Effect of atorvastatin on behavioral alterations and neuroinflammation during epileptogenesis

Epilepsy & Behavior 78 (2018) 109–117

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Effect of atorvastatin on behavioral alterations and neuroinflammation during epileptogenesis Clarissa Vasconcelos de Oliveira a, Jéssica Grigoletto a, Julia Marion Canzian a, Marta Maria Medeiros Frescura Duarte c, Thiago Duarte a, Ana Flávia Furian a,b, Mauro Schneider Oliveira a,⁎ a b c

Graduate Program in Pharmacology, Federal University of Santa Maria, Santa Maria, RS, Brazil Graduate Program in Food and Science Technology, Federal University of Santa Maria, Santa Maria, RS, Brazil Department of Health Sciences, Lutheran University of Brazil, Santa Maria, RS, Brazil

a r t i c l e

i n f o

Article history: Received 21 June 2017 Revised 6 September 2017 Accepted 13 October 2017 Available online xxxx Keywords: Epileptogenesis Pilocarpine Statin Behavior Neuroinflammation

a b s t r a c t Temporal lobe epilepsy (TLE) is the most frequent and medically refractory type of epilepsy in humans. In addition to seizures, patients with TLE suffer from behavioral alterations and cognitive deficits. Poststatus epilepticus model of TLE induced by pilocarpine in rodents has enhanced the understanding of the processes leading to epilepsy and thus, of potential targets for antiepileptogenic therapies. Clinical and experimental evidence suggests that inflammatory processes in the brain may critically contribute to epileptogenesis. Statins are inhibitors of cholesterol synthesis, and present pleiotropic effects that include antiinflammatory properties. We aimed the present study to test the hypothesis that atorvastatin prevents behavioral alterations and proinflammatory state in the early period after pilocarpine-induced status epilepticus. Male and female C57BL/6 mice were subjected to status epilepticus induced by pilocarpine and treated with atorvastatin (10 or 100 mg/kg) for 14 days. Atorvastatin slightly improved the performance of mice in the open-field and object recognition tests. In addition, atorvastatin dose-dependently decreased basal and status epilepticus-induced levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ) and increased interleukin-10 (IL-10) levels in the hippocampus and cerebral cortex. The antiinflammatory effects of atorvastatin were qualitatively identical in both sexes. Altogether, these findings extend the range of beneficial actions of atorvastatin and indicate that its antiinflammatory effects may be useful after an epileptogenic insult. © 2017 Elsevier Inc. All rights reserved.

1. Introduction Epilepsy is a brain disease in which affected individuals have an enduring predisposition to present unprovoked recurrent seizures [1]. With a worldwide prevalence of 0.5–1% in the general population [2] and affecting at least 50 million people worldwide [3], epilepsy is a major worldwide public health problem, being one of the most frequent neurological conditions [4]. In addition to seizures, behavioral comorbidities of epilepsy like anxiety, psychosis, depression, and cognitive deficits exist in many patients with epilepsy, worsening their quality of life [5,6]. Temporal lobe epilepsy (TLE) is a common type of this disease, and it is often elicited by a brain insult [7]. Such event triggers a myriad of cellular and molecular changes that increase the chance of developing epilepsy [7]. The period between the initial insult and the appearance of ⁎ Corresponding author at: Universidade Federal de Santa Maria, Centro de Ciências da Saúde, Departamento de Fisiologia e Farmacologia, Av. Roraima, n° 1000, Prédio 21, sala 5207, CEP 97105-900 Santa Maria, RS, Brazil. E-mail address: [email protected] (M.S. Oliveira).

https://doi.org/10.1016/j.yebeh.2017.10.021 1525-5050/© 2017 Elsevier Inc. All rights reserved.

spontaneous seizures is called epileptogenesis, and may represent the best window of opportunity to modify the disease progression [8]. One of the possible targets of epilepsy-preventing strategies is the inflammatory response that establishes in the brain after the initial epileptogenic injury [9]. In fact, compelling evidence from experimental and clinical studies has suggested that inflammatory mediators in the brain play an etiological role in epileptogenesis, as well as in the accompanying comorbidities and neuropathology of epilepsy [10]. Accordingly, strategies aiming to reduce the levels of inflammatory cytokines and other mediators may constitute a potential antiepileptogenic therapy [9]. Atorvastatin is the leading drug of the class of statins, the firstline medications for the treatment of hypercholesterolemia and prevention of associated cardiovascular burden [11]. Evidence of antiinflammatory and neuroprotective effects of atorvastatin has been obtained in many experimental models of neurological diseases, including epilepsy [12–14]. Moreover, atorvastatin protects from behavioral comorbidities of epilepsy [14,15]. Therefore, considering the need for developing disease-modifying strategies for epileptogenesis, in the present study, we investigated whether atorvastatin improves

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Table 1 Data about animals used in the study.

