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Low-intensity physical training recovers object recognition memory impairment in rats after early-life induced Status epilepticus
Int. J. Devl Neuroscience 31 (2013) 196–201
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Low-intensity physical training recovers object recognition memory impairment in rats after early-life induced Status epilepticus Sandro Daniel Córdova ∗ , Cássio Morais Loss, Diogo Losch de Oliveira Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
a r t i c l e
i n f o
Article history: Received 15 August 2012 Received in revised form 2 January 2013 Accepted 3 January 2013 Keywords: Status epilepticus Pilocarpine Physical training Learning Memory
a b s t r a c t When it occurs early in life, Status epilepticus (SE) can cause behavioural and cognitive impairments in adulthood. Here, we evaluated the putative benefits of low-intensity treadmill training on long-standing cognitive impairment in rats submitted to SE early in life. Wistar rats were submitted to LiCl-pilocarpineinduced SE at P16. Animals from the trained group underwent a low-intensity treadmill protocol for 5 days per week for 4 weeks. At adulthood, rats subjected to early-life SE displayed impairment in long-term memory in an object recognition task, while the training protocol completely reversed this deficit. This result was associated with neither locomotor alterations nor changes in emotional behaviour; there were no differences between groups in the distance travelled, grooming or rearing in the open field test; there were also no differences between groups in the number of risk assessment, time spent in open arms in an elevated plus maze and number of entries into the open arms. These data suggest that physical exercise can ameliorate the long-standing recognition memory deficit induced by early-life SE, suggesting that it may be useful as a putative intervention for patients who suffered SE during infancy. © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Status epilepticus (SE) is a common childhood neurological emergency characterised as a seizure or repeated seizures that last more than 30 min (Chen and Wasterlain, 2006). Although SE can occur at all ages, the highest incidence is observed before 2 years of age (Shinnar et al., 1997; Singh et al., 2010). When it occurs early in life, SE can be potentially harmful to the brain (Wasterlain et al., 1993, 2002) and is associated with a higher incidence of neurological disorders later in life (Kwong et al., 2004). The most frequent neurological dysfunctions associated with early-life SE include alterations in sociolinguistic and psychomotor development, cognitive impairments and alterations in emotional behaviour (Roy et al., 2011). Neurological disabilities observed in humans are very similar to those found in animal models of SE. Male rats that suffered SE at 16–20 days old showed long-term deficits in spatial learning in the water maze task (Cilio et al., 2003) and showed an impairment in aversive memory learning in an inhibitory avoidance task (de Oliveira et al., 2008). Furthermore, animals submitted to LiCl-pilocarpine-induced SE between the 2nd and 3rd weeks of life presented increased levels of anxiety in the
∗ Corresponding author at: Departamento de Bioquímica, ICBS, UFRGS, Rua Ramiro Barcelos 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil. Tel.: +55 51 33085555, fax: +55 51 33085540. E-mail address: [email protected] (S.D. Córdova). 0736-5748/$36.00 © 2013 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2013.01.002
elevated plus maze, as well as impaired performance in the water maze task 3 months after the insult (Kubová et al., 2004). Animal studies and clinical trials have demonstrated that treatment of SE with traditional or new antiepileptic drugs (AEDs), such as phenytoin and levetiracetam, may stop seizure activity but fails to prevent the above-mentioned SE-induced cognitive and behavioural alterations later in life (Brandt et al., 2007; Temkin, 2001).It has been demonstrated that physical training may have benefits for the brain and can be used as a potential therapeutic strategy for different degenerative brain diseases (Ahlskog et al., 2011; Cotman and Berchtold, 2002; Lafenêtre et al., 2010; O’Callaghan et al., 2009). Aerobic training promoted reduction in the fall risk, increased mobility, and improved the quality of life in Parkinson’s disease (PD) patients (Fisher et al., 2008; Herman et al., 2007). In addition, PD patients who participated in a training programme exhibited cognitive benefits in frontal lobe-dependent tasks (Cruise et al., 2011). For patients with Alzheimer’s disease, 12 weeks of physical training led to an improvement in the performance of daily activities (Santana-Sosa et al., 2008). Benefits were also observed in several animal models, with improvements in cognitive function, elevation of hippocampal BDNF (Ke et al., 2011b; Sartori et al., 2009; Vaynman et al., 2004), and increased BrdUlabelled cells in the dentate gyrus of the hippocampus (Chae et al., 2009). Although studies have demonstrated the beneficial effects of physical training on SNC, little is known about the effects of an exercise programme on cognitive changes in individuals who
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suffered SE early in life. The aim of this study was to investigate the potential benefits of a low-intensity treadmill exercise protocol on behavioural alterations in adult rats subjected to LiCl-pilocarpineinduced SE early in life.
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(b) Temporal organisation of locomotion: time in home-base (the square where animal remained for more time during the test), number of trips (departures from the home-base), trip length (distance/number of trips) and stops/trip (number of stops/number of trips). (c) Spatial distribution of locomotion: distance travelled along the vicinity and locomoting time spent along the vicinity of the arena walls.
2. Experimental procedure 2.1. Materials Pilocarpine hydrochloride was purchased from Sigma–Aldrich (USA). FluoroJade B was purchased from Chemicon, Inc. (USA). Other chemicals were purchased from Nuclear (Brazil). 2.2. Subjects Male Wistar rats (15 days postnatal) were obtained from a local breeding facility. The litters were culled to 8 pups. The day of birth was defined as day 0, and the animals were weaned on postnatal day 21. After weaning, animals were housed in standard polypropylene cages (4–5 animals per cage) with food and water ad libitum (21 ± 1 ◦ C room temperature). Rats were kept under a 12:12 h light/dark cycle (lights on at 7:00 a.m.). The handling and care of the animals was conducted according to the Guide for Care and Use of Laboratory Animals from the National Institutes of Health (NIH Publications No. 80-23, revised 1996). All procedures in the present study were approved by the Committee of Ethics of the Universidade Federal do Rio Grande do Sul.
