Neurochem Res DOI 10.1007/s11064-014-1350-y
ORIGINAL PAPER
Synergic Effect of Exercise and Lipoic Acid on Protection Against Kainic Acid Induced Seizure Activity and Oxidative Stress in Mice Hee-jae Kim • Wook Song • Jin-Soo Kim • Eun Hee Jin • Moon-Seok Kwon • Sok Park
Received: 18 March 2014 / Revised: 26 May 2014 / Accepted: 27 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Anti-convulsant effects of physical exercise and lipoic acid (LA), also referred to as thioctic acid with antioxidant activity, were investigated using chemical induced seizure model. We investigated the synergic effect of physical exercise and LA on kainic acid-induced seizure activity caused by oxidative stress. After 8 weeks of swimming training, body weight decreased and endurance capacity increased significantly compared to sedentary mice. Kainic acid (30 mg/kg, i.p.) evoked seizure activity 5 min after injection, and seizure activity peaked approximately 80 min after kainic acid treatment. Median seizure activity score in KA only treated group was 4.55 (range 0.5–5), 3.45 for ‘‘LA ? KA’’ group (range 0.5–4.3), 3.12 for ‘‘EX ? KA’’ group (range 0.05–3.4, p \ 0.05 vs. ‘‘KA only’’ group), 2.13 for ‘‘EX ? LA ? KA’’ group (range 0.5–3.0, p \ 0.05 vs. ‘‘EX ? KA’’ group). Also, there was a synergic cooperation of exercise and LA in lowering the mortality in kainic acid treated mice (v2 = 5.45, p = 0.031; ‘‘EX ? KA’’ group vs. H. Kim W. Song J.-S. Kim Health and Exercise Science Laboratory, Institute of Sports Science, Seoul National University, Seoul, Korea W. Song Institute on Aging, Seoul National University, Seoul, Korea E. H. Jin Education of Physical Recreation, Konkuk University, Seoul, Korea M.-S. Kwon Sports and Wellness Research Center, Yongin University, Yongin, Korea S. Park (&) Division of Sports Industry and Science, Mokwon University, Daejeon, Korea e-mail: [email protected]
‘‘LA ? EX ? KA’’ group). In addition, the synergic effect of exercise and LA was found in PGx activity compared to separated treatment (‘‘LA ? EX ? KA’’: 37.3 ± 1.36; p \ 0.05 vs. ‘‘LA ? KA’’ and ‘‘EX ? KA’’ group). These results indicate that physical exercise along with LA could be a more efficient method for modulating seizure activity and oxidative stress. Keywords Lipoic acid Exercise Physical activity Oxidative stress Kainate-induced seizure
Introduction Epilepsy is common chronic neurological condition which is major or minor symptoms of various neurological diseases with an incidence of 0.5–1 % in the general population [1]. For epilepsy, several reasons have been identified, yet, oxidative stress has been identified as a main possible mechanism for the seizures activity. Cell respiration normally produces free radicals and reactive species of oxygen and nitrogen, which are neutralized by the antioxidant defenses. Reactive species are used in gene expressions for various enzymes and inflammation related responses [2]. Among body organs, because brain processes the most amount of O2 in small mass and abundant for oxidation available substrates with low antioxidant activities, therefore it is extremely vulnerable to oxidative damage [3–5]. Alpha-lipoic acid (ALA), also referred to as thioctic acid, has recently gained considerable attention as an antioxidant. It is known to be play essential role in mitochondrial dehydrogenase reaction. LA, as growth factor, first isolated in the late 1940s, with ability to react with ROS [6], it was determined to possess antioxidant
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properties by acting as coenzyme for pyruvate dehydrogenase and alpha-ketoglutarate in mitochondrial metabolism. With current anticonvulsant evidence of LA on epilepsy induced by pilocarpine, clinical experiments test LA as a drug for potential treatment for neurological disorders such as depression, Parkinson’s disease, and epilepsy [7]. Recently, number of evidences has suggested that physical exercise has the capacity to enhance oxidative stress-associated diseases, such as epilepsy [8–10]. Physical exercise reversed electrophysiological changes in the hippocampus in pilocarpine induced seizures model and promoted positive plastic changes in hippocampus formation [11]. In addition, Physical exercise significantly decreased seizure activity and mortality rate [12]. Exercise prior to seizure has been found to produce neuro-protective effects against brain damage and decreased the duration of PTZ-induced seizure [13]. In addition, in the chronic phase in the pilocarpine model, physical exercise not only attenuate the frequency of seizures [14, 15], but also reduced seizure occurrence and susceptibility to seizure [13, 14]. Taken together, previous researchers clearly demonstrated the effect of physical exercise and LA on brain damage or seizure activity by attenuating oxidative stresses. However potential synergic effect of physical exercise and LA on preventing seizure development has not been explored yet. Therefore, in the present study, we investigated the synergic effect of physical exercise and LA on kainic acid-induced seizure activity caused by oxidative stress.