Number of animals used to status epilepticus inductiona Mice with status epilepticusb Mice resistant to status epilepticusc Mortality during status epilepticusd Total mortalitye Control animalsf Control animals mortalityg Total a b c d e f g

Male

Female

83 39 12 32 15 27 3 110

82 43 10 29 11 28 0 110

Number of animals submitted to pilocarpine injections. Number of animals that entered status epilepticus. Number of animals that did not enter in status epilepticus after 6 pilocarpine injections. Number of animals that died within the 60-minute status epilepticus period. Number of animals in status epilepticus that died in the 14-day follow-up. Number of animals submitted to NaCl 0.9% (pilocarpine vehicle) injections. Number of control animals that died in the 14-day follow-up.

short-term inflammatory response and behavioral alterations after pilocarpine-induced status epilepticus (SE). Given the importance of including both sexes in the preclinical research [16], and the need to test new drugs to treat epilepsy in both sexes [17,18], we performed the present study in male and female mice. 2. Materials and methods

animals, revised in 2011), and with the approval of the Ethics Committee for Animal Research of our University (Process #6165230415/2015). We made every possible effort to limit animal's suffering and to keep their number to a minimum.

2.2. Pilocarpine-induced status epilepticus Epileptogenesis was elicited by a single status epilepticus induced by pilocarpine, using a multiple low dose protocol as described previously [19]. Because of the severe peripheral adverse cholinergic associated with pilocarpine, mice received a previous injection of methylscopolamine (1 mg/kg ip; Sigma-Aldrich). After 30 min, pilocarpine hydrochloride (Sigma-Aldrich) (100 mg/kg, ip) was injected every 20 min until the onset of status epilepticus. The maximum number of pilocarpine injections per animal was 6. Status epilepticus was stopped after 60 min with diazepam (10 mg/kg, ip, Santisa). Age- and weightmatched control animals also received methylscopolamine and diazepam, but NaCl 0.9% instead of pilocarpine. During the 3 days after status epilepticus induction, all mice received special attention for welfare purposes. Special care included hand-offering of softened chow, inserting fresh fruits (apples and bananas) into their homecages, and injections of Ringer-lactate solution containing 5% dextrose. Complete data about animals and status epilepticus are shown in Table 1.

2.1. Animals 2.3. Treatment with atorvastatin One hundred ten C57BL/6 mice (25–35 g; 30–60 day-old) of each sex were used. They were kept under appropriated environmental conditions (12 h light–dark cycle, in a room temperature of 22 ± 1 °C). Standard rodent chow (Puro Lab 22 PB, Puro Trato) and filtered tap water were provided ad libitum. All experimental procedures were conducted in accordance with national (Guidelines of the Brazil's National Council for the Control of Animal Experimentation, revised in 2016) and international legislations (Guidelines of the National Institutes of Health of United States of America for the care and use of laboratory

Treatment with atorvastatin started 3 h after diazepam injection and lasted 14 days. Control and status epilepticus mice received daily doses of vehicle (0.9% NaCl) or atorvastatin (10 or 100 mg/kg) by intragastric gavage. Atorvastatin solution was freshly prepared by dissolving commercial tablets (Lipitor®; Pfizer, SP, Brazil) to 1 or 10 mg/ml [20,21] in 0.9% NaCl. All solutions were administered at 10 ml/kg. The selection of atorvastatin dosing was based on previous studies [22] and on pilot studies.

Fig. 1. Schematic illustration of the experimental protocol used in this study. Numbers in parentheses indicate the number of animals in each experimental group.

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Table 2 Effect of status epilepticus and atorvastatin on behavioral parameters. Parameter

Female Open-field test Latency to start exploration (s) Number of crossings Object recognition test Time spent in object exploration (s) Rotarod test Latency to fall (s) Sucrose preference test Sucrose consumption (%) Forced swim test Immobility time (s) Male Open-field test Latency to start exploration (s) Number of crossings Object recognition test Time spent in object exploration (s) Rotarod test Latency to fall (s) Sucrose preference test Sucrose consumption (%) Forced swim test Immobility time (s)