2.7. Object recognition task The arena for the object recognition test was the same used in the open field task. Two glass objects of comparable size (10 cm high) were fixed to the apparatus 10 cm from the wall and 15 cm from each other. In the training session, animals were allowed to explore two identical objects (object A and A’) for 5 min. Object exploration was considered to be any investigative behaviour (head orientation or sniffing), deliberate contact with the object at a distance of ≤2 cm, or when the animal touched the object with its nose. To evaluate short-term memory (STM), animals were allowed to explore one familiar and one novel object (object B) for 5 min at 3 h after the training session. Twenty-four hours after the training session, long-term memory (LTM) was assessed by changing object B for a new form (object C) and allowing the animals to explore them for 5 min (Bevins and Besheer, 2006). Memory was operationally defined through the discrimination index, which was calculated in absolute values as follows: (A/A + A’)-(A’/A + A’) for training session, (B/A + B) − (A/A + B) for STM, and (C/A + C) − (A/A + C) for LTM. Average speed, total time exploring objects and the distance travelled in the apparatus were also recorded. 2.8. Elevated plus-maze
2.3. Induction of S. epilepticus Rat pups were injected with a solution of LiCl (3 mEq/kg, i.p.) 12–18 h prior to pilocarpine hydrochloride administration (60 mg/kg, i.p.) on postnatal day 16 (Hirsch et al., 1992). Control animals were handled and housed in the same manner and received an equal volume of saline solution (0.9% NaCl). Rats were kept in individual plastic cages at nest temperature for seizure observation. The duration of SE was evaluated only by behavioural measures where SE was defined, according to Hirsch et al. (1992), as sustained orofacial automatisms, salivation, chewing, forelimb clonus, loss of the righting reflex and falling. The rats were allowed to spontaneously recover from SE. Each experimental group contained pups from several litters. According to Priel et al. (1996), the induction of SE at this age does not induce spontaneous seizures in adulthood. 2.4. Exercise protocol Animals were divided into 4 groups: control (C) (n = 10), trained (T) (n = 10), S. epilepticus (SE) (n = 11) and S. epilepticus+trained (SE+T) (n = 11). Seven days after SE induction, animals from the T and SE+T groups were submitted to a low-intensity exercise protocol over 4 weeks, starting the familiarisation at P25 and the training programme at P27. Rats ran 30 min daily on one four-line treadmill for 5 days a week. The warm-up consisted of 2 m/min for the first 5 min, followed by 5 m/min for the next 5 min and 8 m/min in the last 20 min (Kim et al., 2003). Control and SE animals were left on the treadmill for 30 min, 5 days a week, with the treadmill stopped. Stimulation was not used to motivate rats to run; animals that refused to run were removed from the experiment (only 1 animal in the T group was excluded). 2.5. Behavioural tasks After the end of the exercise protocol, animals were subjected to behavioural tasks. Open-field, object recognition and elevated plus maze tasks were conducted on P56, P57-58 and P60, respectively. For all behavioural procedures, animals were placed in the testing room (temperature 21 ± 2◦ C) 1 h before the beginning of the task to allow them to habituate to the environment and the researchers. Because rats are nocturnal, all tasks were performed between 6:00 p.m. and 10:00 p.m. The behavioural profile was recorded and analysed using the ANY-Maze® video-tracking system (Stoelting, CO). Between every trial, all apparatuses were cleaned with a 70% ethanol solution.
The maze consisted of two open arms (50 cm × 10 cm) and two closed arms (50 cm × 10 cm × 40 cm), with arms of each type opposite to each other (Pellow et al., 1985). The maze was elevated to a height of 50 cm from the floor. The experiment was conducted in a room illuminated by red light. The light intensity in the centre of apparatus was 5 lx. Rats were placed into the centre of the maze facing a closed arm and were left free to explore it for 5 min. Number of arm entries, time spent in the arms, number of risk assessment behaviours (when the rodent is motionless in the centre or closed zone, but is stretched forward into the open arms with some but not all paws, returning then to the initial position) and total distance travelled were registered. 2.9. Data analysis All data were expressed as the mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post hoc test for unequal samples. For all parameters, p < 0.05 was considered significant.
3. Results 3.1. Characterisation of S. epilepticus Within 2–5 min after pilocarpine administration, all LiClpilocarpine-treated animals started having behavioural changes consisting of defecation, salivation, body tremor, and scratching. This behavioural pattern progressed within 8–12 min to increased levels of motor activity and culminated in a convulsive SE in all LiCl-pilocarpine-treated animals. Convulsive SE consisted of sustained head nodding, orofacial automatisms, hyperextension of tail, elevated levels of salivation, chewing, and repetitive forelimb and hindlimb clonus. Two hours after SE onset, animals cycled through periods of forelimb clonus and periods of loss of the righting reflex with falling. All behavioural manifestations reported here for convulsive SE are in agreement with previous descriptions from Hirsch et al. (1992) and de Oliveira et al. (2008).
2.6. Open field The test consisted of a circular wooden black arena with the dimensions of 60 cm × 50 cm (diameter × height). The floor was divided virtually into 28 squares (12 central and 16 peripheral). The testing room was illuminated by two lamps directed towards the ceiling. The light intensity was 15 lx in the centre of the arena. Each animal was individually placed in the periphery of the arena and was left to explore it freely for 15 min. Based on the findings of Eilam (2003), the following behavioural parameters were quantified: (a) Levels of locomotor and exploratory activities: number of rearing and wholebody grooming behaviours, total distance travelled, distance travelled across time and total time spent in locomotion.
