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Effects of different physical exercise programs on susceptibility to pilocarpine-induced seizures in female rats
Epilepsy & Behavior 64 (2016) 262–267
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Effects of different physical exercise programs on susceptibility to pilocarpine-induced seizures in female rats Diego Vannucci Campos a, Glauber Menezes Lopim a, Vanessa Santos de Almeida a, Débora Amado b, Ricardo Mario Arida a,⁎ a b
Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), São Paulo, — SP, Brazil Departamento de Neurologia e Neurocirurgia, Universidade Federal de São Paulo (UNIFESP), São Paulo, — SP, Brazil
a r t i c l e
i n f o
Article history: Received 9 May 2016 Revised 12 August 2016 Accepted 13 August 2016 Available online xxxx Keywords: Epilepsy Seizures susceptibility Pilocarpine Exercise Female rats
a b s t r a c t In epilepsy, the most common serious neurological disorder worldwide, several investigations in both humans and animals have shown the effectiveness of physical exercise programs as a complementary therapy. Among the benefits demonstrated, regular exercise can decrease the number of seizures as well as improve cardiovascular and psychological health in people with epilepsy. While many studies in animals have been performed to show the beneficial effects of exercise, they exclusively used male animals. However, females are also worthy of investigation because of their cyclical hormonal fluctuations and possible pregnancy. Considering the few animal studies concerning seizure susceptibility and exercise programs in females, this study aimed to verify whether exercise programs can interfere with seizure susceptibility induced by pilocarpine in adult female Wistar rats. Animals were randomly divided into three groups: control, forced, and voluntary (animals kept in a cage with a wheel). After the final exercise session, animals received a pilocarpine hydrochloride (350 mg/kg i.p.; Sigma) injection to induce seizures. To measure the intensity of pilocarpine-induced motor signs, we used a scale similar to that developed by Racine (1972) in the kindling model. During a 4-h period of observation, we recorded latency for first motor signs, latency for reaching SE, number of animals that developed SE, and intensity of pilocarpine-induced motor signs. No difference was observed among groups in latency for first motor signs and in the number of animals that developed SE. Although the voluntary group presented more intense motor signs, an increased latency for developing SE was observed compared with that in forced and control groups. Our behavioral results are not enough to explain physiological and molecular pathways, but there are mechanisms described in literature which may allow us to propose possible explanations. Voluntary exercise increased latency to SE development. Further investigation is necessary to elucidate the pathways involved in these results, while more studies should be performed regarding gender specific differences. © 2016 Elsevier Inc. All rights reserved.
1. Introduction In epilepsy, the most common serious neurological disorder worldwide, several investigations in both humans and animals have shown the effectiveness of physical exercise programs as a complementary therapy [1–7]. Among these benefits, regular exercise can decrease the number of seizures as well as improve cardiovascular and psychological health in people with epilepsy. Although the favorable effects of exercise after epilepsy have been established [2,8–10], very few studies have verified whether exercise programs might protect against or prevent the development of epilepsy. Findings from these studies have indicated that exercise may be able to modulate neuronal vulnerability to epileptogenic insults [6,11,12]. ⁎ Corresponding author at: Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu 862, Ed. Ciências Biomédicas, 5° andar, Vila Clementino, CEP: 04023-900 São Paulo, — SP, Brazil. E-mail address: [email protected] (R.M. Arida).
http://dx.doi.org/10.1016/j.yebeh.2016.08.011 1525-5050/© 2016 Elsevier Inc. All rights reserved.
Physical activity programs before precipitating insults (such as pilocarpine convulsant dose injection or amygdala electrical stimulation) have shown reduced brain susceptibility to seizures [6,12]. Gomes da Silva et al. [11] also presented results indicating that an aerobic exercise program during adolescence in rats can attenuate the effects of pilocarpine injection in adult life. In that study, even after a 90-day detraining period, the effects of an early physical activity program were able to ameliorate the intensity and increase the latency for motor signs. These effects were attributed to a possible formation of neural reserve. Although many studies on animals have been performed to show the beneficial effects of exercise, all used male animals. The female population is also worthy of attention because of its cyclical hormonal fluctuations and possible pregnancy. In the literature, there are studies which show the notable influence of sexual hormones on phenomena linked to the epilepsies [13–16]. In women with epilepsy, reproductive dysfunctions and endocrine disorders are commonly observed [17–19], mainly in temporal lobe epilepsy.
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Amado and Cavalheiro [20] have shown that the estrous cycle of Wistar female rats was altered after status epilepticus induced by pilocarpine, and this alteration remained for many weeks. Progesterone, luteinizing hormone, and follicle stimulant hormone was at lower levels compared with control animals, while estradiol levels exceeded healthy animal levels during the period of spontaneous and recurrent seizures. Considering the few animal studies concerning seizure susceptibility and exercise programs in females, this study aimed to verify whether exercise programs can interfere with seizure susceptibility induced by pilocarpine in adult female Wistar rats. 2. Methods 2.1. Animals Female Wistar rats were submitted to vaginal smears for 15 days before the experiments to verify the regularity of the estrous cycle. Only rats presenting a regular cycle were included in the procedures. Animals were randomly divided into three groups: control (animals not submitted to physical activity) (n = 16), forced (animals submitted to treadmill training) (n = 16), and voluntary (animals kept in a cage with a wheel) (n = 12). The colony room was maintained at 21 ± 2 °C with a 12-h light/dark cycle and food and water ad libitum throughout the experimental procedures. During all experimental procedures, vaginal smears from all animals were verified daily. All experimental protocols were conducted in accordance with the ethical committee of the university. 2.2. Forced exercise procedure Animals from the treadmill group were familiarized with the apparatus for three days by placing them on a treadmill (Columbus) for 10 min/day at speed 12 m/min at a 0% degree incline. Electric shocks (0.3 mA; two discharges/second) were used sparingly to motivate the animals to run. The trainability of rats was measured according to their performance on a scale of 1–5 [21]. The parameters of the scale were as follows: 1 — refused to run; 2 — below average runner (sporadic, stop and go, wrong direction); 3 — average runner; 4 — above average runner (consistent runner occasionally fell back on the treadmill); 5 — good runner (consistently stayed at the front of the treadmill). Animals rating 3 or higher were included in the group. These procedures were used to exclude possible different levels of stress among animals; however, no animal in this study had to be excluded from the exercise group (all animals submitted to physical training were good runners). Following familiarization, they were submitted to 30 sessions of an aerobic exercise program for 30 min/day reaching 22 m/min in the final week. The training protocol was performed five days/week between 9:00 and 12:00 (diurnal period). Daily running distance for this group was quantified by multiplying the speed programmed in the treadmill by the time that the animals were kept running during every session. Subsequently, the daily values that were obtained were added and divided by 30 (total number of sessions). 2.3. Voluntary exercise procedure Animals submitted to voluntary exercise were placed in a cage with voluntary wheel running (Panlab Harvard Apparatus) for 30 days with free access to food and water. Daily running distance for this group was quantified using an odometer, which registered the number of wheel revolutions. The daily odometer value which was obtained was multiplied by 2πR, considering R as the radius measured from the center of the wheel to its inner surface. Daily values were added and divided by 30 (total number of days that rats had free access to the wheel).