Control

status epilepticus

Vehicle

Atorvastatin 10 mg/kg

Atorvastatin 100 mg/kg

Vehicle

Atorvastatin 10 mg/kg

Atorvastatin 100 mg/kg

10.15 ± 2.83 62.23 ± 4.90

7.87 ± 5.08 80.62 ± 11.57

6.85 ± 2.25 73.28 ± 13.88

141.07⁎ ± 41.40 35.07 ± 10.24

53.0 ± 35.81 52.75 ± 19.10

58.18 ± 28.36 61.90 ± 13.54

36.84 ± 6.55

47.12 ± 6.89

39.00 ± 3.39

8.23⁎ ± 3.25

8.37⁎ ± 5.17

9.36⁎ ± 5.41

270.50 ± 12.87

224.06 ± 20.96

193.92 ± 35.75

219.38 ± 29.32

251.18 ± 20.12

246.86 ± 17.10

74.60 ± 3.85

69.8 ± 1.10

65.38 ± 4.56

64.30 ± 3.22

73.03 ± 4.77

69.74 ± 4.31

181.38 ± 12.77

203.37 ± 16.25

200.42 ± 11.72

160.07 ± 28.03

210.37 ± 21.87

190.45 ± 21.60

11.00 ± 3.58 55.23 ± 8.78

10.33 ± 4.33 51.66 ± 5.27

14.20 ± 4.79 31.00 ± 16.81

106.66⁎ ± 40.33 56.22 ± 21.31

92.77⁎ ± 34.73 47.66 ± 18.47

23.16 ± 18.81 164.83⁎ ± 41.19

32.76 ± 6.67

29.83 ± 6.40

28.80 ± 13.30

5.33⁎ ± 3.90

1.11⁎ ± 1.11

26.16 ± 19.73

244.73 ± 17.15

185.41 ± 47.29

198.40 ± 52.50

174.88 ± 46.21

215.70 ± 25.89

243.91 ± 25.53

68.39 ± 2.99

69.96 ± 3.36

63.94 ± 2.38

68.05 ± 1.88

66.43 ± 6.76

63.88 ± 2.63

205.69 ± 12.89

215.33 ± 15.39

183.00 ± 34.07

129.77⁎ ± 26.33

112.88⁎ ± 24.23

136.16 ± 35.11

Data are mean ± SEM. The number of animals in each group is given in Fig. 1. ⁎ Indicates a significant difference (p b 0.05) versus the respective treated control.

Fig. 2. Effect of oral administration of increasing doses of atorvastatin (10 or 100 mg/kg; ig) on IL-1β levels in the hippocampus and cerebral cortex of female and male mice 14 days after pilocarpine-induced status epilepticus. Data are mean + status epilepticusM for n = 4 each group. Statistical analyses were carried out with two-way ANOVA followed by Newman–Keuls test. *indicates p b 0.05 versus the vehicle-treated control; #indicates p b 0.05 versus vehicle-treated status epilepticus; and δindicates p b 0.05 versus other nonstatus epilepticus controls.

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2.4. Behavioral tests A set of behavioral tests was employed to evaluate the effect of atorvastatin on behavioral comorbidities of epilepsy [23]. Tests were carried out according to previously described protocols in the following sequence: open-field [23], object recognition [23], rotarod [23], sucrose preference [24], and forced swim [23]. Behavioral testing occurred between days 7 and 13 after status epilepticus (Fig. 1 shows a timeline depicting the complete experimental design). Daily treatment with atorvastatin was done at the end of each day in order to minimize possible effects of acute statin on behavior. Animals that died before completing all testing trials were excluded from all analyses.

kit for rodents, commercially provided by R&D Systems® (Minneapolis, MN, USA), and the results are expressed in pg/mg of protein. 2.6. Statistical analyses Data were analyzed by two-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparisons test for post hoc analyses. A probability of p b 0.05 was considered significant. For cytokine analyses, the number of samples was 4 per group because we have used the strategy of sequential sampling, in which an experiment may be interrupted if a significant effect is found in a planned statistical analysis of partial data [25]. Since we found statistically significant differences with the first set of samples (4 per group, randomly selected), the experiment was ended.

2.5. Cytokine analysis 3. Results The content of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interferon-γ (INF-γ), and interleukin-10 (IL-10) was determined in both hippocampi and cerebral cortex at day 14 poststatus epilepticus. Brain tissue was homogenized in phosphate buffered saline (10 mM PBS, pH 7.4) containing 1 mM EDTA and 0.1 mM PMSF. The homogenates were centrifuged at 10,000 g for 10 min at 4 °C. Protein content was measured in supernatant and adjusted for 1 mg/ml, and bovine serum albumin (0.5% BSA) was added to the samples, according to the kit manufacturer instructions. Cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA)

Mice subjected to status epilepticus, regardless of sex, presented increased latency to explore the open-field arena (Table 2). Treatment with atorvastatin attenuated this deficit, being effective at the doses of 10 and 100 mg/kg in females and only at the dose of 100 mg/kg in males (Table 2). Statistical analysis of the number of crossings revealed a significant interaction between status epilepticus condition and pharmacological treatment in males. Post hoc analysis showed that male mice subjected to status epilepticus and treated with 100 mg/kg of atorvastatin presented increased locomotion scores (Table 2). However,

Fig. 3. Effect of oral administration of increasing doses of atorvastatin (10 or 100 mg/kg; ig) on IL-6 levels in the hippocampus and cerebral cortex of female and male mice 14 days after pilocarpine-induced status epilepticus. Data are mean + status epilepticusM for n = 4 each group. Statistical analyses were carried out with two-way ANOVA followed by Newman–Keuls test. *indicates p b 0.05 versus the vehicle-treated control; #indicates p b 0.05 versus vehicle-treated status epilepticus; and δindicates p b 0.05 versus other nonstatus epilepticus controls.