Methods All experimental procedures were approved by the animal care and use committee of Ajou University. Adult male ICR mice weighing approximately 35 g each were obtained from Koatech (Gyunggi, South Korea). Three to four animals were housed in each cage with free access to food and water; the room in which the cages were located was controlled for temperature (22–23 °C) and light (12-h light/dark cycle), which was turned on at 0700 hours daily. The mice were randomly assigned into groups as follow: Control (n = 9), KA only (n = 9), LA ? KA (n = 9), EX ? KA (n = 9), LA ? EX ? KA (n = 9). All mice were adapted to water before beginning training to reduce stress without promoting adaption to swimming training. The adaptation involved keeping the animals in shallow water at 32 °C. The swimming training period lasted 8 weeks and consisted of 60-min sessions daily (three times per week). Swimming was always performed in water maintained at a temperature of 32 °C.
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During the first week of training, all mice experienced a period of swimming adaptation without tail weights, as described above. After the water adaptation period, the mice were subjected to swimming with working burden (5–10 % of body weight) for improvement of endurance exercise capacity [13, 16]. Each of the mice had a weight attached (10 % body weight) to the tail for the duration of the swimto-exhaustion exercise. The mice were assessed to be fatigued when they failed to rise to the surface of the water to breathe within 5 s. Swimming time was recorded as minute for each mice with blind test that researcher could not know the group of mice. The swimming exercise was carried out in a plastic tank (32 9 50 9 35 cm), filled with water to 25 cm depth and maintained at a temperature of 33 ± 1 °C. Lipoic acid was purchased from Sigma (St. Louis, MO, USA) and LA solution was freshly prepared in saline solution, and pH was adjusted to 7.4. Before systemic KA injection (30 mg/kg), mice received a 2-day pretreatment with LA (40 mg/kg body weight, i.p.), or saline solution (control group) [13]. Animals were evaluated continuously for 2 h after KA injection according to the following rating scale: 0 for normal, rare wet dog shakes (WDS), or no convulsions; 1 for intermediate number of WDS or rare focal convulsions affecting the head and extremities; 2 for frequent WDS, frequent focal convulsions, or the appearance of generalized convulsions (i.e., no rearing, no salivation); 3 for frequent WDS, focal convulsions, or frequent appearance of generalized convulsions with rearing (but not falling); 4 for frequent WDS, focal convulsions, or frequent generalized convulsions with falling, jumping, and salivation; and 5 for continuous generalized seizures and death within 2 h [17]. In our experiment, death was defined as the cessation of heartbeat and of breathing at least for 3 min. After the behavioral evaluation (2 h after kainate administration or death), the animals were killed by decapitation and their brain was exposed by removing the parietal bone. To verify glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) activity, a slice from the cerebral cortex was adequately homogenized in 50 volumes (w/v) with Tris–HCl 10 mM (pH 7.4) and an assay was performed by methods of Souza et al. [18] and Pederzolli et al. [19]. The SOD activity was expressed as units/g of protein, GPx activity was represented as GPx units/mg of protein and CAT activity was expressed in units (1 U decomposes 1 lmol of H2O2/min at pH 7.0). The data are expressed as the mean ± mean error (ME). Median scores of seizure activity were calculated and Mann–Whitney U test was done to determine statistical significance. Comparisons were made using v2-test for mortality and ANOVA for body weight and endurance
Neurochem Res
Fig. 1 Effect of 8 weeks of exercise training on body weight (a) and endurance capacity (b). Values are mean ± ME for n = 9 in each group. *p \ 0.05 compared to control group (independent t test)
capacity test. Differences of GPx, SOD and CAT activity between groups were tested with t test. SPSS 15.0 and Origin 8.0 were used to do all statistical analysis. p values \0.05 were considered to be significant. Results As shown in Fig. 1a, the body weights of all experimental animals were increased at the end of 8 weeks of exercise training program compared to levels of baseline. In addition, the trained animals (both of EX group and LA ? EX group) showed the lower body weight (p \ 0.05) compared to sedentary animals (both of Con group and LA group) at the end of training (Fig. 1a). In this study, there was no difference among all experimental groups in levels of food intake (data was not shown). The test of swim-to-exhaustion was performed to evaluate the endurance capacity at the end of training program. Swimming time of the trained animals (both of EX group and LA ? EX group) was significantly increased (p \ 0.05) compared to sedentary animals (both of EX group and LA ? EX group) (Fig. 1b).
