A strength exercise program in rats with epilepsy is protective against seizures
Luiz Fernando Peixinho-Penaa
, Jansen Fernandesa
, Alexandre Aparecido de Almeidaa
Fabiano Guimarães Novaes Gomesa
, Ricardo Cassilhasb
, Daniel Paulino Venanciob
, Marco Tulio de Mellob
Fulvio Alexandre Scorzac
, Esper Abrão Cavalheiroc
, Ricardo Mario Aridaa,
aDepartamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP), São Paulo (SP), Brazil bDepartamento de Psicobiologia, Universidade Federal de São Paulo (UNIFESP), São Paulo (SP), Brazil
cDisciplina de Neurologia Experimental, Universidade Federal de São Paulo/Escola Paulista de Medicina (UNIFESP/EPM), São Paulo, Brazil
a b s t r a c t
a r t i c l e
i n f o
Article history: Received 29 June 2012 Revised 3 August 2012 Accepted 6 August 2012 Available online 24 October 2012
Keywords: Exercise Epilepsy Pilocarpine Seizure Strength training Resistance training
The beneﬁcial effects of physical exercise on epilepsy, such as a decreased seizure frequency, have been ob-served following aerobic exercise programs in both clinical and experimental studies. However, it is not well clariﬁed whether other types of exercise, including strength exercise, can provide similar beneﬁts for epilepsy. Forty four animals with epilepsy were continuously monitored 24 h a day for 60 days and divided into two periods of 30 days. Theﬁrst period was used to determine the number of seizures before beginning the phys-ical exercise program, and the second period was utilized to determine the number of seizures during the strength training. The mean frequency of seizures in the control and SHAM groups increased signiﬁcantly from period 1 to period 2. Although the frequency of seizures did not change signiﬁcantly between the two periods of 30 days of observation in the strength exercise group, a signiﬁcant reduction in the seizure frequen-cy was observed compared with the control and SHAM groups in period 2. Our study demonstrated that a strength exercise program exerted a signiﬁcant inﬂuence on the seizure frequency in animals with epilepsy and strengthens the observed beneﬁcial effect of exercise on epilepsy that has been demonstrated in animal studies. Theﬁnding of this nonclinical study can open a new window to verify the beneﬁcial contribution of strength exercise in epilepsy. Further experimental and clinical investigations are necessary to explore the extent to which strength exercise interferes with the epileptic condition.
© 2012 Elsevier Inc.
Evidence reported in the literature has demonstrated the beneﬁ -cial effects of physical exercise on epilepsy. These positive effects have been observed after aerobic exercise programs in both animal and human studies. In the human investigations, a long-term aerobic exercise program (15 weeks) decreased the seizure frequency in women with intractable epilepsy . In another study, however, four weeks of aerobic training program did not change the average frequency of seizures but led to improved cardiovascular and psycho-logical health of persons with uncontrolled epilepsy. An investiga-tion conducted by McAuley and coworkers reported no impact of seizure frequency after 12 weeks of a physical exercise program. In addition, studies have explored whether intensive exercise alters the seizure susceptibility in people with epilepsy, and, from the few studies that explored this subject, people with temporal lobe epilepsy
did not experience seizures after incremental physical exercise to ex-haustion or during the recovery period of an ergometric test[4,5].
From an experimental point of view, aerobic exercise training was able to retard the development of amygdala kindling, reduce sei-zure frequency, and promote positive plastic changes in the hip-pocampal formation, such as decreased CA1 hyperresponsiveness
 and pronounced changes in the staining of parvalbumin, a calcium-binding protein, in the dentate gyrus(for a review, see Arida et al.[10,11]).