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hydrochloride (350 mg/kg i.p.; Sigma) injection to induce seizures. Animals in a different phase of the cycle were submitted to extra training days until they entered the estrus phase. This was done with the intention of avoiding the varying hormonal rates observed in different parts of the estrus cycle. Scopolamine methylnitrate (1 mg/kg s.c.; Sigma) was administered 30 min before pilocarpine injection to limit peripheral cholinergic effects [22]. The systemic administration of the potent muscarinic agonist pilocarpine in rats promotes sequential behavioral and electrographic changes that build up progressively into a limbic status epilepticus (SE) which lasts 24 h [22]. Therefore, in this study, we observed the behavioral manifestations of pilocarpine-induced SE. To measure the intensity of pilocarpine-induced motor signs, we used a scale describing progressive evolution of seizures similar to that developed by Racine [23] in the kindling model (0.5 — immobility, piloerection, salivation, narrowing of eyes, face and vibrissae twitching, ear rubbing with forepaws; 1.0 — head nodding and chewing movements; 1.5 — clonic movements of forelimbs and mild whole body convulsions, exophthalmia, aggressive behavior; 2.0 — rearing and running with stronger tonic–clonic motions including hindlimbs, tail hypertension, and lockjaw; 2.5 — rearing and falling, eye congestion; 3.0 — loss of postural tone with general body rigidity). Rats were observed for a 4-hour period and each motor sign described above was quantified during this period by a treatment-blinded observer. Following these observations, intensity was calculated by multiplying the number of behavioral manifestations in each step of Racine's scale by the grade given to this step. This was performed for all rats, and the mean and standard deviation were obtained with regard to all analyzed animals from each group. During the observation period, we recorded the following: (a) latency of the first motor signs, (b) latency for reaching SE, (c) number of animals that developed SE, and (d) intensity of pilocarpine-induced motor signs. Latency for the development of status epilepticus (SE) was evaluated by considering SE as a condition characterized by an epileptic seizure that is sufficiently prolonged or repeated at sufficiently brief intervals so as to produce an unvarying and enduring epileptic condition. Thus, animals were considered to be in SE when this continuous seizing condition was observed for at least 30 min. Animals became unresponsive to physical stimuli during this phase. 2.5. Statistical analysis The statistical analysis among voluntary, forced, and control groups was performed using the Kruskal–Wallis test followed by Mann– Whitney U-test and Chi-square test. Values were expressed as mean ± SD and considered significant when p b 0.05. Estrous cycle and animal weight were evaluated using one-way ANOVA followed by Bonferroni post hoc. Mann–Whitney was also performed to compare the average running distance between animals from the exercise groups. We also performed Pearson's correlation between different parameters in the voluntary group. 3. Results 3.1. Influence of physical activity on the estrous cycle In this study, we performed vaginal smears daily in all animals in order to verify possible alterations caused by the different types of physical activity. As shown in Fig. 1, we observed the same percentage of each phase in all groups (p N 0.05). 3.2. Animal weight
2.4. Pilocarpine injection After the final exercise session, animals presenting in the estrus phase of the estrous cycle on that day received a pilocarpine
Weights of animals from all groups were measured before pilocarpine injection to verify whether the exercise programs would alter this factor. We observed that animals from the voluntary group weighed
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3.5. Pearson's correlation for the voluntary group Noting the differences in weight, running distance, and behavioral parameter observed in the voluntary group compared with those in the other groups, we performed Pearson's correlation in order to verify whether weight and running distance were correlated with latency to SE development and/or motor sign intensity. Our results showed a weak positive correlation between motor sign intensity and animal weight (r = 0.436) and negligible negative correlation between motor sign intensity and running distance (r = −0.272). Concerning latency to SE, we observed moderate negative correlation between this latency and animal weight (r = −0.587) and moderate positive correlation between latency and running distance (r = 0.538) (Fig. 4). No significant correlation was observed in the p-value of these analyses (p N 0.05 for all correlations performed). Fig. 1. Percentage of each phase of the estrous cycle in each group. P — proestrus; E — estrus; M — metaestrus; D — diestrus; CTL — control group; FOR — forced group; VOL — voluntary group. No significant difference was observed in each phase of the estrous cycle among groups.
significantly less than animals from the control (p = 0.018) and forced (p b 0.000) groups (Fig. 2).