Fig. 2 Effect of exercise training and LA treatment on kainic acid induced seizure activity (a) and mortality (b). Kainic acid was given in a single dose of 30 mg/kg (i.p.). a Median scores for seizure activity during 2 h after kainic acid injection. b Mortality was estimated with dead mice within 2 h after kainic acid injection. Values mean ± ME for n = 9 in each group. *p \ 0.05 compared to ‘‘KA only’’ group, and #p \ 0.05 compared to ‘‘Ex ? KA’’ or ‘‘LA ? KA’’ group. a Kruskal–Wallis test with Dunn’s test and b v2 test
We measured the seizure rating scores every 5 min (up to 2 h or death) after kainic acid administration. Kainic acid (30 mg/kg) evoked seizure activity 5 min after injection, and seizure activity peaked approximately 80 min after kainic acid treatment. The maximal seizure activity of ‘‘EX ? KA’’ group (rating score = 3.25 ± 0.14) and ‘‘LA ? EX ? KA’’ group (rating score = 2.31 ± 0.11) displayed lower scores than the KA only treated group (rating score = 5.0 ± 0). Median seizure activity score in KA only treated group was 4.55 (range 0.5–5), 3.45 for ‘‘LA ? KA’’ group (range 0.5–4.3), 3.12 (range 0.05–3.4, p \ 0.05 vs. ‘‘KA only’’ group), 2.13 (range 0.5–3.0, p \ 0.05 vs. ‘‘KA only’’ group, p \ 0.05 vs. ‘‘EX ? KA’’ group) (Fig. 2a). Taken together, we found the combined treatment of exercise and LA has synergic effect on attenuating the seizure activity than separated treatment. Systematic injection of kainic acid
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exercise and LA in lowering the mortality in kainic acid treated mice (v2 = 5.45, p = 0.031; ‘‘EX ? KA’’ group vs. ‘‘LA ? EX ? KA’’ group (Fig. 2b). The activity of GPx, SOD and CAT indicates the antioxidant status and the generation of oxidative stress in kainic acid damaged brain. The PGx activity was significantly decreased after kainic acid injection (Fig. 3a, Con: 37.5 ± 1.43; KA: 19.4 ± 2.54, 48.2 % reduction compared to Con). However, exercise training and LA treated mice showed increment of GPx activity compared to KA only treated mice (Fig. 3a, ‘‘LA ? KA’’: 31.3 ± 1.35, ‘‘EX ? KA’’: 30.4 ± 1.56, p \ 0.05 vs. ‘‘KA only’’ group respectively). In addition, the synergic effect of exercise and LA was found in PGx activity compared to separated treatment (Fig. 3a, ‘‘LA ? EX ? KA’’:37.3 ± 1.36; p \ 0.05 vs. ‘‘LA ? KA’’ and ‘‘EX ? KA’’ group). Also, we found significant reduction of SOD (p \ 0.05) and CAT (p \ 0.05) activity in kainic acid (30 mg/kg) treated group (Fig. 3b, c, SOD: 22.9 % reduction; CAT: 50.3 % reduction compared to Con). The SOD and CAT activity of LA treated or trained mice were significant increased compared to KA only treated mice, however, there were no synergistic effects of combined treatment of exercise and LA (Fig. 3b, c). Thus, these results indicate that there is a synergic anti-oxidant effect of physical exercise and LA especially in PGx activity.
Discussion
Fig. 3 Effect of exercise training and LA treatment on GPx (a), SOD (b), and CAT (c) activity in cerebral cortex. Values mean ± ME for n = 9 in each group. *p \ 0.05 compared to ‘‘KA only’’ group, and # p \ 0.05 compared to ‘‘EX ? KA’’ or ‘‘LA ? KA’’ group (independent t test)
(30 mg/kg) induced generalized seizure leading to death within 2 h [mortality = 100 % (9 mice among 9)]. The trained mice showed lower mortality compared to ‘‘KA only’’ group (v2 = 7.74, p = 0.021; ‘‘KA only’’ group vs. ‘‘EX ? KA’’ group), but not between ‘‘KA only’’ and ‘‘LA ? KA’’ group. Also, there was a synergic cooperation of
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In the present study, exercise and LA significantly reduced kainic acid-induced seizure activity and mortality. In addition, a synergic effect of exercise and LA on kainic acid-induced seizure was observed with alternation of antioxidative status especially in GPx activity. On our knowledge, this is the first study to report the synergic effect of exercise and LA on chemical induced seizure and brain damage to provide the evidence for the more efficient neuroprotective treatment. As shown in Fig. 1, the decrease of body weight and improvement of endurance capacity were found following 8 weeks of swimming exercise. And total food intake was not difference among experimental groups including exercise and sedentary mice and there was no visible symptom of under-nutrition or excessive training throughout entire training period. These results indicate that the exercise training was well designed and performed with improvement of endurance capacity. In addition, although there was a substantial body weight reduction following exercise training compared to sedentary mice, this phenomenon would not be related to seizure generation since there have been no previous literatures reporting association between reduction of body weight and seizure susceptibility.