It is important to note that people usually combine both aerobic and strength training (also known as resistance training or weight lifting) into their exercise program routine. Scientiﬁc research has shown that strength training improves muscle strength and increases muscle and bone mass[12–14],ﬂexibility, dynamic balance, mood, self-conﬁdence, and self-esteem. Thus, the symptoms of several chronic diseases, such as arthritis, depression, type-2 diabetes, and osteoporosis, have been reduced after strength training
[14,18–21], reduced falls have also been reported in older adults with functional limitations . From the experimental point of view, only one study has investigated the impact of a strength exer-cise program on brain plasticity. For instance, an elegant work conducted by Cassilhas and collaborators demonstrated that strength
⁎Corresponding author at: Departamento de Fisiologia, Universidade Federal de São Paulo (UNIFESP) Rua Botucatu 862, Ed. Ciências Biomédicas, 5° andar, Vila Clementino 04023‐900, São Paulo (SP), Brazil. Fax: +55 11 55739304.
E-mail address:email@example.com(R.M. Arida).
1525-5050 © 2012 Elsevier Inc.
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Epilepsy & Behavior
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exercise induced an increased expression of hippocampal IGF-1 and receptor (IGF-1R), AKT, synapsin 1, and synaptophysin in the hippo-campus.
Although the beneﬁcial effects of aerobic exercise on epilepsy have been highlighted, it is not well clariﬁed whether other types of exercise, such as strength exercise, can provide similar beneﬁts. In-deed, some studies that analyzed the effect of supervised exercise programs in people with epilepsy included sessions of cardiovascular, strength, andﬂexibility training[3,24]. Nevertheless, the favorable ef-fect of exercise on seizure frequency might be inﬂuenced by combina-tions of the above mentioned stimuli. Clearly, only the aerobic exercise has been investigated as an isolated variable. Accordingly, the aim of this study was to analyze the effect of a strength training program on spontaneous recurrent seizures (SRS) in rats with epilep-sy using the pilocarpine model of epilepepilep-sy.
2. Materials and methods
2.1. Animal care
Seventy male Wistar rats, (age: 60-day old, weight: 256 ± 3.1 g) provided by the Center for Development of Experimental Models for Medicine and Biology (CEDEME/UNIFESP), were housed under envi-ronmentally controlled conditions (7:00–19:00 h of light in a light/ dark cycle; 22–24 °C) and permitted free access to food and water throughout the experiment. All experimental procedures were ap-proved by the University Ethics Committee.
2.2. Pilocarpine-induced epilepsy model and behavioral analyses
Sustained seizures were induced by a single i.p. administration of pilocarpine hydrochloride (350 mg/kg; Sigma, St. Louis, MO). Scopol-amine methylnitrate (Sigma) was injected (1 mg:kg, s.c.) 30 min be-fore the pilocarpine to reduce peripheral cholinergic effects . Following the status epilepticusperiod, the surviving animals (44/ 70) were continuously monitored for 24 h using a video system to de-tect theﬁrst spontaneous recurrent seizure (SRS). Infrared emitting lights were used during the dark periods to allow for the video re-cording of the animals' activity during this time. After theﬁrst SRS had been detected, the animals were continuously monitored 24 h a
day for 60 days. The animals with epilepsy were divided into three groups: the strength group (n = 17), SHAM group (n = 13), and con-trol group (n = 14). The behavioral analysis was divided into two pe-riods of 30 days, period 1 (1–30 days) and period 2 (31–60 days). For the strength group, theﬁrst period was used to quantify the number of seizures before the strength exercise program, and the second pe-riod was used to analyze the number of seizures during the strength exercise program.
2.3. Strength exercise training
2.3.1. Familiarization with the vertical ladder
The animals were subjected to a familiarization protocol for an 80° inclined vertical ladder apparatus (110 cm high × 18 cm wide, with 2-cm grid steps). A housing chamber (L × W × H = 20 × 20 × 20 cm) was located at the top of the ladder and served as a shelter during the resting period (Fig. 1a)[23,26–30]. The familiarization protocol consisted of three trials per day for three days. In theﬁrst trial, the rats were kept in the housing chamber for 60 s and then placed on the ladder, 35 cm from the top. In the second trial, the rats were placed in the middle of the ladder. In the third trial, the rats were placed at the bottom of the ladder as previously described.