3.2. Effects of S. epilepticus and treadmill exercise on open field task There was no significant effect of exercise, as well as SE, in all evaluated parameters of locomotor and exploratory activities (Fig. 1A). All groups travelled similar distances (F(1,39) = 0.1059; p < 0.7466) and performed similar numbers of whole-body grooming behaviours (F(1,39) = 0.006574; p < 0.9358) and rearing (F(1,39) = 0.006574; p < 0.9658). Moreover, there were no differences in the distance travelled across time (F(42,280) = 1.018;
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Fig. 1. Effects of Status epilepticus and treadmill exercise in open field task. The task was performed on P56. Data show end points (A) and spatio-temporal analysis of behaviour (B). N = 10–11 animals/group. * indicates p < 0.05. Two-way ANOVA followed by Bonferroni’s post-test.
p < 0.4466) nor in the locomoting time (F(1,39) = 0.01175; p < 0.9142) in all groups tested. There was a significant effect of exercise on the time spent in the home-base (Fig. 1A), which was higher for trained and SE+trained groups compared with non-trained animals (F(1,38) = 7.720; p < 0.0084) (Fig. 1A). Moreover, training had a significant effect on all evaluated parameters for temporal organisation of locomotion (Fig. 1B). Trained and SE+trained groups performed fewer trips during the test as compared to the SE group (F(1,39) = 11.21; p < 0.0018). In addition, the trained group travelled longer distances and performed more stops/trip when compared with the SE group (F(1,39) = 12.69; p < 0.0011). There was no difference in the time (F(1,39) = 3.097; p < 0.0863), distance travelled (F(1,39) = 2.463; p < 0.1247), or number of stops along the vicinity of arena wall (F(1,39) = 0.7050; p < 0.4062) (Table 1) between the groups, indicating no difference in the spatial distribution of locomotion. 3.3. Effects of S. epilepticus and treadmill exercise on object recognition task During the training session, animals from all groups explored the two stimuli objects equally (F(1,34) = 0.01520; p < 0.9026)
(Fig. 2). For the STM test session, all groups spent a similar amount of time exploring the novel object (F(1,34) = 0.3406; p < 0.5633). However, there was a strong interaction between exercise and SE for the object recognition long-term memory (LTM) evaluation. Animals from the SE group presented a decreased discrimination index when compared with animals from the control, trained and SE+trained groups (F(1,34) = 6.051; p < 0.0191) (Fig. 2). No differences were found in the total time spent exploring objects, distance travelled and average speed among groups (data not shown).
Table 1 Effects of Status epilepticus and treadmill exercise on spatial distribution of exploratory activity in open field task. Group Control Trained SE SE+trained
Time along the vicinity (%) 93.9 ± 1.2 91.1 ± 2.0 90.1 ± 2.5 87.6 ± 2.0
Distance travelled along the vicinity (%) 84.7 ± 2.0 78.5 ± 2.3 81.7 ± 3.4 79.4 ± 2.7
Stops along the vicinity 72.2 ± 5.6 77.3 ± 6.8 74.0 ± 7.9 81.0 ± 8.0
Data are expressed as mean ± SEM of percentages of time along the vicinity, distance along the vicinity and the number of stops along the vicinity of the arena wall. N = 10–11 animals/group.
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Fig. 2. Effects of Status epilepticus and treadmill exercise in object recognition. The task was performed on P57 and P58. Data show training section (A), short-term memory test (B) and long-term memory test (C). N = 10–11 animals/group. * indicates p < 0.05. Two-way ANOVA followed by Bonferroni’s post-test.
3.4. Effects of S. epilepticus and treadmill exercise on elevated plus maze All measures of anxiety-like behaviours were similar between groups, with no difference in the time spent in the open arms (F(1,39) = 0.5270; p < 0.4722) and number of entries into the open arms (F(1,39) = 1.598; p < 0.2137). In addition, there was no difference in the number of risk assessment behaviours (Fig. 3) for all groups. 4. Discussion When it occurs during critical periods of brain development, SE may induce cognitive and behavioural abnormalities in adulthood (Kaindl and Ikonomidou, 2007). Following this type of insult to the brain, there is a cascade of morphological and functional changes that take place in the injured area over a span of weeks to months. This happens before the occurrence of spontaneous seizures and cognitive impairments. This latent period may offer a therapeutic window for the prevention of epileptogenesis and the development of behavioural abnormalities. However, in clinical trials, administration of conventional antiepileptic drugs (AEDs) such as phenytoin, carbamazepine, valproate and levetiracetam has so far failed to prevent epileptogenesis and/or SE-induced cognitive deficits (Brandt et al., 2007; Temkin, 2001). In addition, it has been shown that naïve animals treated with classical AEDs during brain development can exhibit an abnormal maturation of the CNS (Chen et al., 2009; Farwell et al., 1990) as well as cognitive and behavioural alterations in adulthood (Hermann et al., 2010; Holmes, 1997). In this way, adjunctive non-pharmacological therapies may represent one alternative intervention to prevent and/or reverse cognitive deficits caused by seizures. Physical exercise can modify brain physiology and cognition (Ang et al., 2003; Arida et al., 2007; Berchtold et al., 2010; Bjørnebekk et al., 2005; Chae et al., 2009; Creer et al., 2010; Griffin et al., 2009). It has been used as a preventive treatment for a variety of brain disabilities (Alaei et al., 2006; Christie et al., 2005; FerrerAlcon et al., 2008; Garza et al., 2004; Gobbo and O’Mara, 2005; van Praag et al., 2005). However, the effects of exercise on brain physiology and pathology vary according to the intensity, duration, modality and type of training. In this context, voluntary and forced exercise protocols seem to have distinct influences on the CNS and cognitive function (Hayes et al., 2008; Ke et al., 2011a,b; Leasure and Jones, 2008). Ke et al. (2011b) demonstrated that voluntary exercise was more effective than forced training to upregulate hippocampal BDNF levels, as well as to promote motor recovery in rats subjected to a model of brain ischaemia. This exercise-induced elevation in hippocampal BDNF levels appears to be the molecular mechanism responsible for the effects of exercise on cognition, whereas blocking BDNF action using specific immuno-adhesive chimeres abolished the ability of exercise to augment learning and