Neurochem Res
Kainic acid, an analog of glutamate, is 30-fold more potent in neurotoxicity than glutamate. The kainic acid induced seizure model is particularly useful for the research of the evolution, propagation, and pathological consequences of epileptic discharge in the limbic system [20]. Systematic injection of kainic acid cause epileptiform seizure in the CA3 region of hippocampus, and these seizures propagate to other limbic structures and are followed by pattern of neuronal cell loss that is similar to that seen in patients suffering from temporal lobe epilepsy (TLE) [21, 22]. And activation of kainate receptors is associated with an increase in the production of ROS [23, 24]. ROS formation occurs when unpaired electrons escape the electron transport chain and react with molecular oxygen, thus generating superoxide which regulates physiological and pathophysiological conditions [20]. The nervous system contains anti-oxidant enzymes, including SOD and GPx, that are expressed in higher quantities than CAT activity [25]. Enhanced hydrogen peroxide is reduced to water by peroxidases, mostly GPx in the brain. GPx levels in neuronal tissue appear to be relatively low for the prevention of peroxide insults [20]. In the present study, our results show that exercise and LA have neuroprotective effect and enhance the PGx activity in the damaged brain. These results including previous literatures suggest that regulating endogenous anti-oxidant enzymes including PGx by exercise or anti-convulsant agents might be an important strategy for seizure prevention. Since brain tissue is particularly vulnerable to oxidative damage because of its high levels of oxygen consumption, it has been suggested that oxidative stress may play an important role in the pathophysiology following seizure [26, 27]. LA is an endogenous thiol and a cofactor for alpha-ketodehydrogenase complexes that has been explored for the treatment of neurodegenerative diseases including seizure and recently gained considerable attention as a potent anti-oxidant [3]. Recent study reported that LA exerts anticonvulsant effects in a pilocarpine-induced epilepsy model [28–30]. In addition, based on the abundant researches, physical exercise has been found to provide neuro-protective effects against brain damage. We previously reported that regular exercise exerts anticonvulsant effect and increase of antioxidant enzyme activity [12]. These results are consistent with other reports showing that physical activity prevents oxidative stress in the brain [31– 33]. In the present study, anti-oxidant enzymes including SOD, CAT, and PGx were significantly decreased in kainic acid treated group, and increase of these enzymes activity was found without change of GPx protein expression (data was not shown) in both of trained group and LA treated group. Also, we found synergistic improvement of GPx activity in exercise training with LA treated mice. Taken together, evaluating the effective alternation of oxidative
statues using combined treatment of anticonvulsant agents and physical activity might be a potent preventive strategy for inhibiting the seizure development. Effect of physical exercise on seizure and brain damage is still debatable, however, demonstrating the relationship between exercise and epilepsy activity became important matter as benefits of regular cardiovascular exercise have become more clearly understood [34]. Since physical exercise increase antioxidant system through different mechanism, trained subjects can manage injuries induced by reactive species of oxygen better than sedentary ones [35, 36]. While many different antioxidant substance that known to reduce seizure activity that induced by oxidative stress, it can only provide optimal seizure control in about 80 % of all patient, seizure activity remains uncontrolled in a significant number of individuals [37]. However, exercise intensity and duration applied for healthy adults are not recommended for patients with epilepsy, since vigorous exercise may cause harmful effect on damaged brain. To bring efficient modulation of seizure activity, efforts to evaluate the effective coordination of physical exercise and potent anti-convulsant agents (e.g., physical exercise and LA) are crucial. Our results showed that treatment with LA in trained mice significantly decreased seizure activity and mortality with increase of PGx activity. These results indicate that physical exercise along with LA could be a more efficient method for modulating seizure activity. Acknowledgments This work was supported by National Research Foundation of Korea Grant funded by Korean Government (NRF2014S1A5A8018765) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by MEST 2011-0030133.
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23. Bleakman D, Lodge D (1998) Neuropharmacology of AMPA and kainate receptors. Neuropharmacology 37:1187–1204 24. Wang Q, Xu J, Rottinghaus GE, Simonyi A, Lubahn D, Sun GY, Sun AY (2002) Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res 958:439–447 25. Shivakumar BR, Anandatheerthavarada HK, Ravindranath V (1991) Free radical scavenging systems in developing rat brain. Int J Dev Neurosci 9:181–185 26. Patel MN (2002) Oxidative stress, mitochondrial dysfunction, and epilepsy. Free Radic Res 36:1139–1146 27. Frantseva MV, Velazquez JL, Hwang PA, Carlen PL (2000) Free radical production correlates with cell death in an in vitro model of epilepsy. Eur J Neurosci 12:1431–1439 28. Santos IM, Tome Ada R, Feitosa CM, de Souza GF, Feng D, de Freitas RM, Jordan J (2010) Lipoic acid blocks seizures induced by pilocarpine via increases in delta-aminolevulinic dehydratase and Na?, K?