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status epilepticus did not alter locomotion scores of male and female mice per se. Mice subjected to status epilepticus spent less time exploring the objects than control animals in the habituation session of the object recognition test (Table 2). Total exploration time was so critically reduced that the analysis of object recognition index at 4 or 24 h was impracticable (data not shown). Interestingly, the time spent exploring objects during the training session was not reduced in poststatus epilepticus males treated with 100 mg/kg of atorvastatin, indicating a protective effect (Table 2). No differences were found between control and poststatus epilepticus mice (of both sexes) in the rotarod and taste preference tests (Table 2). However, we found that poststatus epilepticus males presented shorter immobility times than their respective controls in the forced swim test (Table 2). This effect was blunted by atorvastatin at the dose of 100 mg/kg. Status epilepticus and atorvastatin did not alter the behavior of females in this task (Table 2). In the current study, we investigated the effect of atorvastatin during the epileptogenic period on the levels of key cytokines in the hippocampi and cerebral cortex and behavior of mice. Statistical analyses revealed an increase in the levels of proinflammatory cytokines IL-1β (Fig. 2), IL-6 (Fig. 3), TNF-α (Fig. 4), and INF-γ (Fig. 5) in the hippocampi and cerebral cortex of female and male mice subjected to status epilepticus. Conversely, status epilepticus downregulated the levels of the antiinflammatory cytokine IL-10 in the hippocampus and cerebral cortex of male and female mice (Fig. 6). The treatment with atorvastatin dose-dependently attenuated the status epilepticus-elicited increase in

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the levels of IL-1β (Fig. 2), IL-6 (Fig. 3), TNF-α (Fig. 4), and INF-γ (Fig. 5). Status epilepticus-induced decrease of IL-10 levels in the hippocampus and cerebral cortex was recovered by treatment with atorvastatin at the doses of 10 and 100 mg/kg in females and only by the higher dose in males (Fig. 6). Interestingly, atorvastatin treatment had a pronounced effect on cytokine levels in control animals (mice not subjected to pilocarpine-induced status epilepticus). In fact, atorvastatin per se decreased IL-1β (Fig. 2), IL-6 (Fig. 3), TNF-α (Fig. 4), and INF-γ (Fig. 5) whereas increased IL-10 levels in the hippocampi and cerebral cortex of mice of both sexes. In a separated analysis, the levels of cytokines between females and males were compared. Regarding this point, an interesting finding is that females presented higher levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and INF-γ) than males. On the other hand, the levels of IL-10 were lower in females than in males. 4. Discussion In the present study, we present evidence that atorvastatin treatment during epileptogenesis attenuates neuroinflammation and decreases behavioral comorbidities in the model of TLE induced by pilocarpine. Atorvastatin decreased basal and status epilepticusinduced increase of IL-1β, IL-6, TNF-α, and INF-γ levels and increased IL-10 levels in the hippocampi and cerebral cortex of mice. Interestingly, the antiinflammatory effect of atorvastatin was qualitatively identical in both sexes.

Fig. 4. Effect of oral administration of increasing doses of atorvastatin (10 or 100 mg/kg; ig) on TNF-α levels in the hippocampus and cerebral cortex of female and male mice 14 days after pilocarpine-induced status epilepticus. Data are mean + status epilepticusM for n = 4 each group. Statistical analyses were carried out with two-way ANOVA followed by Newman–Keuls test. *indicates p b 0.05 versus the vehicle-treated control; #indicates p b 0.05 versus vehicle-treated status epilepticus; and δindicates p b 0.05 versus other nonstatus epilepticus controls.

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Fig. 5. Effect of oral administration of increasing doses of atorvastatin (10 or 100 mg/kg; ig) on IFN-γ levels in the hippocampus and cerebral cortex of female and male mice 14 days after pilocarpine-induced status epilepticus. Data are mean + status epilepticusM for n = 4 each group. Statistical analyses were carried out with two-way ANOVA followed by Newman–Keuls test. *indicates p b 0.05 versus the vehicle-treated control; #indicates p b 0.05 versus vehicle-treated status epilepticus; and δindicates p b 0.05 versus other nonstatus epilepticus controls.