2.3.2. Strength exercise group
The animals of the strength group were subjected to a progressive strength exercise, 5 sessions per week for 4 weeks. Each session consisted of 8 climbing series with a progressively heavier load
ﬁxed to the proximal part of the animal's tail with a Coastlock Snap Swivel and Scotch 23 Rubber Tape (Scotch 3 M). For each series, the animals had to make 8–12 dynamic repetitive movements to reach the housing chamber. In theﬁrst two series, the load was 50% of the animal's total body mass; in subsequent series, the load was progres-sively increased to aﬁnal load of 100% (Fig. 1b). The average body weight was 492 g (range 470–550). The rest interval between the se-ries was 60 s. The training sessions for each animal were between 20 and 30 min. Animals from SHAM group spent 20 min in the training apparatus (at the side top of the vertical ladder,Fig. 1a). This proce-dure was performedﬁve times per week for 4 weeks.
20cm SHAM 20cm SHAM
2.4. Tissue collection and processing
Twenty-four hours after the last training session, the rats were eu-thanized by decapitation. The striated skeletal muscleﬂexor digitorum longus (FDL — an ankle extensor muscle that participates in the climbing movement), of the right leg was dissected, and each FDL muscle was coated with embedding medium (Tissue-Tek OCT, Miles, Naperville, IL, USA) and immersed in liquid nitrogen-cooled isopentane. All of the tissues and sera were stored at−80 °C until use.
2.4.1. Measurement ofﬁber cross-sectional areas of the FDL
Transverse sections (8μm thick) were cut from the mid-bellies in a
cryostat at −20 °C, melted onto poly-L-lysine-coated microscopic slides (Superfrost, Fisher Scientiﬁc) and stained with Meyer's hematoxylin-eosin (H&E). Digital images of the H&E-stained sections were captured using an Olympus brightﬁeld microscope BX50, camera DP71 (Melville, NY) with a 40× objective. The blinded analysis of the cross-sectional areas of 100ﬁbers per muscle sample was performed using the software Axio Vision 4.6 (Carl Zeiss MicroImaging GmbH).
Immunohistochemical studies were performed in cryostat sections (8μm) from FDL skeletal muscle. Cryostat sections (8μm) from FDL
were incubated for 10 min in phosphate buffer (PBS: 137 mM NaCl, 2.68 mM KCl, 8.03 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4). The sec-tions were incubated with blocking solution (3% albumin, w/v and 0.01% saponin, w/v, in PBS) for 1 h at room temperature. After a PBS wash, sections were incubated overnight at 4 °C with primary anti-body against slow myosin and fast myosin (1:100), respectively dilut-ed in blocking solution. Following six 5‐min washes in blocking solution, sections were incubated for 1 h at room temperature with the appropriated secondary antibody conjugated to peroxidase (1:200) diluted in blocking solution. Peroxidase activity was revealed using PBS containing 3,3-diaminobenzidine (DAB, 0.05%, w/v) and hy-drogen peroxide (0.01%, v/v) for 3 min at room temperature. The sec-tions were washed in PBS and then air-dried and coverslipped with Permount (Fisher Scientiﬁc, Pittsburgh, PA, USA). Negative control ex-periments were performed by omission of primary antibody. Sections were visualized with Olympus B50 microscope (Olympus, Japan, JP). Images were digitalized using a CoolSNAP-Pro charge-coupled device digital camera and Image-Pro Express Software (both from Media Cy-bernetics, Silver Spring, MD, 40× USA).
2.5. Statistical analysis
The Statistica 5.0 software was used for all analyses. The Shapiro–
Wilks W test was used to verify data normality. To determine whether the frequency of seizures varied or not between the two periods of
30 days of observation for the three groups, to investigate whether any difference could be found between the same periods of the three groups, and to examine whether sleep duration (measuring“freezing”) varied or not between the two periods of 30 days of observation for the three groups, the ANOVA for repeated measurements followed by Fish-er post-hoc test was adopted. To vFish-erify the diffFish-erence of cross-sectional areaﬁbers between groups, the one-way ANOVA followed by Fisher post-hoc test was adopted. The signiﬁcance level was set at 5%, and the data were presented as the mean±standard error.
