Epilepsy & Behavior 20 (2011) 607–612
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Epilepsy & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ye b e h
Melatonin administration after pilocarpine-induced status epilepticus: A new way to prevent or attenuate postlesion epilepsy? Eliângela Lima a, Francisco R. Cabral a,c, Esper A. Cavalheiro a, Maria da Graça Naffah-Mazzacoratti b, Débora Amado a,⁎ a b c
Disciplina de Neurologia Experimental, Escola Paulista de Medicina, Universidade Federal de São Paulo, SãoPaulo, Brazil Disciplina de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil Instituto do Cérebro, Hospital Israelita Albert Einstein, SãoPaulo, Brazil
a r t i c l e
i n f o
Article history: Received 25 August 2010 Revised 11 January 2011 Accepted 12 January 2011 Available online 30 March 2011 Keywords: Epilepsy Melatonin N-acetylserotonin Status epilepticus Neuroprotection Experimental model Pilocarpine
a b s t r a c t Objective: The goal of this study was to verify the effects of treatment with melatonin and N-acetylserotonin on the pilocarpine-induced epilepsy model. Methods: The animals were divided into four groups: (1) animals treated with saline (Saline); (2) animals that received pilocarpine and exhibited SE (SE); (3) animals that exhibited SE and were treated with N-acetylserotonin (30 minutes and 1, 2, 4, 6, 12, 24, 36, and 48 hours) after SE onset (SE + NAS); (4) animals that exhibited SE and were treated with melatonin at the same time the SE + NAS group (SE + MEL). Behavioral (latency to first seizure, frequency of seizures, and mortality) and histological (Nissl and neo-Timm) parameters were analyzed. Results: The animals treated with melatonin (SE + MEL) had a decreased number of spontaneous seizures during the chronic period (P b 0.05), a reduction in mossy fiber sprouting, and less cell damage than the SE group. Animals treated with N-acetylserotonin did not exhibit any kind of significant change. Conclusion: Melatonin exerts an important neuroprotective effect by attenuating SE-induced postlesion and promoting a decrease in the number of seizures in epileptic rats. This suggests, for the first time, that melatonin could be used co-therapeutically in treatment of patients exhibiting SE to minimize associated injuries in these situations. © 2011 Published by Elsevier Inc.
1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) is the main hormone synthesized and released by the pineal gland when stimulated by a lack of light. It has been linked to the regulation of many biological functions including sleep, circadian rhythm, mood, sex maturation, and immune responses [1]. In addition, melatonin has been considered a neuroprotective molecule because of its antioxidant and anti-inflammatory effects [2–4] and its inhibitory effects on the central nervous system [5]. In the same context, melatonin aside, several related pineal constituents also possess antioxidant and anti-inflammatory properties, indicating neuroprotection [6,7]. One of these is N-acetylserotonin (NAS), the melatonin precursor [8,9]. Although the antioxidant and anti-inflammatory capacity of NAS implies that it may slow neuroprotective effects, more studies are necessary [4,10]. On the basis of
⁎ Corresponding author at: Disciplina de Neurologia Experimental, Rua Botucatu, UNIFESP–EPM, 862-Ed. Leal Prado, SãoPaulo-SP, Brasil, CEP: 04023–900. Fax: + 55 11 55494743. E-mail address:
[email protected] (D. Amado). 1525-5050/$ – see front matter © 2011 Published by Elsevier Inc. doi:10.1016/j.yebeh.2011.01.018
these properties, several studies show a correlation between epilepsy and melatonin. Epilepsy is the most frequent neurodegenerative disease after stroke [11]. It is defined as “a disorder of the brain characterized by a lasting predisposition to epileptic seizures and by the neurobiologic, cognitive, psychological, and social consequences of this condition” [12]. In this context, the use of neuroprotective molecules to prevent this kind of condition is very important. Peled et al. [13] showed that melatonin, in association with an antiepileptic drug (AED), can decrease the frequency of seizures in children with severe and intractable mixed-type seizures, especially those with tonic–clonic characteristics. In addition, Gupta et al. [14] observed that incorporation of melatonin into the treatment of children with epilepsy on valproate monotherapy improved their quality of life (QOL) when compared with the placebo group. This result can be attributed to several properties of melatonin, such as its antioxidant properties, free radical-sharing behavior, and favorable effects on sleep. In addition, compared with the control group, relatively lower levels of salivary melatonin were measured during the interictal period in patients with temporal lobe epilepsy. In contrast, high levels of salivary melatonin were observed during the 24-hour period after a seizure in these patients [15]. Furthermore,
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melatonin has been described as acting as an anticonvulsant in chemically [16–18] and electrically [19,20] induced seizures, in experimental models. In vitro data have shown that melatonin is able to protect neurons from excitotoxicity mediated by kainatesensitive glutamate receptors and from oxidative stress-induced DNA damage and apoptosis [21]. In vivo, this hormone has been considered neuroprotective against excitotoxicity induced with kainate when administered in pharmacological doses [22]. Temporal lobe epilepsy (TLE) is among the most frequent types of drug-resistant epilepsy [23,24] and is considered to be a relevant medical and social issue. Our team's recent data, using the pilocarpine model of epilepsy, show that prior removal of the pineal gland (pinealectomy) promotes increased neuronal excitability by facilitating the epileptogenic process. In addition, we also found that this facilitation of epileptogenesis can be reversed by subsequent administration of melatonin [25]. In this context, animals without a pineal gland are more susceptible to amygdaline and cortical inflammation, requiring a decreased number of stimuli before attaining stage 5 according to Racine´s scale [26,27]. This suggests that melatonin has endogenous neuroprotective