3.3. Average running distance All animals from the forced group ran exactly 650 m/day, while average running distance for the voluntary group was 10,260 ± 2580 m/day. The best runner ran 13,820 m/day and the worst, 5480 m/day. The voluntary group clearly ran more than the forced group (U b 0.000).
3.4. Behavioral analysis after pilocarpine The behavioral manifestations of pilocarpine-induced seizures for all groups were similar to those previously reported [22]. Briefly, pilocarpine injection sequentially induced behavioral changes such as akinesia, facial automatisms, and limbic seizures consisting of forelimb clonus with rearing, salivation, and masticatory jaw movements and falling. These behaviors built up progressively into motor limbic seizures that recurred repeatedly and finally developed into SE. No difference was observed among groups in latency for first motor signs and in the number of animals that developed SE. Although the voluntary group presented more intense motor signs, an increased latency for developing SE was observed in this group compared with that in the forced and control groups (Fig. 3).
Fig. 2. Weight of animal from control (CTL), forced (FOR), and voluntary (VOL) groups. Animals from VOL group weighed less than animals from other groups. CTL = 241 ± 15 g; FOR = 252 ± 14 g; VOL = 223 ± 20 g. *VOL group presented reduced weight compared with CTL (p = 0.018) and FOR (p b 0.000) groups.
4. Discussion In this study, we aimed to investigate the influence of two different modalities of exercise programs on susceptibility to pilocarpineinduced seizures in female adult rats. Our findings showed that only voluntary exercise promoted alterations in seizure susceptibility. Studies performed in female humans, especially in athletes, have already shown that intense physical activity can induce menstrual cycle irregularities, with increased incidence of anovulatory cycles. This may occur because of alterations in the balance of neurosteroid hormones triggered by stress, following endogenous cortisol release [24,25]. Therefore, in this study, it was extremely important to observe the animals' estrous cycle during the physical activity programs in order to evaluate whether the training could be considered a stress factor or not. Our results demonstrated that the two types of physical exercise were not able to modify the estrous cycle of the rats. Yet studies conducted in women presenting pathological conditions linked to menstrual cycle alterations have demonstrated that moderate physical activity programs can ameliorate regularity of the cycle [26,27]. Moreover, previous experimental studies performed by our group have already shown that the estrous cycle presented alterations (an increase in the occurrence of the diestrus phase and a decrease in occurrence of the proestrus phase) in the chronic phase of the pilocarpine model [28,29]. These findings are suggestive of increased anovulatory cycles and an inadequate luteal phase. This anovulation in epileptic animals reflects an altered release of sex hormones. In fact, an increased estradiol level and a decreased progesterone level were found in epileptic rats when compared with nonepileptic rats [29]. Thus, studies concerning females must always consider observations in cyclicality, as this may indicate important physiological alterations. Another issue observed in this investigation was the difference of weight among groups. Voluntary group animals presented a reduced weight compared with the other groups. Although the pilocarpine injection dose was calculated based on animal weight, the body composition of animals may alter the pharmacodynamic and pharmacokinetic aspects of the drug. This may be considered a limitation of this study, since these aspects were beyond our scope and therefore not evaluated. Concerning the behavioral results obtained after pilocarpine injection, it is interesting to observe that the physical exercise programs presented different responses. Forced exercise on a treadmill did not alter behavioral parameters compared with the control group. Different studies conducted in male rats have shown that a treadmill exercise program can act positively on behavioral parameters before pilocarpine-induced seizures [6,11]. Gomes da Silva et al. [11], using the same pilocarpine-induced seizure method, submitted young male rats to this type of exercise program for 40 days and observed that exercise increased the latency to and reduced the intensity of first motor signs later, following a detraining period, in adult life. Yet Setkowicz and Mazur [6], who submitted animals to treadmill running and swimming for 45 days, observed that all evaluated parameters were
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B
(AU)
A *
D
C *
Fig. 3. Behavioral analysis after pilocarpine injection. Latency for motor signs (in seconds) (A) and number of animals that progressed to SE (C) did not differ among groups. Voluntary (VOL) group presented more intense motor signs (B) and increased latency for SE (D) development compared with control (CTL) and forced (FOR) groups. *Kruskal–Wallis performed for motor sign intensity presented H = 0.001, followed by Mann–Whitney presenting U b 0.000 between VOL and CTL; and U = 0.015 between VOL and FOR. For SE latency, H = 0.002; U = 0.009 between CTL and VOL; U = 0.003 between VOL and FOR. Motor sign intensity is expressed as an arbitrary unit (AU).
ameliorated after pilocarpine injection. It may be possible to conclude, therefore, that treadmill running may act differently in male and female rats. This type of physical activity should be tested in different levels of intensity and duration to better understand how it works in females.
Regarding voluntary exercise, our results were somewhat mixed. Intensity of motor signs was increased in this group; however, SE development was delayed. It is important to mention that pilocarpine injection is a devastating insult and is a drug with a specific mechanism
Fig. 4. Pearson's correlation for voluntary group. Motor sign intensity was correlated with animals' weight (n = 12; r = 0.436) and running distance (n = 12; r = −0.272). SE latency was correlated with animals' weight (n = 8; r = −0.587) and running distance (n = 8; r = 0.538). No significant correlation was observed in these analyses.