The behavioral features of pilocarpine-induced seizures during the acute period were similar to those reported previously[25,31]. The behavioral pattern of SRS showed the same characteristics described by Cavalheiro et al.. Brieﬂy, the SRS consisted of facial automa-tisms, forelimb clonus, rearing, loss of postural control, and general-ized clonic seizures.
3.2. Seizure frequency
The ﬁrst spontaneous seizure was observed between 3 and 71 days (control group, between 27 and 71 days; SHAM group, be-tween 3 and 29 days; and strength group, bebe-tween 14 and 64 days) after the pilocarpine administration, and the mean seizure silent peri-od lasted for 39.0 ± 3.3 days, 13.0 ± 2.6 days, and 33.0 ± 6.3 days (mean ± S.E.) for the control, SHAM, and strength groups, respective-ly. The mean frequency of seizures in the control group increased sig-niﬁcantly from period 1 (mean 11.3 ± 2.5) to period 2 (mean 19.1 ± 2.4) (pb0.05). Similarly, the mean frequency of seizures in the
SHAM group increased signiﬁcantly from period 1 (mean 11.9 ± 1.3) to period 2 (mean 19.4 ± 3.4) (pb0.05). In the group of animals sub-mitted to the strength training program, the frequency of seizures did not change signiﬁcantly from period 1 (mean 13.0 ± 1.8) to period 2 (mean 12.0 ± 1.9). When the same periods of the three groups were analyzed together, a signiﬁcant reduction in the seizure frequency was observed when comparing the training group with the control and sham groups during period 2 (Fig. 2).
The analysis of the SRS over the light/dark cycle showed an increase in the seizure frequency during the diurnal period (07:00–18:59 h), compared with the nocturnal period (19:00–06:59 h) for all groups (control group, pb0.001; strength group, pb0,001; and SHAM group, pb0.001). However, when seizure frequency per unit time (6 hour
blocks) was analyzed separately for the active and inactive periods, an-imals from the strength group had a reduction of seizures from 13:00 h to 18:59 during the training program (second period) compared to the
0 5 10 15 20 25
CONTROL GROUP SHAM GROUP STRENGTH GROUP
Period 1 Period 2
Number of seizures
control and SHAM groups. No signiﬁcant difference was found between the control and SHAM groups (Fig. 3). Thus, we carried out a blind rater score for sleepiness to verify whether exercised rats had the same sleep duration as non-exercised rats. For this purpose, we performed 24 h/day video recording in our dataset (observing rats in their home cage) using video tracking software capable of measuring“freezing”or movement. The mean freezing (control:ﬁrst period=818.6±25.5 min and second period=808.2±24.6 min; SHAM:ﬁrst period=834.8±26.4 min and second period=798.2±24.7 min; and strength group:ﬁrst period= 825.1±26.0 min and second period=794.9±25.8 min; mean±SE) did not differ statistically among groups (p>0.05).
To ensure the effectiveness of the exercise protocol, we performed a cross-sectional area analysis of the FDL. As shown inFig. 4, the mus-cleﬁber area of the strength group was increased compared with the control and SHAM groups (F[2,6] = 11,266; pb0.05), indicating that the protocol applied during the four weeks of the experiment was sufﬁcient to cause muscle hypertrophy. Post hoc Fisher analysis re-vealed differences between control group vs strength group (p = 0.01) and SHAM group vs strength group (p = 0.004). However, the hypertrophy was evident in fast twitchﬁbers which were marked by the immunohistochemical method against type II myosin.Flexor digitorum longusmuscle is compounded by fast twitchﬁbers predom-inantly, which are more responsive to strength training (Fig. 5).