memory in rats (Vaynman et al., 2004). While voluntary exercise is known to benefit spatial learning and memory, the effects of forced exercise are still highly controversial. Recently data from Ang et al. (2006) demonstrated that forced exercise could also significantly improve spatial learning and memory in rats. Such differing outcomes might be accounted by differences between rat strains and the age of the animals used because these are known to affect outcomes (Wyss et al., 2000). In addition, differences in the water maze protocol administered to the rats could also explain the differences found between studies. The forced exercise regime is advantageous over the voluntary one because of the outcome measurement and because manipulation of exercise speed, frequency, duration and intensity can be controlled. In this context, we chose a low-intensity forced exercise protocol in treadmill to investigate the putative beneficial effects of a training programme on the long-standing cognitive alterations induced by SE in young rats. SE induced early in life caused a significant impairment in longterm memory for object recognition 42 days after LiCl-pilocarpine administration (Fig. 2). These data indicate that prolonged seizures early in life can cause long-standing cognitive impairments in object recognition memory, which corroborates with another work that observed that SE can induce memory impairments in animals subjected to the Morris water maze and inhibitory avoidance tasks (de Oliveira et al., 2008; Kubová et al., 2004; Liu et al., 2007). SE induces massive neurodegeneration in several brain regions, including the hippocampus and perirhinal cortex (Wang et al., 2008). Therefore, cognitive deficits found in the present work may be related to neuronal damage in these vital areas for learning and memory formation (Winters et al., 2004). When SE-induced animals were submitted to a low-intensity forced treadmill exercise protocol, the impairment in object recognition memory was completely reversed (Fig. 2). The neurochemical and physiological mechanisms involved in this effect are still under investigation. O’Callaghan et al. (2007) demonstrated that 1 week of forced physical training on a treadmill increases long-term potentiation and BDNF levels in rats. In addition, an intraventricular infusion of BDNF led to improvement in the performance of the object substitution task (Griffin et al., 2009); BDNF-dependent activation of ERK1/2 cascade was required for this memory formation (Alonso et al., 2002). Moreover, BDNF is also implicated in the persistence of memory storage because blocking endogenous BDNF activity through the delivery of a functionblocking antibody caused amnesia at 7 days after an inhibitory avoidance task in rats (Bekinschtein et al., 2007). The exercise protocol used in the present study did not alter the performance of the trained group as compared to the sedentary control animals. Barnes et al. (1991) and Rachetti et al. (2012) found similar results, where trained rats did not show improvements in spatial and recognition memory compared to sedentary animals. These authors hypothesised that the absence of a training effect
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Fig. 3. Effects of Status epilepticus and treadmill exercise in elevated plus maze. The task was performed on P60. Data show time spent in the open arms (A), number of entries into the open arms (B), and number of risk assessment behaviours (C). N = 10–11 animals/group.
might be due to sedentary control animals achieving maximal performance during the memory tasks, thereby making it difficult to distinguish improvement in the performance of the trained groups. In addition because learning and memory are dynamic processes, the failure to find memory improvement in these trained control animals does not indicate that exercise cannot change cognition; when the animals suffered a brain insult, the training protocol was able to reverse the cognitive deficit. To eliminate the possibility of an effect of impaired locomotion on the performance of the animals in the object recognition task, the animals were tested in the open field task. Both SE and treadmill training did not alter gross exploratory activity, such as total distance travelled or number of rearings and groomings (Fig. 1A). Nevertheless, the spatio-temporal organisation of locomotor and exploratory activities was altered in all exercised animals when compared to the SE group. These data suggest that low-intensity exercise does not induce alterations in locomotion, but rather, adaptations in spatial organisation of exploration, which persists even in animals that suffered SE early in life. Data from elevated plus maze (EPM) tasks suggest that behavioural alterations observed in SE in object recognition and open field tasks are not related to altered anxiety levels because there was no difference among groups (Fig. 3) in EPM. These data are in agreement with the work of Salim et al. (2010), which shows that rats subjected to a moderate intensity treadmill exercise protocol did not present anxiety-like behavioural alterations. In summary, the present study showed that animals submitted to SE during early periods of life (P16) showed a significant impairment in recognition memory at adulthood, which was reversed by a low-intensity treadmill protocol. These observations suggest that exercise may be considered as a potential alternative and complementary nonpharmacological therapy for patients who suffered SE during childhood. Acknowledgements This work was supported by the Brazilian funding agencies, CNPq, FAPERGS, and CAPES and by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” #01.06.0842-00. References Ahlskog, J.E., Geda, Y.E., Graff-Radford, N.R., Petersen, R.C., 2011. Physical exercise as a preventive or disease-modifying treatment of dementia and brain aging. Mayo Clinic Proceedings 86, 876–884. Alaei, H., Borjeian, L., Azizi, M., Orian, S., Pourshanazari, A., Hanninen, O., 2006. Treadmill running reverses retention deficit induced by morphine. European Journal of Pharmacology 536, 138–141. Alonso, M., Vianna, M.R., Depino, A.M., Mello e Souza, T., Pereira, P., Szapiro, G., Viola, H., Pitossi, F., Izquierdo, I., Medina, J.H., 2002. BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation. Hippocampus 12, 551–560.