-ATPase activity in rat brain. Pharmacol Biochem Behav 95:88–91 29. Freitas RM, Nascimento VS, Vasconcelos SM, Sousa FC, Viana GS, Fonteles MM (2004) Catalase activity in cerebellum, hippocampus, frontal cortex and striatum after status epilepticus induced by pilocarpine in Wistar rats. Neurosci Lett 365:102–105 30. Freitas RM (2009) The evaluation of effects of lipoic acid on the lipid peroxidation, nitrite formation and antioxidant enzymes in the hippocampus of rats after pilocarpine-induced seizures. Neurosci Lett 455:140–144 31. Cechetti F, Worm PV, Elsner VR, Bertoldi K, Sanches E, Ben J, Siqueira IR, Netto CA (2012) Forced treadmill exercise prevents oxidative stress and memory deficits following chronic cerebral hypoperfusion in the rat. Neurobiol Learn Mem 97:90–96 32. Coskun S, Gonul B, Guzel NA, Balabanli B (2005) The effects of vitamin C supplementation on oxidative stress and antioxidant content in the brains of chronically exercised rats. Mol Cell Biochem 280:135–138 33. Hrncic D, Rasic-Markovic A, Lekovic J, Krstic D, Colovic M, Macut D, Susic V, Djuric D, Stanojlovic O (2013) Exercise decreases susceptibility to homocysteine seizures: the role of oxidative stress. Int J Sports Med. doi:10.1055/s-0033-1357162 34. Arida RM, Scorza FA, Scorza CA, Cavalheiro EA (2009) Is physical activity beneficial for recovery in temporal lobe epilepsy? Evidences from animal studies. Neurosci Biobehav Rev 33:422–431 35. Aksu I, Topcu A, Camsari UM, Acikgoz O (2009) Effect of acute and chronic exercise on oxidant-antioxidant equilibrium in rat hippocampus, prefrontal cortex and striatum. Neurosci Lett 452:281–285 36. Radak Z, Kumagai S, Taylor AW, Naito H, Goto S (2007) Effects of exercise on brain function: role of free radicals. Appl Physiol Nutr Metab 32:942–946 37. Dichter MA, Hauser WA, Vinters HV, Pedley TA (2007) Epidemiology, pathology and genetics of epilipcy. In: Engel J, Pedley TA, Aicardi J, Dichter MA, Moshe SL (eds) Epilepsy: comprehensive textbook, vol 1, 2nd edn. Lippinoctt Williams & Wilkins, Philadelphia, pp 9–211
ORIGINAL PAPER
Synergic Effect of Exercise and Lipoic Acid on Protection Against Kainic Acid Induced Seizure Activity and Oxidative Stress in Mice Hee-jae Kim • Wook Song • Jin-Soo Kim • Eun Hee Jin • Moon-Seok Kwon • Sok Park
Received: 18 March 2014 / Revised: 26 May 2014 / Accepted: 27 May 2014 Ó Springer Science+Business Media New York 2014
Abstract Anti-convulsant effects of physical exercise and lipoic acid (LA), also referred to as thioctic acid with antioxidant activity, were investigated using chemical induced seizure model. We investigated the synergic effect of physical exercise and LA on kainic acid-induced seizure activity caused by oxidative stress. After 8 weeks of swimming training, body weight decreased and endurance capacity increased significantly compared to sedentary mice. Kainic acid (30 mg/kg, i.p.) evoked seizure activity 5 min after injection, and seizure activity peaked approximately 80 min after kainic acid treatment. Median seizure activity score in KA only treated group was 4.55 (range 0.5–5), 3.45 for ‘‘LA ? KA’’ group (range 0.5–4.3), 3.12 for ‘‘EX ? KA’’ group (range 0.05–3.4, p \ 0.05 vs. ‘‘KA only’’ group), 2.13 for ‘‘EX ? LA ? KA’’ group (range 0.5–3.0, p \ 0.05 vs. ‘‘EX ? KA’’ group). Also, there was a synergic cooperation of exercise and LA in lowering the mortality in kainic acid treated mice (v2 = 5.45, p = 0.031; ‘‘EX ? KA’’ group vs. H. Kim W. Song J.-S. Kim Health and Exercise Science Laboratory, Institute of Sports Science, Seoul National University, Seoul, Korea W. Song Institute on Aging, Seoul National University, Seoul, Korea E. H. Jin Education of Physical Recreation, Konkuk University, Seoul, Korea M.-S. Kwon Sports and Wellness Research Center, Yongin University, Yongin, Korea S. Park (&) Division of Sports Industry and Science, Mokwon University, Daejeon, Korea e-mail: [email protected]
‘‘LA ? EX ? KA’’ group). In addition, the synergic effect of exercise and LA was found in PGx activity compared to separated treatment (‘‘LA ? EX ? KA’’: 37.3 ± 1.36; p \ 0.05 vs. ‘‘LA ? KA’’ and ‘‘EX ? KA’’ group). These results indicate that physical exercise along with LA could be a more efficient method for modulating seizure activity and oxidative stress. Keywords Lipoic acid Exercise Physical activity Oxidative stress Kainate-induced seizure
Introduction Epilepsy is common chronic neurological condition which is major or minor symptoms of various neurological diseases with an incidence of 0.5–1 % in the general population [1]. For epilepsy, several reasons have been identified, yet, oxidative stress has been identified as a main possible mechanism for the seizures activity. Cell respiration normally produces free radicals and reactive species of oxygen and nitrogen, which are neutralized by the antioxidant defenses. Reactive species are used in gene expressions for various enzymes and inflammation related responses [2]. Among body organs, because brain processes the most amount of O2 in small mass and abundant for oxidation available substrates with low antioxidant activities, therefore it is extremely vulnerable to oxidative damage [3–5]. Alpha-lipoic acid (ALA), also referred to as thioctic acid, has recently gained considerable attention as an antioxidant. It is known to be play essential role in mitochondrial dehydrogenase reaction. LA, as growth factor, first isolated in the late 1940s, with ability to react with ROS [6], it was determined to possess antioxidant