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of action. Although many studies in the literature have demonstrated beneficial effects of exercise programs in both the healthy brain and neurological diseases [9,30–33], we have to consider that, in this model, exercise programs for short periods may not be enough to diminish the negative behavioral aspects of pilocarpine-induced seizures in females. Our results contrast with those of Setkowicz and Mazur [6], since they observed a significant beneficial influence of physical activity on male rats submitted to pilocarpine injection. Moreover, results from other studies regarding kindling development after physical activity also seem contradictory. A study conducted by our group demonstrated that physical activity was effective in delaying amygdala kindling development [12] in adult male animals, but when animals were submitted to physical training during development, this delay was not observed [34]. Thus, as we can observe in the literature, beneficial effects of physical exercise on healthy animals submitted to a later insult are inconclusive and need to be carefully evaluated. Several mechanisms may be involved in a behavioral manifestation, and extra variables such as differences in age, training level, and gender need to be considered. Physical activity exerts effects on different aspects of brain function including enhancement of different neurotransmitter systems [35–40]. Regarding the pilocarpine insult, physical activity is also able to modulate the cholinergic system. This neurotransmission system is of special interest since the drug acts on muscarinic receptors. Fordyce and Farrar [41] have demonstrated that aerobic physical activity can increase the density of muscarinic receptors and reduce choline uptake in the hippocampus of animals. Increased density of muscarinic receptors may be a plausible explanation for the increased intensity of motor signs. Comparing results between forced and voluntary groups, it is important to bear in mind that different kinds of exercise may exert different effects on brain health. Stress is among the most frequently selfreported precipitants of seizures in people with epilepsy, and especially emotional stress [42]. To this end, we observed the cyclicality of the estrous cycle in order to avoid induction of stress in animals from both exercise groups. Although all animals were good runners, it is not possible to exclude the influence of minor stress from the results, as after all, animals submitted to the treadmill performed this physical activity during the diurnal period (the resting period for rats) and, unlike the voluntary exercise group, they were obligated to run. However, we would like to point out that recent studies have indicated that even exhaustive exercise (separate from moderate exercise, already established in literature) seems not to be a seizure-inducing factor [43,44]. Another possible reason for the difference found between groups, and this may be the main issue, is the difference in average running distance. Animals from the voluntary group ran almost 20 times further than animals from the forced group. This may suggest not only that there may be a different brain response due to exercise intensity but also that, for females, a different protocol for a moderate physical activity program might be required, as perhaps our protocol only represented a light exercise program for female rats. Yamamoto et al. [45] have already shown that female Wistar rats ran 10 times further than male Wistar rats when both were submitted to voluntary wheel running. Finally, in order to further investigate the voluntary group, which presented different responses and extra variables compared with control and forced groups, we performed Pearson's correlation statistics to elucidate whether running distance and animal weight could be considered confounding variables. Our results have shown that motor sign intensity was not necessarily influenced by running distance and weight, since the correlation values obtained indicate a weak positive correlation between intensity of motor signs and animal weight and a negligible negative correlation between intensity and running distance. We also correlated the latency for SE development with weight and running distance: moderate negative correlation was observed between latency and weight, while moderate positive correlation was observed for latency and running distance. These data suggest that the animals' weight might be associated with the results obtained for latency.
We verified through this correlation analysis that animals presenting greater weight also presented reduced latency for SE development, and this result is in accordance with our comparisons among groups, since the group presenting the lowest weight also presented increased latency to SE development. However, running distance might also be involved in this increased latency. Animals which ran longer distances presented increased latency for SE development, and this is also in accordance with our results among groups, since the voluntary group presented increased latency and animals from this group ran further than the other exercise group. It is important to mention that we are considering these possible influences by weight and running distance based on the r-value presented in the correlation statistics, which indicated a moderate correlation. However, the p-value was not significant in any Pearson's correlation performed. 5. Conclusion Although our behavioral results are not enough to explain the physiological and molecular pathways, there are mechanisms described in literature which may allow us to propose possible explanations. Clinical and experimental findings have already demonstrated that exercise can enhance neurogenesis in the hippocampus, reduce cell death, modulate neurotransmitter release, increase brain plasticity, and ameliorate or prevent parameters related to different neurological conditions, such as Alzheimer's, Parkinson's, and epilepsy [9,30–33,46,47]. Although females present biological differences, mainly related to hormonal levels and neurosteroid action, it is possible that exercise may also exert positive effects for them as clinical studies have already been published which show benefits to women with epilepsy [8]. Our results indicate a positive effect of voluntary exercise increasing latency to SE development. Although voluntary exercise exerted beneficial effects on female rats and forced exercise did not exert negative effects, it seems that exercise acts differently according to gender, and neurosteroids may have a crucial role to play in these differences. Valente et al. [48] demonstrated that castrated female animals presented an increased number of seizures compared with noncastrated animals, suggesting central involvement of neurosteroids. Thus, further investigation is necessary to elucidate the pathways involved in these results. Furthermore, different exercise programs should also be investigated for the female gender; some effects may be gender-specific, and recognizing gender differences is an emerging necessity that deserves more attention. Acknowledgments Research was supported by CAPES, FAPESP (2015/19256-0), and CNPq (300605/2013-07). Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] Esquivel E, Chaussain M, Plouin P, Ponsot G, Arthuis M. Physical exercise and voluntary hyperventilation in childhood absence epilepsy. Electroencephalogr Clin Neurophysiol 1991;79(2):127–32. [2] Nakken KO. Physical exercise in outpatients with epilepsy. Epilepsia 1999;40(5): 643–51. [3] Arida RM, Fernandes MJ, Scorza FA, Preti SC, Cavalheiro EA. Physical training does not influence interictal LCMRglu in pilocarpine-treated rats with epilepsy. Physiol Behav 2003;79(4–5):789–94. [4] Arida RM, Scorza FA, de Albuquerque M, Cysneiros RM, de Oliveira RJ, Cavalheiro EA. Evaluation of physical exercise habits in Brazilian patients with epilepsy. Epilepsy Behav 2003;4(5):507–10. [5] Arida RM, Sanabria ER, da Silva AC, Faria LC, Scorza FA, Cavalheiro EA. Physical training reverts hippocampal electrophysiological changes in rats submitted to the pilocarpine model of epilepsy. Physiol Behav 2004;83(1):165–71. [6] Setkowicz Z, Mazur A. Physical training decreases susceptibility to subsequent pilocarpine-induced seizures in the rat. Epilepsy Res 2006;71(2–3):142–8.