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International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Low-intensity physical training recovers object recognition memory impairment in rats after early-life induced Status epilepticus Sandro Daniel Córdova ∗ , Cássio Morais Loss, Diogo Losch de Oliveira Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
a r t i c l e
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Article history: Received 15 August 2012 Received in revised form 2 January 2013 Accepted 3 January 2013 Keywords: Status epilepticus Pilocarpine Physical training Learning Memory
a b s t r a c t When it occurs early in life, Status epilepticus (SE) can cause behavioural and cognitive impairments in adulthood. Here, we evaluated the putative benefits of low-intensity treadmill training on long-standing cognitive impairment in rats submitted to SE early in life. Wistar rats were submitted to LiCl-pilocarpineinduced SE at P16. Animals from the trained group underwent a low-intensity treadmill protocol for 5 days per week for 4 weeks. At adulthood, rats subjected to early-life SE displayed impairment in long-term memory in an object recognition task, while the training protocol completely reversed this deficit. This result was associated with neither locomotor alterations nor changes in emotional behaviour; there were no differences between groups in the distance travelled, grooming or rearing in the open field test; there were also no differences between groups in the number of risk assessment, time spent in open arms in an elevated plus maze and number of entries into the open arms. These data suggest that physical exercise can ameliorate the long-standing recognition memory deficit induced by early-life SE, suggesting that it may be useful as a putative intervention for patients who suffered SE during infancy. © 2013 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction Status epilepticus (SE) is a common childhood neurological emergency characterised as a seizure or repeated seizures that last more than 30 min (Chen and Wasterlain, 2006). Although SE can occur at all ages, the highest incidence is observed before 2 years of age (Shinnar et al., 1997; Singh et al., 2010). When it occurs early in life, SE can be potentially harmful to the brain (Wasterlain et al., 1993, 2002) and is associated with a higher incidence of neurological disorders later in life (Kwong et al., 2004). The most frequent neurological dysfunctions associated with early-life SE include alterations in sociolinguistic and psychomotor development, cognitive impairments and alterations in emotional behaviour (Roy et al., 2011). Neurological disabilities observed in humans are very similar to those found in animal models of SE. Male rats that suffered SE at 16–20 days old showed long-term deficits in spatial learning in the water maze task (Cilio et al., 2003) and showed an impairment in aversive memory learning in an inhibitory avoidance task (de Oliveira et al., 2008). Furthermore, animals submitted to LiCl-pilocarpine-induced SE between the 2nd and 3rd weeks of life presented increased levels of anxiety in the
∗ Corresponding author at: Departamento de Bioquímica, ICBS, UFRGS, Rua Ramiro Barcelos 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil. Tel.: +55 51 33085555, fax: +55 51 33085540. E-mail address: [email protected] (S.D. Córdova). 0736-5748/$36.00 © 2013 ISDN. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijdevneu.2013.01.002
elevated plus maze, as well as impaired performance in the water maze task 3 months after the insult (Kubová et al., 2004). Animal studies and clinical trials have demonstrated that treatment of SE with traditional or new antiepileptic drugs (AEDs), such as phenytoin and levetiracetam, may stop seizure activity but fails to prevent the above-mentioned SE-induced cognitive and behavioural alterations later in life (Brandt et al., 2007; Temkin, 2001).It has been demonstrated that physical training may have benefits for the brain and can be used as a potential therapeutic strategy for different degenerative brain diseases (Ahlskog et al., 2011; Cotman and Berchtold, 2002; Lafenêtre et al., 2010; O’Callaghan et al., 2009). Aerobic training promoted reduction in the fall risk, increased mobility, and improved the quality of life in Parkinson’s disease (PD) patients (Fisher et al., 2008; Herman et al., 2007). In addition, PD patients who participated in a training programme exhibited cognitive benefits in frontal lobe-dependent tasks (Cruise et al., 2011). For patients with Alzheimer’s disease, 12 weeks of physical training led to an improvement in the performance of daily activities (Santana-Sosa et al., 2008). Benefits were also observed in several animal models, with improvements in cognitive function, elevation of hippocampal BDNF (Ke et al., 2011b; Sartori et al., 2009; Vaynman et al., 2004), and increased BrdUlabelled cells in the dentate gyrus of the hippocampus (Chae et al., 2009). Although studies have demonstrated the beneficial effects of physical training on SNC, little is known about the effects of an exercise programme on cognitive changes in individuals who
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suffered SE early in life. The aim of this study was to investigate the potential benefits of a low-intensity treadmill exercise protocol on behavioural alterations in adult rats subjected to LiCl-pilocarpineinduced SE early in life.
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(b) Temporal organisation of locomotion: time in home-base (the square where animal remained for more time during the test), number of trips (departures from the home-base), trip length (distance/number of trips) and stops/trip (number of stops/number of trips). (c) Spatial distribution of locomotion: distance travelled along the vicinity and locomoting time spent along the vicinity of the arena walls.
2. Experimental procedure 2.1. Materials Pilocarpine hydrochloride was purchased from Sigma–Aldrich (USA). FluoroJade B was purchased from Chemicon, Inc. (USA). Other chemicals were purchased from Nuclear (Brazil). 2.2. Subjects Male Wistar rats (15 days postnatal) were obtained from a local breeding facility. The litters were culled to 8 pups. The day of birth was defined as day 0, and the animals were weaned on postnatal day 21. After weaning, animals were housed in standard polypropylene cages (4–5 animals per cage) with food and water ad libitum (21 ± 1 ◦ C room temperature). Rats were kept under a 12:12 h light/dark cycle (lights on at 7:00 a.m.). The handling and care of the animals was conducted according to the Guide for Care and Use of Laboratory Animals from the National Institutes of Health (NIH Publications No. 80-23, revised 1996). All procedures in the present study were approved by the Committee of Ethics of the Universidade Federal do Rio Grande do Sul.
