1, SOD-2, and CAT expressions were observed in hippocampus compared with the KA group (Fig. 3).