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Effects of different physical exercise programs on susceptibility to pilocarpine-induced seizures in female rats Diego Vannucci Campos a, Glauber Menezes Lopim a, Vanessa Santos de Almeida a, Débora Amado b, Ricardo Mario Arida a,⁎ a b
Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), São Paulo, — SP, Brazil Departamento de Neurologia e Neurocirurgia, Universidade Federal de São Paulo (UNIFESP), São Paulo, — SP, Brazil
a r t i c l e
i n f o
Article history: Received 9 May 2016 Revised 12 August 2016 Accepted 13 August 2016 Available online xxxx Keywords: Epilepsy Seizures susceptibility Pilocarpine Exercise Female rats
a b s t r a c t In epilepsy, the most common serious neurological disorder worldwide, several investigations in both humans and animals have shown the effectiveness of physical exercise programs as a complementary therapy. Among the benefits demonstrated, regular exercise can decrease the number of seizures as well as improve cardiovascular and psychological health in people with epilepsy. While many studies in animals have been performed to show the beneficial effects of exercise, they exclusively used male animals. However, females are also worthy of investigation because of their cyclical hormonal fluctuations and possible pregnancy. Considering the few animal studies concerning seizure susceptibility and exercise programs in females, this study aimed to verify whether exercise programs can interfere with seizure susceptibility induced by pilocarpine in adult female Wistar rats. Animals were randomly divided into three groups: control, forced, and voluntary (animals kept in a cage with a wheel). After the final exercise session, animals received a pilocarpine hydrochloride (350 mg/kg i.p.; Sigma) injection to induce seizures. To measure the intensity of pilocarpine-induced motor signs, we used a scale similar to that developed by Racine (1972) in the kindling model. During a 4-h period of observation, we recorded latency for first motor signs, latency for reaching SE, number of animals that developed SE, and intensity of pilocarpine-induced motor signs. No difference was observed among groups in latency for first motor signs and in the number of animals that developed SE. Although the voluntary group presented more intense motor signs, an increased latency for developing SE was observed compared with that in forced and control groups. Our behavioral results are not enough to explain physiological and molecular pathways, but there are mechanisms described in literature which may allow us to propose possible explanations. Voluntary exercise increased latency to SE development. Further investigation is necessary to elucidate the pathways involved in these results, while more studies should be performed regarding gender specific differences. © 2016 Elsevier Inc. All rights reserved.
1. Introduction In epilepsy, the most common serious neurological disorder worldwide, several investigations in both humans and animals have shown the effectiveness of physical exercise programs as a complementary therapy [1–7]. Among these benefits, regular exercise can decrease the number of seizures as well as improve cardiovascular and psychological health in people with epilepsy. Although the favorable effects of exercise after epilepsy have been established [2,8–10], very few studies have verified whether exercise programs might protect against or prevent the development of epilepsy. Findings from these studies have indicated that exercise may be able to modulate neuronal vulnerability to epileptogenic insults [6,11,12]. ⁎ Corresponding author at: Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), Rua Botucatu 862, Ed. Ciências Biomédicas, 5° andar, Vila Clementino, CEP: 04023-900 São Paulo, — SP, Brazil. E-mail address: [email protected] (R.M. Arida).
http://dx.doi.org/10.1016/j.yebeh.2016.08.011 1525-5050/© 2016 Elsevier Inc. All rights reserved.
Physical activity programs before precipitating insults (such as pilocarpine convulsant dose injection or amygdala electrical stimulation) have shown reduced brain susceptibility to seizures [6,12]. Gomes da Silva et al. [11] also presented results indicating that an aerobic exercise program during adolescence in rats can attenuate the effects of pilocarpine injection in adult life. In that study, even after a 90-day detraining period, the effects of an early physical activity program were able to ameliorate the intensity and increase the latency for motor signs. These effects were attributed to a possible formation of neural reserve. Although many studies on animals have been performed to show the beneficial effects of exercise, all used male animals. The female population is also worthy of attention because of its cyclical hormonal fluctuations and possible pregnancy. In the literature, there are studies which show the notable influence of sexual hormones on phenomena linked to the epilepsies [13–16]. In women with epilepsy, reproductive dysfunctions and endocrine disorders are commonly observed [17–19], mainly in temporal lobe epilepsy.
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Amado and Cavalheiro [20] have shown that the estrous cycle of Wistar female rats was altered after status epilepticus induced by pilocarpine, and this alteration remained for many weeks. Progesterone, luteinizing hormone, and follicle stimulant hormone was at lower levels compared with control animals, while estradiol levels exceeded healthy animal levels during the period of spontaneous and recurrent seizures. Considering the few animal studies concerning seizure susceptibility and exercise programs in females, this study aimed to verify whether exercise programs can interfere with seizure susceptibility induced by pilocarpine in adult female Wistar rats. 2. Methods 2.1. Animals Female Wistar rats were submitted to vaginal smears for 15 days before the experiments to verify the regularity of the estrous cycle. Only rats presenting a regular cycle were included in the procedures. Animals were randomly divided into three groups: control (animals not submitted to physical activity) (n = 16), forced (animals submitted to treadmill training) (n = 16), and voluntary (animals kept in a cage with a wheel) (n = 12). The colony room was maintained at 21 ± 2 °C with a 12-h light/dark cycle and food and water ad libitum throughout the experimental procedures. During all experimental procedures, vaginal smears from all animals were verified daily. All experimental protocols were conducted in accordance with the ethical committee of the university. 2.2. Forced exercise procedure Animals from the treadmill group were familiarized with the apparatus for three days by placing them on a treadmill (Columbus) for 10 min/day at speed 12 m/min at a 0% degree incline. Electric shocks (0.3 mA; two discharges/second) were used sparingly to motivate the animals to run. The trainability of rats was measured according to their performance on a scale of 1–5 [21]. The parameters of the scale were as follows: 1 — refused to run; 2 — below average runner (sporadic, stop and go, wrong direction); 3 — average runner; 4 — above average runner (consistent runner occasionally fell back on the treadmill); 5 — good runner (consistently stayed at the front of the treadmill). Animals rating 3 or higher were included in the group. These procedures were used to exclude possible different levels of stress among animals; however, no animal in this study had to be excluded from the exercise group (all animals submitted to physical training were good runners). Following familiarization, they were submitted to 30 sessions of an aerobic exercise program for 30 min/day reaching 22 m/min in the final week. The training protocol was performed five days/week between 9:00 and 12:00 (diurnal period). Daily running distance for this group was quantified by multiplying the speed programmed in the treadmill by the time that the animals were kept running during every session. Subsequently, the daily values that were obtained were added and divided by 30 (total number of sessions). 2.3. Voluntary exercise procedure Animals submitted to voluntary exercise were placed in a cage with voluntary wheel running (Panlab Harvard Apparatus) for 30 days with free access to food and water. Daily running distance for this group was quantified using an odometer, which registered the number of wheel revolutions. The daily odometer value which was obtained was multiplied by 2πR, considering R as the radius measured from the center of the wheel to its inner surface. Daily values were added and divided by 30 (total number of days that rats had free access to the wheel).