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properties by acting as coenzyme for pyruvate dehydrogenase and alpha-ketoglutarate in mitochondrial metabolism. With current anticonvulsant evidence of LA on epilepsy induced by pilocarpine, clinical experiments test LA as a drug for potential treatment for neurological disorders such as depression, Parkinson’s disease, and epilepsy [7]. Recently, number of evidences has suggested that physical exercise has the capacity to enhance oxidative stress-associated diseases, such as epilepsy [8–10]. Physical exercise reversed electrophysiological changes in the hippocampus in pilocarpine induced seizures model and promoted positive plastic changes in hippocampus formation [11]. In addition, Physical exercise significantly decreased seizure activity and mortality rate [12]. Exercise prior to seizure has been found to produce neuro-protective effects against brain damage and decreased the duration of PTZ-induced seizure [13]. In addition, in the chronic phase in the pilocarpine model, physical exercise not only attenuate the frequency of seizures [14, 15], but also reduced seizure occurrence and susceptibility to seizure [13, 14]. Taken together, previous researchers clearly demonstrated the effect of physical exercise and LA on brain damage or seizure activity by attenuating oxidative stresses. However potential synergic effect of physical exercise and LA on preventing seizure development has not been explored yet. Therefore, in the present study, we investigated the synergic effect of physical exercise and LA on kainic acid-induced seizure activity caused by oxidative stress.
Methods All experimental procedures were approved by the animal care and use committee of Ajou University. Adult male ICR mice weighing approximately 35 g each were obtained from Koatech (Gyunggi, South Korea). Three to four animals were housed in each cage with free access to food and water; the room in which the cages were located was controlled for temperature (22–23 °C) and light (12-h light/dark cycle), which was turned on at 0700 hours daily. The mice were randomly assigned into groups as follow: Control (n = 9), KA only (n = 9), LA ? KA (n = 9), EX ? KA (n = 9), LA ? EX ? KA (n = 9). All mice were adapted to water before beginning training to reduce stress without promoting adaption to swimming training. The adaptation involved keeping the animals in shallow water at 32 °C. The swimming training period lasted 8 weeks and consisted of 60-min sessions daily (three times per week). Swimming was always performed in water maintained at a temperature of 32 °C.
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During the first week of training, all mice experienced a period of swimming adaptation without tail weights, as described above. After the water adaptation period, the mice were subjected to swimming with working burden (5–10 % of body weight) for improvement of endurance exercise capacity [13, 16]. Each of the mice had a weight attached (10 % body weight) to the tail for the duration of the swimto-exhaustion exercise. The mice were assessed to be fatigued when they failed to rise to the surface of the water to breathe within 5 s. Swimming time was recorded as minute for each mice with blind test that researcher could not know the group of mice. The swimming exercise was carried out in a plastic tank (32 9 50 9 35 cm), filled with water to 25 cm depth and maintained at a temperature of 33 ± 1 °C. Lipoic acid was purchased from Sigma (St. Louis, MO, USA) and LA solution was freshly prepared in saline solution, and pH was adjusted to 7.4. Before systemic KA injection (30 mg/kg), mice received a 2-day pretreatment with LA (40 mg/kg body weight, i.p.), or saline solution (control group) [13]. Animals were evaluated continuously for 2 h after KA injection according to the following rating scale: 0 for normal, rare wet dog shakes (WDS), or no convulsions; 1 for intermediate number of WDS or rare focal convulsions affecting the head and extremities; 2 for frequent WDS, frequent focal convulsions, or the appearance of generalized convulsions (i.e., no rearing, no salivation); 3 for frequent WDS, focal convulsions, or frequent appearance of generalized convulsions with rearing (but not falling); 4 for frequent WDS, focal convulsions, or frequent generalized convulsions with falling, jumping, and salivation; and 5 for continuous generalized seizures and death within 2 h [17]. In our experiment, death was defined as the cessation of heartbeat and of breathing at least for 3 min. After the behavioral evaluation (2 h after kainate administration or death), the animals were killed by decapitation and their brain was exposed by removing the parietal bone. To verify glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) activity, a slice from the cerebral cortex was adequately homogenized in 50 volumes (w/v) with Tris–HCl 10 mM (pH 7.4) and an assay was performed by methods of Souza et al. [18] and Pederzolli et al. [19]. The SOD activity was expressed as units/g of protein, GPx activity was represented as GPx units/mg of protein and CAT activity was expressed in units (1 U decomposes 1 lmol of H2O2/min at pH 7.0). The data are expressed as the mean ± mean error (ME). Median scores of seizure activity were calculated and Mann–Whitney U test was done to determine statistical significance. Comparisons were made using v2-test for mortality and ANOVA for body weight and endurance
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Fig. 1 Effect of 8 weeks of exercise training on body weight (a) and endurance capacity (b). Values are mean ± ME for n = 9 in each group. *p \ 0.05 compared to control group (independent t test)
capacity test. Differences of GPx, SOD and CAT activity between groups were tested with t test. SPSS 15.0 and Origin 8.0 were used to do all statistical analysis. p values \0.05 were considered to be significant. Results As shown in Fig. 1a, the body weights of all experimental animals were increased at the end of 8 weeks of exercise training program compared to levels of baseline. In addition, the trained animals (both of EX group and LA ? EX group) showed the lower body weight (p \ 0.05) compared to sedentary animals (both of Con group and LA group) at the end of training (Fig. 1a). In this study, there was no difference among all experimental groups in levels of food intake (data was not shown). The test of swim-to-exhaustion was performed to evaluate the endurance capacity at the end of training program. Swimming time of the trained animals (both of EX group and LA ? EX group) was significantly increased (p \ 0.05) compared to sedentary animals (both of EX group and LA ? EX group) (Fig. 1b).