2.7. Object recognition task The arena for the object recognition test was the same used in the open field task. Two glass objects of comparable size (10 cm high) were fixed to the apparatus 10 cm from the wall and 15 cm from each other. In the training session, animals were allowed to explore two identical objects (object A and A’) for 5 min. Object exploration was considered to be any investigative behaviour (head orientation or sniffing), deliberate contact with the object at a distance of ≤2 cm, or when the animal touched the object with its nose. To evaluate short-term memory (STM), animals were allowed to explore one familiar and one novel object (object B) for 5 min at 3 h after the training session. Twenty-four hours after the training session, long-term memory (LTM) was assessed by changing object B for a new form (object C) and allowing the animals to explore them for 5 min (Bevins and Besheer, 2006). Memory was operationally defined through the discrimination index, which was calculated in absolute values as follows: (A/A + A’)-(A’/A + A’) for training session, (B/A + B) − (A/A + B) for STM, and (C/A + C) − (A/A + C) for LTM. Average speed, total time exploring objects and the distance travelled in the apparatus were also recorded. 2.8. Elevated plus-maze
2.3. Induction of S. epilepticus Rat pups were injected with a solution of LiCl (3 mEq/kg, i.p.) 12–18 h prior to pilocarpine hydrochloride administration (60 mg/kg, i.p.) on postnatal day 16 (Hirsch et al., 1992). Control animals were handled and housed in the same manner and received an equal volume of saline solution (0.9% NaCl). Rats were kept in individual plastic cages at nest temperature for seizure observation. The duration of SE was evaluated only by behavioural measures where SE was defined, according to Hirsch et al. (1992), as sustained orofacial automatisms, salivation, chewing, forelimb clonus, loss of the righting reflex and falling. The rats were allowed to spontaneously recover from SE. Each experimental group contained pups from several litters. According to Priel et al. (1996), the induction of SE at this age does not induce spontaneous seizures in adulthood. 2.4. Exercise protocol Animals were divided into 4 groups: control (C) (n = 10), trained (T) (n = 10), S. epilepticus (SE) (n = 11) and S. epilepticus+trained (SE+T) (n = 11). Seven days after SE induction, animals from the T and SE+T groups were submitted to a low-intensity exercise protocol over 4 weeks, starting the familiarisation at P25 and the training programme at P27. Rats ran 30 min daily on one four-line treadmill for 5 days a week. The warm-up consisted of 2 m/min for the first 5 min, followed by 5 m/min for the next 5 min and 8 m/min in the last 20 min (Kim et al., 2003). Control and SE animals were left on the treadmill for 30 min, 5 days a week, with the treadmill stopped. Stimulation was not used to motivate rats to run; animals that refused to run were removed from the experiment (only 1 animal in the T group was excluded). 2.5. Behavioural tasks After the end of the exercise protocol, animals were subjected to behavioural tasks. Open-field, object recognition and elevated plus maze tasks were conducted on P56, P57-58 and P60, respectively. For all behavioural procedures, animals were placed in the testing room (temperature 21 ± 2◦ C) 1 h before the beginning of the task to allow them to habituate to the environment and the researchers. Because rats are nocturnal, all tasks were performed between 6:00 p.m. and 10:00 p.m. The behavioural profile was recorded and analysed using the ANY-Maze® video-tracking system (Stoelting, CO). Between every trial, all apparatuses were cleaned with a 70% ethanol solution.
The maze consisted of two open arms (50 cm × 10 cm) and two closed arms (50 cm × 10 cm × 40 cm), with arms of each type opposite to each other (Pellow et al., 1985). The maze was elevated to a height of 50 cm from the floor. The experiment was conducted in a room illuminated by red light. The light intensity in the centre of apparatus was 5 lx. Rats were placed into the centre of the maze facing a closed arm and were left free to explore it for 5 min. Number of arm entries, time spent in the arms, number of risk assessment behaviours (when the rodent is motionless in the centre or closed zone, but is stretched forward into the open arms with some but not all paws, returning then to the initial position) and total distance travelled were registered. 2.9. Data analysis All data were expressed as the mean ± SEM and analysed by two-way ANOVA followed by Bonferroni post hoc test for unequal samples. For all parameters, p < 0.05 was considered significant.
3. Results 3.1. Characterisation of S. epilepticus Within 2–5 min after pilocarpine administration, all LiClpilocarpine-treated animals started having behavioural changes consisting of defecation, salivation, body tremor, and scratching. This behavioural pattern progressed within 8–12 min to increased levels of motor activity and culminated in a convulsive SE in all LiCl-pilocarpine-treated animals. Convulsive SE consisted of sustained head nodding, orofacial automatisms, hyperextension of tail, elevated levels of salivation, chewing, and repetitive forelimb and hindlimb clonus. Two hours after SE onset, animals cycled through periods of forelimb clonus and periods of loss of the righting reflex with falling. All behavioural manifestations reported here for convulsive SE are in agreement with previous descriptions from Hirsch et al. (1992) and de Oliveira et al. (2008).
2.6. Open field The test consisted of a circular wooden black arena with the dimensions of 60 cm × 50 cm (diameter × height). The floor was divided virtually into 28 squares (12 central and 16 peripheral). The testing room was illuminated by two lamps directed towards the ceiling. The light intensity was 15 lx in the centre of the arena. Each animal was individually placed in the periphery of the arena and was left to explore it freely for 15 min. Based on the findings of Eilam (2003), the following behavioural parameters were quantified: (a) Levels of locomotor and exploratory activities: number of rearing and wholebody grooming behaviours, total distance travelled, distance travelled across time and total time spent in locomotion.