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hydrochloride (350 mg/kg i.p.; Sigma) injection to induce seizures. Animals in a different phase of the cycle were submitted to extra training days until they entered the estrus phase. This was done with the intention of avoiding the varying hormonal rates observed in different parts of the estrus cycle. Scopolamine methylnitrate (1 mg/kg s.c.; Sigma) was administered 30 min before pilocarpine injection to limit peripheral cholinergic effects [22]. The systemic administration of the potent muscarinic agonist pilocarpine in rats promotes sequential behavioral and electrographic changes that build up progressively into a limbic status epilepticus (SE) which lasts 24 h [22]. Therefore, in this study, we observed the behavioral manifestations of pilocarpine-induced SE. To measure the intensity of pilocarpine-induced motor signs, we used a scale describing progressive evolution of seizures similar to that developed by Racine [23] in the kindling model (0.5 — immobility, piloerection, salivation, narrowing of eyes, face and vibrissae twitching, ear rubbing with forepaws; 1.0 — head nodding and chewing movements; 1.5 — clonic movements of forelimbs and mild whole body convulsions, exophthalmia, aggressive behavior; 2.0 — rearing and running with stronger tonic–clonic motions including hindlimbs, tail hypertension, and lockjaw; 2.5 — rearing and falling, eye congestion; 3.0 — loss of postural tone with general body rigidity). Rats were observed for a 4-hour period and each motor sign described above was quantified during this period by a treatment-blinded observer. Following these observations, intensity was calculated by multiplying the number of behavioral manifestations in each step of Racine's scale by the grade given to this step. This was performed for all rats, and the mean and standard deviation were obtained with regard to all analyzed animals from each group. During the observation period, we recorded the following: (a) latency of the first motor signs, (b) latency for reaching SE, (c) number of animals that developed SE, and (d) intensity of pilocarpine-induced motor signs. Latency for the development of status epilepticus (SE) was evaluated by considering SE as a condition characterized by an epileptic seizure that is sufficiently prolonged or repeated at sufficiently brief intervals so as to produce an unvarying and enduring epileptic condition. Thus, animals were considered to be in SE when this continuous seizing condition was observed for at least 30 min. Animals became unresponsive to physical stimuli during this phase. 2.5. Statistical analysis The statistical analysis among voluntary, forced, and control groups was performed using the Kruskal–Wallis test followed by Mann– Whitney U-test and Chi-square test. Values were expressed as mean ± SD and considered significant when p b 0.05. Estrous cycle and animal weight were evaluated using one-way ANOVA followed by Bonferroni post hoc. Mann–Whitney was also performed to compare the average running distance between animals from the exercise groups. We also performed Pearson's correlation between different parameters in the voluntary group. 3. Results 3.1. Influence of physical activity on the estrous cycle In this study, we performed vaginal smears daily in all animals in order to verify possible alterations caused by the different types of physical activity. As shown in Fig. 1, we observed the same percentage of each phase in all groups (p N 0.05). 3.2. Animal weight
2.4. Pilocarpine injection After the final exercise session, animals presenting in the estrus phase of the estrous cycle on that day received a pilocarpine
Weights of animals from all groups were measured before pilocarpine injection to verify whether the exercise programs would alter this factor. We observed that animals from the voluntary group weighed
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3.5. Pearson's correlation for the voluntary group Noting the differences in weight, running distance, and behavioral parameter observed in the voluntary group compared with those in the other groups, we performed Pearson's correlation in order to verify whether weight and running distance were correlated with latency to SE development and/or motor sign intensity. Our results showed a weak positive correlation between motor sign intensity and animal weight (r = 0.436) and negligible negative correlation between motor sign intensity and running distance (r = −0.272). Concerning latency to SE, we observed moderate negative correlation between this latency and animal weight (r = −0.587) and moderate positive correlation between latency and running distance (r = 0.538) (Fig. 4). No significant correlation was observed in the p-value of these analyses (p N 0.05 for all correlations performed). Fig. 1. Percentage of each phase of the estrous cycle in each group. P — proestrus; E — estrus; M — metaestrus; D — diestrus; CTL — control group; FOR — forced group; VOL — voluntary group. No significant difference was observed in each phase of the estrous cycle among groups.
significantly less than animals from the control (p = 0.018) and forced (p b 0.000) groups (Fig. 2).
3.3. Average running distance All animals from the forced group ran exactly 650 m/day, while average running distance for the voluntary group was 10,260 ± 2580 m/day. The best runner ran 13,820 m/day and the worst, 5480 m/day. The voluntary group clearly ran more than the forced group (U b 0.000).