properties [20]. Experimental models of limbic status epilepticus have provided insight into the possible mechanisms involved in epileptogenesis. As it is understood today, prolonged seizures can cause brain injury at both early and late stages of development [28,29]. Sloviter (1994) [30] has previously suggested and has since reiterated (2009) [31] the hypothesis that acquired epilepsy is an immediate network defect caused primarily by the initial loss of neurons. We observed in a previous study that animals treated with melatonin just before and 2 hours continuously after SE onset exhibited an increased latency to SE, a survival rate of 100% after SE and a decreased number of seizures in the chronic period. The animals treated with N-acetylserotonin also exhibited an increased latency to SE. Neither the MEL + SE nor NAS + SE group showed any changes in latency to the first seizure, which marks the beginning of the chronic period. Another interesting point to be considered is that the animals treated with melatonin exhibited neuronal preservation in the hilus, CA1, and CA3 subfields of the hippocampus in association with a minor grade of mossy fiber sprouting when compared with the other groups [32]. However, to simulate what happens in clinical situations, we need to analyze the potential effect of melatonin after the onset of status epilepticus, not before. Patients arrive at the emergency room with SE and we need to prevent this condition as soon as possible as SE causes lesions that could be responsible for intractable epilepsy. Thus, the aim of this study was to verify the effects of melatonin and N-acetylserotonin administration after onset of status epilepticus and determine if its neuroprotective activity attenuates the consequences of the pilocarpine-induced epilepsy model induced in male rats with respect to behavior and morphology.
2. Methods 2.1. Animals All experimental protocols were approved by the ethics committee of the Universidade Federal de São Paulo (UNIFESP) and all efforts were made to minimize animal suffering following the proposal of the International Ethical Guideline for Biomedical Research (CIOMS/OMS, 1985) [33]. Wistar adult male rats, 200–250 g, housed under environmentally controlled conditions on a 12/12-hour light/dark cycle with free access to food and water were used. These animals were divided in four groups: control animals (Saline group, n = 5); animals that received pilocarpine and exhibited SE (SE group, n = 19); animals that received N-acetylserotonin (2.5 mg/kg ip) 30 minutes and 1, 2, 4, 6, 12, 24, 36, and 48 hours after onset of SE (SE + NAS group, n = 10); and animals that received melatonin (2.5 mg/kg ip)
30 minutes and 1, 2, 4, 6, 12, 24, 36, and 48 hours after onset of SE (SE + MEL onset, n = 10). 2.2. Pilocarpine administration These animals received a systemic injection of pilocarpine HCl (350 mg/kg ip, Merck S.A.) or saline instead of pilocarpine (control group). To prevent peripheral cholinergic effects, scopolamine methylnitrate (1 mg/kg sc, Sigma Co., St. Louis, MO, USA) was injected 30 minutes before pilocarpine or saline. 2.3. Behavioral analysis Behavioral parameters such as latency to the first spontaneous seizure (duration of latent period), seizure frequency in the chronic phase, and mortality during all periods were analyzed. After SE onset, the animals were continuously monitored via video surveillance (Stellate system) to observe the duration of the latent period and the occurrence of the first spontaneous seizure inaugurating the chronic period. These animals were observed 24 hours/day for 60 days after the first seizure to verify seizure frequency. 2.4. Melatonin or N-acetylserotonin administration Melatonin or N-acetylserotonin (2.5 mg/kg ip, Sigma Co.) was initially dissolved in 100% ethanol followed by dilution in saline (final ethanol 5%) and administered as previously described. 2.5. Morphological analysis: Neo-Timm and Nissl staining Sixty days after the first spontaneous seizure, animals were euthanized with a lethal dose of sodium pentobarbital and transcardially perfused with 0.1% sodium sulfide fixative in 0.12 M Millonig's buffer with 0.002% CaCl2 (250 mL per animal) and 3% glutaraldehyde in phosphate buffer 0.2 M (100 mL per animal). Brains were immediately removed and immersed in 30% sucrose and 2% glutaraldehyde solution overnight. Coronal frozen sections (40 μm thick) were then mounted on slides and processed for Neo-Timm staining. For this, a solution containing 240 mL of 50% arabic gum with 10.25 g of citric acid, 9.45 g sodium citrate in 30 mL of H2O, 3.73 g hydroquinone in 60 mL of H2O, and a 2-mL volume of 0.51 g silver nitrate with 3 mL H2O was used. The slides were washed in distilled water twice for 5 minutes, dehydrated through alcohol to xylene, and coverslipped with Canada balsam. The distribution of Timm granules in the supragranular layer (SGL) of the dentate gyrus was blindly analyzed by three observers, using a scale of 0– 5 and the scoring system previously described by Cavazos [34], where a (mean) score of 0 = absence of Timm granules in the supragranular layer; 1 = occasional granules or sparse distribution in the SGL; 2 = several granules not continuously distributed in the SGL; 3 = Timm granules almost continuously distributed in the SGL; 4 = dense laminar band of granules almost continuous in the SGL; and 5 = a dense band continuously distributed in the SGL of the dentate gyrus. Altogether, 12 sections from each animal (5 animals per group) were assessed across different levels (septum–temporal axis) of the hippocampus, bilaterally, by three observers blind to the experimental condition. Adjacent sections were stained with Nissl. In Nissl-stained sections, we analyzed the hilus, CA1, and CA3 areas of both hippocampi and three adjacent coronal sections per animal were taken at the following stereotaxic coordinates (−3.3/–3.8 mm from Bregma), and five animals per group were analyzed. Cell density, evaluated by Nissl staining, was measured under 400× magnification on a Zeiss Axiolab optical microscope, and representative images of the slices were digitalized using the NIH Image 1.61 system. Cells were counted by three observers blinded to the conditions, and the results were expressed as means±SD.