Statins constitute the first-choice prescription drugs for the treatment of many dyslipidemias [26]. By inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase, these drugs block hepatic synthesis of cholesterol and elicit an indirect increase in the expression of lowdensity lipoprotein receptors [27]. It is interesting to note, however, that in addition to their cholesterol-lowering actions, statins exhibit cholesterol-independent effects. These include immunomodulatory, antiinflammatory, and antiexcitotoxic properties [27–30]. Accordingly, increasing evidence of different nature confirms the protective actions of statins in several neuropathological conditions, including epilepsy [12–14]. Here, we tested the effects of atorvastatin, the best studied statin in epilepsy models [13], on mice behavior and neuroinflammatory status after an epileptogenic insult. Previous studies have shown that atorvastatin presents anticonvulsant effects against kainate- [20] and pentylenetetrazole-induced seizures [22]. Moreover, atorvastatin protects from audiogenic seizures in DBA/2 susceptible mice [31] and decreases spontaneous spike–wave discharge seizures in WAG/Rij rats [15]. Moreover, statins seem to reduce the risk of anxiety, depression, hostility [32], and dementia [33]. However, no study has investigated whether atorvastatin attenuates the status epilepticus-induced behavioral and inflammatory changes during epileptogenic period of the pilocarpine model of TLE. The current study showed that vehicle-treated status epilepticus mice of both sexes present higher latency to explore the open-field arena when compared with their respective controls. Given that a

lower propensity for exploration correlates with a higher level of anxiety [34], our results indicate an increased anxiety-like behavior occurring at an early timepoint after status epilepticus. Moreover, atorvastatin treatment brought start latencies of both status epilepticus male (100 mg/kg atorvastatin) and female (10 or 100 mg/kg atorvastatin) mice to the levels of their respective controls, indicating an anxiolytic-like behavior of this statin. In the present study, we did not detect status epilepticus-induced deficits in spontaneous exploration (number of crossings in the open field) or fine motor coordination (latency to fall from rotarod). These results suggest that motor deficits did not occur at an early stage of the epileptogenic process in the pilocarpine model. In fact, changes in motor performance seem not to occur in this model even at later time points [19,23]. Interestingly, status epilepticus male mice treated with a high dose of atorvastatin (100 mg/kg) presented an increased locomotor activity when compared with all other groups. At the present, the significance and mechanisms involved in this effect are not understood. However, to some extent, these results are in line with those studies that have shown that atorvastatin improves motor performance in animal models of Huntington's and Parkinson's disease [28,30,35], but does not alter any behavioral parameter in the open-field test per se [36]. Antidepressant-like effects of atorvastatin have been demonstrated in mice [37,38], as well as in WAG/Rij rats with absence epilepsy [15]. In the present study, we investigated whether status epilepticus mice alter sucrose preference (an index of anhedonia) or swimming immobility, and the potential effects of atorvastatin on these parameters.

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Fig. 6. Effect of oral administration of increasing doses of atorvastatin (10 or 100 mg/kg; ig) on IL-10 levels in the hippocampus and cerebral cortex of female and male mice 14 days after pilocarpine-induced status epilepticus. Data are mean + status epilepticusM for n = 4 each group. Statistical analyses were carried out with two-way ANOVA followed by Newman–Keuls test. *indicates p b 0.05 versus the vehicle-treated control; #indicates p b 0.05 versus vehicle-treated status epilepticus; and δindicates p b 0.05 versus other nonstatus epilepticus controls.

Anhedonic behavior has been reported in the chronic phase of the pilocarpine model [23,39]. However, we did not detect changes 14 days after status epilepticus, suggesting that anhedonia symptoms may take more time to develop after pilocarpine-induced status epilepticus. On the other hand, we found that status epilepticus decreases the immobility time of male mice in the forced swim test. Similar findings at later time points were described in the pilocarpine model [23,40]. Such reduced immobility of the epileptic mice is thought to result from a problem in understanding the context (i.e., the inescapable situation) [19]. The high dose of atorvastatin appeared to improve this parameter, since the immobility time of atorvastatin (100 mg/kg)-treated controls and poststatus epilepticus males was similar. Clinical and experimental evidence suggests that some inflammatory processes within the central nervous system may either contribute to or be a consequence of epileptogenesis [41]. Experimentally-induced status epilepticus triggers an inflammatory response in brain areas recruited in the onset and propagation of epileptic activity in rodents [41,42]. In this context, the current study presents evidence of an antiinflammatory effect of atorvastatin treatment in mice of both sexes during epileptogenesis triggered by pilocarpine-induced status epilepticus. Accordingly, atorvastatin reduced the levels of proinflammatory cytokines IL-1β, TNF-α, INF-γ, and IL-6 in the hippocampi and cerebral cortex of mice subjected to status epilepticus. Moreover, atorvastatin increased the levels of the antiinflammatory cytokine IL-10 in poststatus epilepticus animals. In this context, the antiinflammatory effects of statins after an epileptogenic insult have been reported elsewhere,

