The mechanism of epileptic seizure has not been identified clearly. Exercise can play a role of antioxidants against oxidative stress. In the present study, the neuroprotective effects of preconditioning exercise on epileptic seizure were investigated with focusing on antioxidants activity in the hippocampus. Rats were allocated to the following groups: saline control group, kainic acid control group, and previous exercise and kainic acid group. Rats in the previous exercise and kainic acid group were subjected to treadmill exercise 5 days a week for 4 weeks. After 48 hr of exercise period, rats in the kainic acid control group and previous exercise and kainic acid group were injected with kainic acid. The number of neuronal nitric oxide synthase-positive cells and the level of nitrite in hippocampus were increased and the expressions of superoxide dismutase-1, superoxide dismutase-2, and catalase in hippocampus were reduced in kainic acid control group compared with saline control group. By contrast, in the previous exercise and kainic acid group, the number of neuronal nitric oxide synthase-positive cells and the level of nitrite were decreased and the expressions of superoxide dismutase-1, superoxide dismutase-2, and catalase were increased compared with the kainic acid control group. Preconditioning exercise may have neuroprotective effects against oxidative stress via increased antioxidant activity in the hippocampus of epileptic seizure.
The epileptic seizure that is neuronal disorder involves involuntary convulsion and the underlying mechanism has not been identified clearly. However, it has reported that neuronal death induced by dysfunction of neurotransmitter is one of crucial cause in epileptic seizure (
Excitotoxicity is induced by dysfunction of glutamate and excitotoxicity is the main mechanism of neuronal disorder by seizure in epilepsy (
In many studies of epileptic seizure, kainic acid seizure model is useful for epileptic activity in the hippocampus (
Exercise has been demonstrated to have positive effects on brain function. Regular exercise improves neurogenesis, inhibits apoptosis, and increases neurotrophins (
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 (
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
In the present results, KA group showed the number of nNOS-positive cells was increased compared with SC group (
In the present results, KA group showed increased nitrite level in hippocampus compared with SC group (
In the present results, KA group showed reduced SOD-1, SOD-2, and CAT expressions in hippocampus compared with SC group (
Epileptic seizure is known to accompany hippocampal neurodegeneration (
The animals injected kainic acid is a model similar to the temporal lobe epilepsy of humans and has been applied in many studies related to epilepsy (
Excitotoxicity is an oxidative neurodegenerative response of increased NO caused by hyper-influx in cellular Ca2+ by over-activity of glutamate (
The brain is known to be highly susceptible to oxidative stress due to many aerobic metabolism and high level of polyunsaturated acid and iron load (
Regular exercise is known to have a positive effect on the structural development of neurons and improvement of damage, and this effect continues for a certain period of time to protect the nervous system from subsequent neurodegenerative conditions (
The study of
Taken together, preconditioning exercise is seen to play a neuroprotective role in oxidative stress by epileptic seizure through the suppression of NO and activation of antioxidant enzymes. Exercise has effectiveness method to prevent an oxidative stress from epilepsy.
This research was supported by the Tongmyong University Research Grants 2016 (2016A033).
No potential conflict of interest relevant to this article was reported.
The nNOS-positive cells in dentate gyrus of SC (A), KA (B), and KE (C) group. Results are represented as the mean±standard deviation. Different letters represent significantly difference as
The number of nNOS-positive cells in dentate gyrus (A) and nitrite level in the hippocampus (B). Results are represented as the mean±standard deviation. Different letters represent significantly difference as
The expression of SOD-1 (A), SOD-2 (B), and CAT (C) in the hippocampus. Results are represented as the mean±standard deviation. Different letters represent significantly difference as