3.4. Behavioral analysis after pilocarpine The behavioral manifestations of pilocarpine-induced seizures for all groups were similar to those previously reported [22]. Briefly, pilocarpine injection sequentially induced behavioral changes such as akinesia, facial automatisms, and limbic seizures consisting of forelimb clonus with rearing, salivation, and masticatory jaw movements and falling. These behaviors built up progressively into motor limbic seizures that recurred repeatedly and finally developed into SE. No difference was observed among groups in latency for first motor signs and in the number of animals that developed SE. Although the voluntary group presented more intense motor signs, an increased latency for developing SE was observed in this group compared with that in the forced and control groups (Fig. 3).
Fig. 2. Weight of animal from control (CTL), forced (FOR), and voluntary (VOL) groups. Animals from VOL group weighed less than animals from other groups. CTL = 241 ± 15 g; FOR = 252 ± 14 g; VOL = 223 ± 20 g. *VOL group presented reduced weight compared with CTL (p = 0.018) and FOR (p b 0.000) groups.
4. Discussion In this study, we aimed to investigate the influence of two different modalities of exercise programs on susceptibility to pilocarpineinduced seizures in female adult rats. Our findings showed that only voluntary exercise promoted alterations in seizure susceptibility. Studies performed in female humans, especially in athletes, have already shown that intense physical activity can induce menstrual cycle irregularities, with increased incidence of anovulatory cycles. This may occur because of alterations in the balance of neurosteroid hormones triggered by stress, following endogenous cortisol release [24,25]. Therefore, in this study, it was extremely important to observe the animals' estrous cycle during the physical activity programs in order to evaluate whether the training could be considered a stress factor or not. Our results demonstrated that the two types of physical exercise were not able to modify the estrous cycle of the rats. Yet studies conducted in women presenting pathological conditions linked to menstrual cycle alterations have demonstrated that moderate physical activity programs can ameliorate regularity of the cycle [26,27]. Moreover, previous experimental studies performed by our group have already shown that the estrous cycle presented alterations (an increase in the occurrence of the diestrus phase and a decrease in occurrence of the proestrus phase) in the chronic phase of the pilocarpine model [28,29]. These findings are suggestive of increased anovulatory cycles and an inadequate luteal phase. This anovulation in epileptic animals reflects an altered release of sex hormones. In fact, an increased estradiol level and a decreased progesterone level were found in epileptic rats when compared with nonepileptic rats [29]. Thus, studies concerning females must always consider observations in cyclicality, as this may indicate important physiological alterations. Another issue observed in this investigation was the difference of weight among groups. Voluntary group animals presented a reduced weight compared with the other groups. Although the pilocarpine injection dose was calculated based on animal weight, the body composition of animals may alter the pharmacodynamic and pharmacokinetic aspects of the drug. This may be considered a limitation of this study, since these aspects were beyond our scope and therefore not evaluated. Concerning the behavioral results obtained after pilocarpine injection, it is interesting to observe that the physical exercise programs presented different responses. Forced exercise on a treadmill did not alter behavioral parameters compared with the control group. Different studies conducted in male rats have shown that a treadmill exercise program can act positively on behavioral parameters before pilocarpine-induced seizures [6,11]. Gomes da Silva et al. [11], using the same pilocarpine-induced seizure method, submitted young male rats to this type of exercise program for 40 days and observed that exercise increased the latency to and reduced the intensity of first motor signs later, following a detraining period, in adult life. Yet Setkowicz and Mazur [6], who submitted animals to treadmill running and swimming for 45 days, observed that all evaluated parameters were
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B
(AU)
A *
D
C *
Fig. 3. Behavioral analysis after pilocarpine injection. Latency for motor signs (in seconds) (A) and number of animals that progressed to SE (C) did not differ among groups. Voluntary (VOL) group presented more intense motor signs (B) and increased latency for SE (D) development compared with control (CTL) and forced (FOR) groups. *Kruskal–Wallis performed for motor sign intensity presented H = 0.001, followed by Mann–Whitney presenting U b 0.000 between VOL and CTL; and U = 0.015 between VOL and FOR. For SE latency, H = 0.002; U = 0.009 between CTL and VOL; U = 0.003 between VOL and FOR. Motor sign intensity is expressed as an arbitrary unit (AU).
ameliorated after pilocarpine injection. It may be possible to conclude, therefore, that treadmill running may act differently in male and female rats. This type of physical activity should be tested in different levels of intensity and duration to better understand how it works in females.
Regarding voluntary exercise, our results were somewhat mixed. Intensity of motor signs was increased in this group; however, SE development was delayed. It is important to mention that pilocarpine injection is a devastating insult and is a drug with a specific mechanism
Fig. 4. Pearson's correlation for voluntary group. Motor sign intensity was correlated with animals' weight (n = 12; r = 0.436) and running distance (n = 12; r = −0.272). SE latency was correlated with animals' weight (n = 8; r = −0.587) and running distance (n = 8; r = 0.538). No significant correlation was observed in these analyses.