3.2. Effects of S. epilepticus and treadmill exercise on open field task There was no significant effect of exercise, as well as SE, in all evaluated parameters of locomotor and exploratory activities (Fig. 1A). All groups travelled similar distances (F(1,39) = 0.1059; p < 0.7466) and performed similar numbers of whole-body grooming behaviours (F(1,39) = 0.006574; p < 0.9358) and rearing (F(1,39) = 0.006574; p < 0.9658). Moreover, there were no differences in the distance travelled across time (F(42,280) = 1.018;
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Fig. 1. Effects of Status epilepticus and treadmill exercise in open field task. The task was performed on P56. Data show end points (A) and spatio-temporal analysis of behaviour (B). N = 10–11 animals/group. * indicates p < 0.05. Two-way ANOVA followed by Bonferroni’s post-test.
p < 0.4466) nor in the locomoting time (F(1,39) = 0.01175; p < 0.9142) in all groups tested. There was a significant effect of exercise on the time spent in the home-base (Fig. 1A), which was higher for trained and SE+trained groups compared with non-trained animals (F(1,38) = 7.720; p < 0.0084) (Fig. 1A). Moreover, training had a significant effect on all evaluated parameters for temporal organisation of locomotion (Fig. 1B). Trained and SE+trained groups performed fewer trips during the test as compared to the SE group (F(1,39) = 11.21; p < 0.0018). In addition, the trained group travelled longer distances and performed more stops/trip when compared with the SE group (F(1,39) = 12.69; p < 0.0011). There was no difference in the time (F(1,39) = 3.097; p < 0.0863), distance travelled (F(1,39) = 2.463; p < 0.1247), or number of stops along the vicinity of arena wall (F(1,39) = 0.7050; p < 0.4062) (Table 1) between the groups, indicating no difference in the spatial distribution of locomotion. 3.3. Effects of S. epilepticus and treadmill exercise on object recognition task During the training session, animals from all groups explored the two stimuli objects equally (F(1,34) = 0.01520; p < 0.9026)
(Fig. 2). For the STM test session, all groups spent a similar amount of time exploring the novel object (F(1,34) = 0.3406; p < 0.5633). However, there was a strong interaction between exercise and SE for the object recognition long-term memory (LTM) evaluation. Animals from the SE group presented a decreased discrimination index when compared with animals from the control, trained and SE+trained groups (F(1,34) = 6.051; p < 0.0191) (Fig. 2). No differences were found in the total time spent exploring objects, distance travelled and average speed among groups (data not shown).
Table 1 Effects of Status epilepticus and treadmill exercise on spatial distribution of exploratory activity in open field task. Group Control Trained SE SE+trained
Time along the vicinity (%) 93.9 ± 1.2 91.1 ± 2.0 90.1 ± 2.5 87.6 ± 2.0
Distance travelled along the vicinity (%) 84.7 ± 2.0 78.5 ± 2.3 81.7 ± 3.4 79.4 ± 2.7
Stops along the vicinity 72.2 ± 5.6 77.3 ± 6.8 74.0 ± 7.9 81.0 ± 8.0
Data are expressed as mean ± SEM of percentages of time along the vicinity, distance along the vicinity and the number of stops along the vicinity of the arena wall. N = 10–11 animals/group.
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Fig. 2. Effects of Status epilepticus and treadmill exercise in object recognition. The task was performed on P57 and P58. Data show training section (A), short-term memory test (B) and long-term memory test (C). N = 10–11 animals/group. * indicates p < 0.05. Two-way ANOVA followed by Bonferroni’s post-test.
3.4. Effects of S. epilepticus and treadmill exercise on elevated plus maze All measures of anxiety-like behaviours were similar between groups, with no difference in the time spent in the open arms (F(1,39) = 0.5270; p < 0.4722) and number of entries into the open arms (F(1,39) = 1.598; p < 0.2137). In addition, there was no difference in the number of risk assessment behaviours (Fig. 3) for all groups. 4. Discussion When it occurs during critical periods of brain development, SE may induce cognitive and behavioural abnormalities in adulthood (Kaindl and Ikonomidou, 2007). Following this type of insult to the brain, there is a cascade of morphological and functional changes that take place in the injured area over a span of weeks to months. This happens before the occurrence of spontaneous seizures and cognitive impairments. This latent period may offer a therapeutic window for the prevention of epileptogenesis and the development of behavioural abnormalities. However, in clinical trials, administration of conventional antiepileptic drugs (AEDs) such as phenytoin, carbamazepine, valproate and levetiracetam has so far failed to prevent epileptogenesis and/or SE-induced cognitive deficits (Brandt et al., 2007; Temkin, 2001). In addition, it has been shown that naïve animals treated with classical AEDs during brain development can exhibit an abnormal maturation of the CNS (Chen et al., 2009; Farwell et al., 1990) as well as cognitive and behavioural alterations in adulthood (Hermann et al., 2010; Holmes, 1997). In this way, adjunctive non-pharmacological therapies may represent one alternative intervention to prevent and/or reverse cognitive deficits caused by seizures. Physical exercise can modify brain physiology and cognition (Ang et al., 2003; Arida et al., 2007; Berchtold et al., 2010; Bjørnebekk et al., 2005; Chae et al., 2009; Creer et al., 2010; Griffin et al., 2009). It has been used as a preventive treatment for a variety of brain disabilities (Alaei et al., 2006; Christie et al., 2005; FerrerAlcon et al., 2008; Garza et al., 2004; Gobbo and O’Mara, 2005; van Praag et al., 2005). However, the effects of exercise on brain physiology and pathology vary according to the intensity, duration, modality and type of training. In this context, voluntary and forced exercise protocols seem to have distinct influences on the CNS and cognitive function (Hayes et al., 2008; Ke et al., 2011a,b; Leasure and Jones, 2008). Ke et al. (2011b) demonstrated that voluntary exercise was more effective than forced training to upregulate hippocampal BDNF levels, as well as to promote motor recovery in rats subjected to a model of brain ischaemia. This exercise-induced elevation in hippocampal BDNF levels appears to be the molecular mechanism responsible for the effects of exercise on cognition, whereas blocking BDNF action using specific immuno-adhesive chimeres abolished the ability of exercise to augment learning and