The present work demonstrated that a strength exercise program exerted signiﬁcant inﬂuence on the seizure frequency of animals with epilepsy. The mean number of seizures in the animals subjected to the strength training did not differ signiﬁcantly from period 1 (before
physical training) to period 2 (the physical training period). Although a reduction in the number of seizures was not observed in these animals after the strength training, our data suggest that strength training can exert a positive effect on the seizure frequency, based on the fact that the control and SHAM groups demonstrated an increased frequency of seizures during the two periods of behavior observation. Indeed, it has already been reported that there is a maturation process during the early stages of recurrent seizures in the pilocarpine model of epilepsy
. The analysis of the same period of the three groups demonstrated a signiﬁcant difference between the training group and the control and SHAM groups in period 2. Ourﬁndings are in accordance with pre-vious studies that analyzed the seizure frequency of animals with epi-lepsy after an aerobic exercise program[7–9,33].
Stress is among the most frequently self-reported precipitants of sei-zures in people with epilepsy[34–36]. Considering that strength exer-cise involves intense muscle contractions, we could presume that it is an exhaustive effort. Although animal and human studies have reported that intensive exercise does not alter seizure susceptibility[4,5,7,37], it remains unclear whether strength exercise is helpful, harmful, or essen-tially has no impact on the frequency of seizures. Strength training or weight training is used by many people and for many different reasons. The obvious beneﬁt of strength training is an increase in strength, which, in turn, can prevent injuries and improve posture and perfor-mance in sports and day-to-day activities, in addition to reducing the symptoms of the several chronic diseases cited above.
While the effects of strength exercise on brain plasticity or cognitive performance are not well explored, some studies have reported positive
ﬁndings. One investigation demonstrated that the signiﬁcant increase in muscular strength in trained elderly subjects was associated with en-hance psychological well-being and cognitive functioning . A meta-analysis reported that older adults with cognitive impairment who participate in exercise rehabilitation programs had similar strength and endurance training outcomes, suggesting the positive im-pact of strength exercise in rehabilitation programs. Furthermore, recent investigations have revealed positiveﬁndings of strength exer-cise on cognition. Cassilhas et al.demonstrated that 6 months of moderate- or high-intensity resistance training improved memory among senior men. Thisﬁnding has been extended by other research groups. Because it is important to indicate the effectiveness of the strength exercise protocol and to demonstrate the positive impact of strength exercise in the present investigation, we conﬁrmed by histo-logical analysis of the FDL muscle that the strength exercise protocol was efﬁcient in causing muscle hypertrophy[23,28,29]as conﬁrmed by H&E analysis.
Although information about the light/dark cycle is well documented
([32,42–45]), one study has demonstrated that the seizure frequency in pilocarpine-treated rats was not related to the circadian rhythm. Accordingly, a literature search has demonstrated that the seizure
4000 4500 5000 5500 6000
CONTROL GROUP SHAM GROUP STRENGTH GROUP
Fiber area (µm
Fig. 4.Cross-sectional area of FDL muscle from control (n =3), SHAM (n= 3), and strength (n=3) groups was measured after exercise intervention. * Different from control and SHAM groups (p≤0.05). One-way ANOVA followed by Fisher post-hoc test. Data are presented as mean ±standard error.
Period 1 (1 – 30 days)
Period 2 (31 – 60 days)
1:00 - 6:59 7:00 - 12:59
13:00 - 18:59 19:00 - 0:59 1:00 - 6:59 7:00 - 12:59 13:00 - 18:59 19:00 - 0:59 2
Number of seizures
Time of day
6 8 10
threshold is reduced during sleep (for a review, see Matos et al.). In our investigation, the animals subjected to the strength exercise exhibited the same pattern of seizure frequency as the control and SHAM groups over the light/dark cycle, which is in accordance with pre-vious experimental studies reporting an increase in seizure frequency during the diurnal period compared with the nocturnal period
[7,48,49]. On the other hand, the strength exercise protocol signiﬁcantly affected the seizure frequency during the light period, suggesting that exercise per se may interfere in seizure susceptibility.
In conclusion, the novelﬁnding presented here reinforces the ben-eﬁcial effect of exercise on epilepsy that has been demonstrated in animal studies[6–9,33]. The mechanisms by which physical training is able to induce such changes are not completely understood and deserve further experimental investigation.
This research was supported by CAPES, FAPESP, CNPq, INNT, and CInAPCe (Brazil).
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