Fig. 2 Effect of exercise training and LA treatment on kainic acid induced seizure activity (a) and mortality (b). Kainic acid was given in a single dose of 30 mg/kg (i.p.). a Median scores for seizure activity during 2 h after kainic acid injection. b Mortality was estimated with dead mice within 2 h after kainic acid injection. Values mean ± ME for n = 9 in each group. *p \ 0.05 compared to ‘‘KA only’’ group, and #p \ 0.05 compared to ‘‘Ex ? KA’’ or ‘‘LA ? KA’’ group. a Kruskal–Wallis test with Dunn’s test and b v2 test
We measured the seizure rating scores every 5 min (up to 2 h or death) after kainic acid administration. Kainic acid (30 mg/kg) evoked seizure activity 5 min after injection, and seizure activity peaked approximately 80 min after kainic acid treatment. The maximal seizure activity of ‘‘EX ? KA’’ group (rating score = 3.25 ± 0.14) and ‘‘LA ? EX ? KA’’ group (rating score = 2.31 ± 0.11) displayed lower scores than the KA only treated group (rating score = 5.0 ± 0). Median seizure activity score in KA only treated group was 4.55 (range 0.5–5), 3.45 for ‘‘LA ? KA’’ group (range 0.5–4.3), 3.12 (range 0.05–3.4, p \ 0.05 vs. ‘‘KA only’’ group), 2.13 (range 0.5–3.0, p \ 0.05 vs. ‘‘KA only’’ group, p \ 0.05 vs. ‘‘EX ? KA’’ group) (Fig. 2a). Taken together, we found the combined treatment of exercise and LA has synergic effect on attenuating the seizure activity than separated treatment. Systematic injection of kainic acid
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exercise and LA in lowering the mortality in kainic acid treated mice (v2 = 5.45, p = 0.031; ‘‘EX ? KA’’ group vs. ‘‘LA ? EX ? KA’’ group (Fig. 2b). The activity of GPx, SOD and CAT indicates the antioxidant status and the generation of oxidative stress in kainic acid damaged brain. The PGx activity was significantly decreased after kainic acid injection (Fig. 3a, Con: 37.5 ± 1.43; KA: 19.4 ± 2.54, 48.2 % reduction compared to Con). However, exercise training and LA treated mice showed increment of GPx activity compared to KA only treated mice (Fig. 3a, ‘‘LA ? KA’’: 31.3 ± 1.35, ‘‘EX ? KA’’: 30.4 ± 1.56, p \ 0.05 vs. ‘‘KA only’’ group respectively). In addition, the synergic effect of exercise and LA was found in PGx activity compared to separated treatment (Fig. 3a, ‘‘LA ? EX ? KA’’:37.3 ± 1.36; p \ 0.05 vs. ‘‘LA ? KA’’ and ‘‘EX ? KA’’ group). Also, we found significant reduction of SOD (p \ 0.05) and CAT (p \ 0.05) activity in kainic acid (30 mg/kg) treated group (Fig. 3b, c, SOD: 22.9 % reduction; CAT: 50.3 % reduction compared to Con). The SOD and CAT activity of LA treated or trained mice were significant increased compared to KA only treated mice, however, there were no synergistic effects of combined treatment of exercise and LA (Fig. 3b, c). Thus, these results indicate that there is a synergic anti-oxidant effect of physical exercise and LA especially in PGx activity.