memory in rats (Vaynman et al., 2004). While voluntary exercise is known to benefit spatial learning and memory, the effects of forced exercise are still highly controversial. Recently data from Ang et al. (2006) demonstrated that forced exercise could also significantly improve spatial learning and memory in rats. Such differing outcomes might be accounted by differences between rat strains and the age of the animals used because these are known to affect outcomes (Wyss et al., 2000). In addition, differences in the water maze protocol administered to the rats could also explain the differences found between studies. The forced exercise regime is advantageous over the voluntary one because of the outcome measurement and because manipulation of exercise speed, frequency, duration and intensity can be controlled. In this context, we chose a low-intensity forced exercise protocol in treadmill to investigate the putative beneficial effects of a training programme on the long-standing cognitive alterations induced by SE in young rats. SE induced early in life caused a significant impairment in longterm memory for object recognition 42 days after LiCl-pilocarpine administration (Fig. 2). These data indicate that prolonged seizures early in life can cause long-standing cognitive impairments in object recognition memory, which corroborates with another work that observed that SE can induce memory impairments in animals subjected to the Morris water maze and inhibitory avoidance tasks (de Oliveira et al., 2008; Kubová et al., 2004; Liu et al., 2007). SE induces massive neurodegeneration in several brain regions, including the hippocampus and perirhinal cortex (Wang et al., 2008). Therefore, cognitive deficits found in the present work may be related to neuronal damage in these vital areas for learning and memory formation (Winters et al., 2004). When SE-induced animals were submitted to a low-intensity forced treadmill exercise protocol, the impairment in object recognition memory was completely reversed (Fig. 2). The neurochemical and physiological mechanisms involved in this effect are still under investigation. O’Callaghan et al. (2007) demonstrated that 1 week of forced physical training on a treadmill increases long-term potentiation and BDNF levels in rats. In addition, an intraventricular infusion of BDNF led to improvement in the performance of the object substitution task (Griffin et al., 2009); BDNF-dependent activation of ERK1/2 cascade was required for this memory formation (Alonso et al., 2002). Moreover, BDNF is also implicated in the persistence of memory storage because blocking endogenous BDNF activity through the delivery of a functionblocking antibody caused amnesia at 7 days after an inhibitory avoidance task in rats (Bekinschtein et al., 2007). The exercise protocol used in the present study did not alter the performance of the trained group as compared to the sedentary control animals. Barnes et al. (1991) and Rachetti et al. (2012) found similar results, where trained rats did not show improvements in spatial and recognition memory compared to sedentary animals. These authors hypothesised that the absence of a training effect
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Fig. 3. Effects of Status epilepticus and treadmill exercise in elevated plus maze. The task was performed on P60. Data show time spent in the open arms (A), number of entries into the open arms (B), and number of risk assessment behaviours (C). N = 10–11 animals/group.
might be due to sedentary control animals achieving maximal performance during the memory tasks, thereby making it difficult to distinguish improvement in the performance of the trained groups. In addition because learning and memory are dynamic processes, the failure to find memory improvement in these trained control animals does not indicate that exercise cannot change cognition; when the animals suffered a brain insult, the training protocol was able to reverse the cognitive deficit. To eliminate the possibility of an effect of impaired locomotion on the performance of the animals in the object recognition task, the animals were tested in the open field task. Both SE and treadmill training did not alter gross exploratory activity, such as total distance travelled or number of rearings and groomings (Fig. 1A). Nevertheless, the spatio-temporal organisation of locomotor and exploratory activities was altered in all exercised animals when compared to the SE group. These data suggest that low-intensity exercise does not induce alterations in locomotion, but rather, adaptations in spatial organisation of exploration, which persists even in animals that suffered SE early in life. Data from elevated plus maze (EPM) tasks suggest that behavioural alterations observed in SE in object recognition and open field tasks are not related to altered anxiety levels because there was no difference among groups (Fig. 3) in EPM. These data are in agreement with the work of Salim et al. (2010), which shows that rats subjected to a moderate intensity treadmill exercise protocol did not present anxiety-like behavioural alterations. In summary, the present study showed that animals submitted to SE during early periods of life (P16) showed a significant impairment in recognition memory at adulthood, which was reversed by a low-intensity treadmill protocol. These observations suggest that exercise may be considered as a potential alternative and complementary nonpharmacological therapy for patients who suffered SE during childhood. Acknowledgements This work was supported by the Brazilian funding agencies, CNPq, FAPERGS, and CAPES and by the FINEP research grant “Rede Instituto Brasileiro de Neurociência (IBN-Net)” #01.06.0842-00. References Ahlskog, J.E., Geda, Y.E., Graff-Radford, N.R., Petersen, R.C., 2011. Physical exercise as a preventive or disease-modifying treatment of dementia and brain aging. Mayo Clinic Proceedings 86, 876–884. Alaei, H., Borjeian, L., Azizi, M., Orian, S., Pourshanazari, A., Hanninen, O., 2006. Treadmill running reverses retention deficit induced by morphine. European Journal of Pharmacology 536, 138–141. Alonso, M., Vianna, M.R., Depino, A.M., Mello e Souza, T., Pereira, P., Szapiro, G., Viola, H., Pitossi, F., Izquierdo, I., Medina, J.H., 2002. BDNF-triggered events in the rat hippocampus are required for both short- and long-term memory formation. Hippocampus 12, 551–560.
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