and our current data are in agreement with those studies. For instance, atorvastatin decreases ED-1 positive cells (marker of monocytes/macrophages) infiltration and messenger RNA expression of TNF-α, IL-1β, and inducible nitric oxide synthase in the rat hippocampus 3 days after systemic injection of kainic acid [20]. Furthermore, simvastatin decreases the hippocampal levels of IL-1β and TNF-α without altering IL-6 levels in the rat hippocampus 3 days after intracerebroventricular injection of kainic acid [43]. In line with this view, lovastatin blunts the increase in the levels of IL-1β and TNF-α in the hippocampus of rats subjected to pilocarpine-induced status epilepticus [44]. On the other hand, no differences were observed in the number of CD68-positive cells (marker of monocytes/macrophages) or CD11b/c positive cells (marker of activated microglia) between vehicle- and atorvastatin-treated rats 6 weeks after electrically-induced status epilepticus [45]. The current results are particularly interesting considering that proinflammatory cytokines may play a role in epileptogenesis, since hippocampal and cortical levels of inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α) are not altered after status epilepticus in the Amazon rodent Proechimys guyannensis, an animal species resistant to epileptogenesis [46]. Moreover, it has been demonstrated that omega-3 fatty acids, which reduce proinflammatory markers during epileptogenesis in rats subjected to the pilocarpine model of epilepsy, are promising disease-modifying agents for epilepsy [47]. In addition, it is remarkable that a statistically significant correlation exists between reduced neuroinflammation and delayed neurocognitive decline in atorvastatin-treated patients with atrial fibrillation [48], suggesting that the beneficial effects of atorvastatin

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during epileptogenesis may also apply to improvement of behavioral alterations. Neuroinflammatory diseases are more prevalent in females than males [49], and there is evidence that sex also affects the type and severity of a central nervous system inflammatory response after epileptogenic brain insult [49,50]. Although many studies have tested antiepileptogenic therapies that target the immune/inflammatory response, none of these studies compared the effect of these drugs between males and females [49]. In the present study, we showed that female mice present higher levels of proinflammatory cytokines and lower levels of antiinflammatory IL-10 than males, which agree with the view that neuroinflammation is more pronounced in females than in males [49]. Therefore, sex-related differences in factors that regulate inflammation could likely contribute to sex-biased differences in epilepsy [51]. Regarding this point, it is interesting that the beneficial effect of atorvastatin on object recognition test training after status epilepticus was seen only in males, which had lower basal and status epilepticusstimulated levels of proinflammatory cytokines than their female counterparts. Indeed, present results agree with the current view that preclinical studies of drugs to treat epilepsy should be tested in both sexes [16,17,52].

Acknowledgments The authors gratefully acknowledge the student fellowships from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) (to C.V.O., J.G., T.D.), A.F.F., J.M.C. and M.S.O. are grantees of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) research productivity fellowships. The authors also thank Dr. C.F. Mello for a critical reading of the manuscript. Conflict of interest statement The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper. Funding sources This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References [1] Fisher RS, Acevedo C, Arzimanoglou A, Bogacz A, Cross JH, Elger CE, et al. Official report: a practical clinical definition of epilepsy. Epilepsia 2014;55:475–82. [2] Goldenberg MM. Overview of drugs used for epilepsy and seizures: etiology, diagnosis, and treatment. Pharm Ther 2010;35:392–415. [3] Thurman DJ, Beghi E, Begley CE, Berg AT, Buchhalter JR, Ding D, et al. Epidemiology ICo. Standards for epidemiologic studies and surveillance of epilepsy. Epilepsia 2011;52(Suppl. 7):2–26. [4] Schmidt D, Sillanpää M. Evidence-based review on the natural history of the epilepsies. Curr Opin Neurol 2012;25:159–63. [5] Boro A, Haut S. Medical comorbidities in the treatment of epilepsy. Epilepsy Behav 2003;4(Suppl. 2):S2–12. [6] Seidenberg M, Pulsipher DT, Hermann B. Association of epilepsy and comorbid conditions. Future Neurol 2009;4:663–8. [7] Engel J. Mesial temporal lobe epilepsy: what have we learned? Neuroscientist 2001; 7:340–52. [8] Sloviter RS, Bumanglag AV, Schwarcz R, Frotscher M. Abnormal dentate gyrus network circuitry in temporal lobe epilepsy. In: AM Noebels JL, Rogawski MA, et al, editors. Jasper's basic mechanisms of the epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012. [9] Löscher W, Brandt C. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research. Pharmacol Rev 2010; 62:668–700. [10] Vezzani A, Lang B, Aronica E. Immunity and inflammation in epilepsy. Cold Spring Harb Perspect Med 2015;6:a022699. [11] Collins R, Reith C, Emberson J, Armitage J, Baigent C, Blackwell L, et al. Interpretation of the evidence for the efficacy and safety of statin therapy. Lancet 2016;388: 2532–61. [12] Banach M, Czuczwar SJ, Borowicz KK. Statins — are they anticonvulsant? Pharmacol Rep 2014;66:521–8.