Discussion
Fig. 3 Effect of exercise training and LA treatment on GPx (a), SOD (b), and CAT (c) activity in cerebral cortex. Values mean ± ME for n = 9 in each group. *p \ 0.05 compared to ‘‘KA only’’ group, and # p \ 0.05 compared to ‘‘EX ? KA’’ or ‘‘LA ? KA’’ group (independent t test)
(30 mg/kg) induced generalized seizure leading to death within 2 h [mortality = 100 % (9 mice among 9)]. The trained mice showed lower mortality compared to ‘‘KA only’’ group (v2 = 7.74, p = 0.021; ‘‘KA only’’ group vs. ‘‘EX ? KA’’ group), but not between ‘‘KA only’’ and ‘‘LA ? KA’’ group. Also, there was a synergic cooperation of
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In the present study, exercise and LA significantly reduced kainic acid-induced seizure activity and mortality. In addition, a synergic effect of exercise and LA on kainic acid-induced seizure was observed with alternation of antioxidative status especially in GPx activity. On our knowledge, this is the first study to report the synergic effect of exercise and LA on chemical induced seizure and brain damage to provide the evidence for the more efficient neuroprotective treatment. As shown in Fig. 1, the decrease of body weight and improvement of endurance capacity were found following 8 weeks of swimming exercise. And total food intake was not difference among experimental groups including exercise and sedentary mice and there was no visible symptom of under-nutrition or excessive training throughout entire training period. These results indicate that the exercise training was well designed and performed with improvement of endurance capacity. In addition, although there was a substantial body weight reduction following exercise training compared to sedentary mice, this phenomenon would not be related to seizure generation since there have been no previous literatures reporting association between reduction of body weight and seizure susceptibility.
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Kainic acid, an analog of glutamate, is 30-fold more potent in neurotoxicity than glutamate. The kainic acid induced seizure model is particularly useful for the research of the evolution, propagation, and pathological consequences of epileptic discharge in the limbic system [20]. Systematic injection of kainic acid cause epileptiform seizure in the CA3 region of hippocampus, and these seizures propagate to other limbic structures and are followed by pattern of neuronal cell loss that is similar to that seen in patients suffering from temporal lobe epilepsy (TLE) [21, 22]. And activation of kainate receptors is associated with an increase in the production of ROS [23, 24]. ROS formation occurs when unpaired electrons escape the electron transport chain and react with molecular oxygen, thus generating superoxide which regulates physiological and pathophysiological conditions [20]. The nervous system contains anti-oxidant enzymes, including SOD and GPx, that are expressed in higher quantities than CAT activity [25]. Enhanced hydrogen peroxide is reduced to water by peroxidases, mostly GPx in the brain. GPx levels in neuronal tissue appear to be relatively low for the prevention of peroxide insults [20]. In the present study, our results show that exercise and LA have neuroprotective effect and enhance the PGx activity in the damaged brain. These results including previous literatures suggest that regulating endogenous anti-oxidant enzymes including PGx by exercise or anti-convulsant agents might be an important strategy for seizure prevention. Since brain tissue is particularly vulnerable to oxidative damage because of its high levels of oxygen consumption, it has been suggested that oxidative stress may play an important role in the pathophysiology following seizure [26, 27]. LA is an endogenous thiol and a cofactor for alpha-ketodehydrogenase complexes that has been explored for the treatment of neurodegenerative diseases including seizure and recently gained considerable attention as a potent anti-oxidant [3]. Recent study reported that LA exerts anticonvulsant effects in a pilocarpine-induced epilepsy model [28–30]. In addition, based on the abundant researches, physical exercise has been found to provide neuro-protective effects against brain damage. We previously reported that regular exercise exerts anticonvulsant effect and increase of antioxidant enzyme activity [12]. These results are consistent with other reports showing that physical activity prevents oxidative stress in the brain [31– 33]. In the present study, anti-oxidant enzymes including SOD, CAT, and PGx were significantly decreased in kainic acid treated group, and increase of these enzymes activity was found without change of GPx protein expression (data was not shown) in both of trained group and LA treated group. Also, we found synergistic improvement of GPx activity in exercise training with LA treated mice. Taken together, evaluating the effective alternation of oxidative
statues using combined treatment of anticonvulsant agents and physical activity might be a potent preventive strategy for inhibiting the seizure development. Effect of physical exercise on seizure and brain damage is still debatable, however, demonstrating the relationship between exercise and epilepsy activity became important matter as benefits of regular cardiovascular exercise have become more clearly understood [34]. Since physical exercise increase antioxidant system through different mechanism, trained subjects can manage injuries induced by reactive species of oxygen better than sedentary ones [35, 36]. While many different antioxidant substance that known to reduce seizure activity that induced by oxidative stress, it can only provide optimal seizure control in about 80 % of all patient, seizure activity remains uncontrolled in a significant number of individuals [37]. However, exercise intensity and duration applied for healthy adults are not recommended for patients with epilepsy, since vigorous exercise may cause harmful effect on damaged brain. To bring efficient modulation of seizure activity, efforts to evaluate the effective coordination of physical exercise and potent anti-convulsant agents (e.g., physical exercise and LA) are crucial. Our results showed that treatment with LA in trained mice significantly decreased seizure activity and mortality with increase of PGx activity. These results indicate that physical exercise along with LA could be a more efficient method for modulating seizure activity. Acknowledgments This work was supported by National Research Foundation of Korea Grant funded by Korean Government (NRF2014S1A5A8018765) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by MEST 2011-0030133.
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