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of action. Although many studies in the literature have demonstrated beneficial effects of exercise programs in both the healthy brain and neurological diseases [9,30–33], we have to consider that, in this model, exercise programs for short periods may not be enough to diminish the negative behavioral aspects of pilocarpine-induced seizures in females. Our results contrast with those of Setkowicz and Mazur [6], since they observed a significant beneficial influence of physical activity on male rats submitted to pilocarpine injection. Moreover, results from other studies regarding kindling development after physical activity also seem contradictory. A study conducted by our group demonstrated that physical activity was effective in delaying amygdala kindling development [12] in adult male animals, but when animals were submitted to physical training during development, this delay was not observed [34]. Thus, as we can observe in the literature, beneficial effects of physical exercise on healthy animals submitted to a later insult are inconclusive and need to be carefully evaluated. Several mechanisms may be involved in a behavioral manifestation, and extra variables such as differences in age, training level, and gender need to be considered. Physical activity exerts effects on different aspects of brain function including enhancement of different neurotransmitter systems [35–40]. Regarding the pilocarpine insult, physical activity is also able to modulate the cholinergic system. This neurotransmission system is of special interest since the drug acts on muscarinic receptors. Fordyce and Farrar [41] have demonstrated that aerobic physical activity can increase the density of muscarinic receptors and reduce choline uptake in the hippocampus of animals. Increased density of muscarinic receptors may be a plausible explanation for the increased intensity of motor signs. Comparing results between forced and voluntary groups, it is important to bear in mind that different kinds of exercise may exert different effects on brain health. Stress is among the most frequently selfreported precipitants of seizures in people with epilepsy, and especially emotional stress [42]. To this end, we observed the cyclicality of the estrous cycle in order to avoid induction of stress in animals from both exercise groups. Although all animals were good runners, it is not possible to exclude the influence of minor stress from the results, as after all, animals submitted to the treadmill performed this physical activity during the diurnal period (the resting period for rats) and, unlike the voluntary exercise group, they were obligated to run. However, we would like to point out that recent studies have indicated that even exhaustive exercise (separate from moderate exercise, already established in literature) seems not to be a seizure-inducing factor [43,44]. Another possible reason for the difference found between groups, and this may be the main issue, is the difference in average running distance. Animals from the voluntary group ran almost 20 times further than animals from the forced group. This may suggest not only that there may be a different brain response due to exercise intensity but also that, for females, a different protocol for a moderate physical activity program might be required, as perhaps our protocol only represented a light exercise program for female rats. Yamamoto et al. [45] have already shown that female Wistar rats ran 10 times further than male Wistar rats when both were submitted to voluntary wheel running. Finally, in order to further investigate the voluntary group, which presented different responses and extra variables compared with control and forced groups, we performed Pearson's correlation statistics to elucidate whether running distance and animal weight could be considered confounding variables. Our results have shown that motor sign intensity was not necessarily influenced by running distance and weight, since the correlation values obtained indicate a weak positive correlation between intensity of motor signs and animal weight and a negligible negative correlation between intensity and running distance. We also correlated the latency for SE development with weight and running distance: moderate negative correlation was observed between latency and weight, while moderate positive correlation was observed for latency and running distance. These data suggest that the animals' weight might be associated with the results obtained for latency.
We verified through this correlation analysis that animals presenting greater weight also presented reduced latency for SE development, and this result is in accordance with our comparisons among groups, since the group presenting the lowest weight also presented increased latency to SE development. However, running distance might also be involved in this increased latency. Animals which ran longer distances presented increased latency for SE development, and this is also in accordance with our results among groups, since the voluntary group presented increased latency and animals from this group ran further than the other exercise group. It is important to mention that we are considering these possible influences by weight and running distance based on the r-value presented in the correlation statistics, which indicated a moderate correlation. However, the p-value was not significant in any Pearson's correlation performed. 5. Conclusion Although our behavioral results are not enough to explain the physiological and molecular pathways, there are mechanisms described in literature which may allow us to propose possible explanations. Clinical and experimental findings have already demonstrated that exercise can enhance neurogenesis in the hippocampus, reduce cell death, modulate neurotransmitter release, increase brain plasticity, and ameliorate or prevent parameters related to different neurological conditions, such as Alzheimer's, Parkinson's, and epilepsy [9,30–33,46,47]. Although females present biological differences, mainly related to hormonal levels and neurosteroid action, it is possible that exercise may also exert positive effects for them as clinical studies have already been published which show benefits to women with epilepsy [8]. Our results indicate a positive effect of voluntary exercise increasing latency to SE development. Although voluntary exercise exerted beneficial effects on female rats and forced exercise did not exert negative effects, it seems that exercise acts differently according to gender, and neurosteroids may have a crucial role to play in these differences. Valente et al. [48] demonstrated that castrated female animals presented an increased number of seizures compared with noncastrated animals, suggesting central involvement of neurosteroids. Thus, further investigation is necessary to elucidate the pathways involved in these results. Furthermore, different exercise programs should also be investigated for the female gender; some effects may be gender-specific, and recognizing gender differences is an emerging necessity that deserves more attention. Acknowledgments Research was supported by CAPES, FAPESP (2015/19256-0), and CNPq (300605/2013-07). Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] Esquivel E, Chaussain M, Plouin P, Ponsot G, Arthuis M. Physical exercise and voluntary hyperventilation in childhood absence epilepsy. Electroencephalogr Clin Neurophysiol 1991;79(2):127–32. [2] Nakken KO. Physical exercise in outpatients with epilepsy. Epilepsia 1999;40(5): 643–51. [3] Arida RM, Fernandes MJ, Scorza FA, Preti SC, Cavalheiro EA. Physical training does not influence interictal LCMRglu in pilocarpine-treated rats with epilepsy. Physiol Behav 2003;79(4–5):789–94. [4] Arida RM, Scorza FA, de Albuquerque M, Cysneiros RM, de Oliveira RJ, Cavalheiro EA. Evaluation of physical exercise habits in Brazilian patients with epilepsy. Epilepsy Behav 2003;4(5):507–10. [5] Arida RM, Sanabria ER, da Silva AC, Faria LC, Scorza FA, Cavalheiro EA. Physical training reverts hippocampal electrophysiological changes in rats submitted to the pilocarpine model of epilepsy. Physiol Behav 2004;83(1):165–71. [6] Setkowicz Z, Mazur A. Physical training decreases susceptibility to subsequent pilocarpine-induced seizures in the rat. Epilepsy Res 2006;71(2–3):142–8.
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