[13] Scicchitano F, Constanti A, Citraro R, Sarro GD, Russo E. Statins and epilepsy: preclinical studies, clinical trials and statin–anticonvulsant drug interactions. Curr Drug Targets 2015;16:747–56. [14] McFarland AJ, Anoopkumar-Dukie S, Arora DS, Grant GD, McDermott CM, Perkins AV, et al. Molecular mechanisms underlying the effects of statins in the central nervous system. Int J Mol Sci 2014;15:20607–37. [15] Citraro R, Chimirri S, Aiello R, Gallelli L, Trimboli F, Britti D, et al. Protective effects of some statins on epileptogenesis and depressive-like behavior in WAG/Rij rats, a genetic animal model of absence epilepsy. Epilepsia 2014;55:1284–91. [16] Clayton JA, Collins FS. Policy: NIH to balance sex in cell and animal studies. Nature 2014;509:282–3. [17] Scharfman HE, NJ MacLusky. Sex differences in the neurobiology of epilepsy: a preclinical perspective. Neurobiol Dis 2014;72:180–92 Pt B. [18] Luef G, Taubøll E. Gender issues in epilepsy—difference in management of epilepsy. Seizure 2015;28:1–2. [19] Gröticke I, Hoffmann K, Löscher W. Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice. Exp Neurol 2007;207:329–49. [20] Lee JK, Won JS, Singh AK, Singh I. Statin inhibits kainic acid-induced seizure and associated inflammation and hippocampal cell death. Neurosci Lett 2008;440:260–4. [21] Zaid AN, Assali M, Zalmout S, et al. Compounding and stability evaluation of atorvastatin extemporaneous oral suspension using tablets or pure powder. Eur J Hosp Pharm 2017;24:157–61. [22] Funck VR, de Oliveira CV, Pereira LM, Rambo LM, Ribeiro LR, Royes LF, et al. Differential effects of atorvastatin treatment and withdrawal on pentylenetetrazol-induced seizures. Epilepsia 2011;52:2094–104. [23] Oliveira CV, Grigoletto J, Funck VR, Ribeiro LR, Royes LF, Fighera MR, et al. Evaluation of potential gender-related differences in behavioral and cognitive alterations following pilocarpine-induced status epilepticus in C57BL/6 mice. Physiol Behav 2015;143:142–50. [24] Grigoletto J, CVd Oliveira, ACB Grauncke, TLd Souza, Souto NS, MLd Freitas, et al. Rosmarinic acid is anticonvulsant against seizures induced by pentylenetetrazol and pilocarpine in mice. Epilepsy Behav 2016;62:27–34. [25] Chamove AS. Reducing animal numbers: sequential sampling. Animal welfare information center (AWIC). Newsletter 1996;7:3–6. [26] Wang TJ, Stafford RS, Ausiello JC, Chaisson CE. Randomized clinical trials and recent patterns in the use of statins. Am Heart J 2001;141:957–63. [27] Schachter M. Chemical, pharmacokinetic and pharmacodynamic properties of statins: an update. Fundam Clin Pharmacol 2005;19:117–25. [28] Ling Q, Tejada-Simon MV. Statins and the brain: new perspective for old drugs. Prog Neuropsychopharmacol Biol Psychiatry 2016;66:80–6. [29] Stüve O, Youssef S, Steinman L, Zamvil SS. Statins as potential therapeutic agents in neuroinflammatory disorders. Curr Opin Neurol 2003;16:393–401. [30] van der Most PJ, Dolga AM, Nijholt IM, Luiten PG, Eisel UL. Statins: mechanisms of neuroprotection. Prog Neurobiol 2009;88:64–75. [31] Russo E, Donato di Paola E, Gareri P, Siniscalchi A, Labate A, Gallelli L, et al. Pharmacodynamic potentiation of antiepileptic drugs' effects by some HMG-CoA reductase inhibitors against audiogenic seizures in DBA/2 mice. Pharmacol Res 2013;70:1–12. [32] Young-Xu Y, Chan KA, Liao JK, Ravid S, Blatt CM. Long-term statin use and psychological well-being. J Am Coll Cardiol 2003;42:690–7. [33] Jick H, Zornberg GL, Jick SS, Seshadri S, Drachman DA. Statins and the risk of dementia. The Lancet 2000;356:1627–31. [34] Palanza P. Animal models of anxiety and depression: how are females different? Neurosci Biobehav Rev 2001;25:219–33. [35] Patassini S, Giampà C, Martorana A, Bernardi G, Fusco FR. Effects of simvastatin on neuroprotection and modulation of Bcl-2 and BAX in the rat quinolinic acid model of Huntington's disease. Neurosci Lett 2008;448:166–9. [36] Gaur V, Kumar A. Neuroprotective potentials of candesartan, atorvastatin and their combination against stroke induced motor dysfunction. Inflammopharmacology 2011;19:205–14. [37] Shahsavarian A, Javadi S, Jahanabadi S, Khoshnoodi M, Shamsaee J, Shafaroodi H, et al. Antidepressant-like effect of atorvastatin in the forced swimming test in mice: the role of PPAR-gamma receptor and nitric oxide pathway. Eur J Pharmacol 2014;745:52–8. [38] Ludka FK, ADE Zomkowski, Cunha MP, Dal-Cim T, ALB Zeni, ALS Rodrigues, et al. Acute atorvastatin treatment exerts antidepressant-like effect in mice via the L-arginine-nitric oxide-cyclic guanosine monophosphate pathway and increases BDNF levels. Eur Neuropsychopharmacol 2013;23:400–12. [39] Mazarati A, Siddarth P, Baldwin RA, Shin D, Caplan R, Sankar R. Depression after status epilepticus: behavioural and biochemical deficits and effects of fluoxetine. Brain 2008;131:2071–83. [40] Banerjee PN, Filippi D, Allen Hauser W. The descriptive epidemiology of epilepsy—a review. Epilepsy Res 2009;85:31–45. [41] Vezzani A, Friedman A, Dingledine RJ. The role of inflammation in epileptogenesis. Neuropharmacology 2013;69:16–24. [42] Vezzani A, Granata T. Brain inflammation in epilepsy: experimental and clinical evidence. Epilepsia 2005;46:1724–43. [43] Xie C, Sun J, Qiao W, Lu D, Wei L, Na M, et al. Administration of simvastatin after kainic acid-induced status epilepticus restrains chronic temporal lobe epilepsy. PLoS One 2011;6:e24966. [44] Gouveia TL, Scorza FA, Iha HA, Frangiotti MI, Perosa SR, Cavalheiro EA, et al. Lovastatin decreases the synthesis of inflammatory mediators during epileptogenesis in the hippocampus of rats submitted to pilocarpine-induced epilepsy. Epilepsy Behav 2014;36:68–73. [45] van Vliet EA, Holtman L, Aronica E, Schmitz LJ, Wadman WJ, Gorter JA. Atorvastatin treatment during epileptogenesis in a rat model for temporal lobe epilepsy. Epilepsia 2011;52:1319–30.

C.V. Oliveira et al. / Epilepsy & Behavior 78 (2018) 109–117 [46] Scorza CA, Marques MJ, Gomes da Silva S, Naffah-Mazzacoratti MD, Scorza FA, Cavalheiro EA. Status epilepticus does not induce acute brain inflammatory response in the Amazon rodent Proechimys, an animal model resistant to epileptogenesis. Neurosci Lett. 2017;S0304-3940(17):30165–9. https://doi.org/10. 1016/j.neulet.2017.02.049. [47] Gouveia TL, Vieira de Sousa PV, de Almeida SS, Nejm MB, Vieira de Brito JM, Cysneiros RM, et al. High serum levels of proinflammatory markers during epileptogenesis. Can omega-3 fatty acid administration reduce this process? Epilepsy Behav 2015;51:300–5. [48] Lappegard KT, Pop-Purceleanu M, van Heerde W, Sexton J, Tendolkar I, Pop G. Improved neurocognitive functions correlate with reduced inflammatory burden in

[49] [50]

[51] [52]

117

atrial fibrillation patients treated with intensive cholesterol lowering therapy. J Neuroinflammation 2013;10:78. Pitkänen A, Huusko N, Ndode-Ekane XE, Kyyriäinen J, Lipponen A, Lipsanen A, et al. Gender issues in antiepileptogenic treatments. Neurobiol Dis 2014;72:224–32 Pt B. Bruce-Keller AJ, Dimayuga FO, Reed JL, Wang C, Angers R, Wilson ME, et al. Gender and estrogen manipulation do not affect traumatic brain injury in mice. J Neurotrauma 2007;24:203–15. Reddy DS. The neuroendocrine basis of sex differences in epilepsy. Pharmacol Biochem Behav 2017;152:97–104. Luef G, Tauboll E. Gender issues in epilepsy—difference in management of epilepsy. Seizure 2015;28:1–2.