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2.6. Statistical analysis Fischer's test was used to compare mortality with appropriate controls. The latency to occurrence of the first spontaneous seizure (duration of silent period in days), seizure frequency in the chronic period, and counts of Nissl-stained cells were evaluated using ANOVA followed by a Tukey post hoc test for comparison of all groups. A P value b0.05 was accepted as significant in all cases. Values were expressed as mean ± SD. Spearman's correlation coefficient was used to determine the relationship between seizure frequency and surviving cells. 3. Results The animals that received pilocarpine exhibited a sequence of behavioral events. Five to ten minutes following the systemic administration of pilocarpine (350 mg/kg), the animals showed stereotypical oral and masticatory movements, hypokinesia, head
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bobbing, and wet-dog shakes. This initial behavior rapidly progressed, evolving into generalized limbic seizures 20–30 minutes after pilocarpine injection. These seizures recurred every 1–3 minutes culminating in status epilepticus SE, and 30 minutes after onset of SE, melatonin or N-acetylserotonin was administered. All animals had the same duration of SE. We blocked SE 4 hours 30 minutes after its onset with diazepam (1 mg/kg). With respect to behavioral parameters, the latency in days to the first spontaneous seizure did not statistically differ among the groups. On the other hand, animals treated with melatonin (SE + MEL group) exhibited a decrease in spontaneous seizure frequency (1.28 ± 0.43/ week) when compared with the SE group (2.53 ± 0.57/week) in the chronic period (P b 0.01). We did not observe any differences in the animals treated with N-acetylserotonin (SE + NAS) that exhibited a spontaneous seizure frequency of 3.3 ± 2.12/week. The F value was 8.141. Hilus, CA1, and CA3 normal-looking neurons in the hippocampal formation were counted under a light microscope after Nissl staining.
Fig. 1. Photomicrographs of Nissl-stained coronal sections of the dorsal hippocampus. (A) Saline group. (B) Animals that received pilocarpine and exhibited SE. (C) Animals that exhibited SE and were treated with N-acetylserotonin. (D) Animals that exhibited SE and were treated with melatonin. Magnification = 400×, scale bar = 260 μm. The arrow indicates the neuronal preservation observed in the CA3 region.
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Table 1 Number of remaining neurons per area in the hippocampus.
Table 3 A. Variance grade within each group.
Region Hilus CA1 CA3
SAL (n = 5) 86.0 ± 16.4 143.8 ± 16.0 48.0 ± 9.6
SE (n = 6) 84.5 ± 17.0 119.8 ± 10.4a 31.0 ± 3.4b
SE + NAS (n = 5) 71.8 ± 6.0 100.0 ± 12.4 38.8 ± 4.8
SE + MEL (n = 5) 89.0 ± 6.6 102.4 ± 8.6 51.2 ± 8.9b
Note. Data are expressed as means ± SD. The MEL + SE and NAS + SE groups were compared with the SE group, and the SE group was compared with the SAL group, using ANOVA followed by Tukey test. a P b 0.05. b P b 0.01.
Animal
1 2 3 4 5 Median SD
Grade Saline group
SE group
SE + NAS group
SE + MEL group
0 0 0 0 0 0
5 4 4 3 4 4 0.7
4 3 4 4 4 3.8 0.44
2 3 3 4 3 3 0.7
B. Grade of mossy fiber sprouting (n = 5/group)
Table 2 Correlation between seizure frequency and number of surviving cells in the SE and SE + MEL groups. Region
Correlation coefficient
P
Hilus CA1 CA3
0.235 0.738 –0.846
0.51 0.015 0.002
Note. Statistical test: Spearman's correlation.
The animals treated with melatonin had an increased number of cells in the CA3 region (51.2 ± 8.9 per area, F = 8.941, P = 0.01) when compared with the animals that received only pilocarpine. The animals treated with N-acetylserotonin did not show any differences compared with the SE group (Fig. 1, Table 1). Spearman correlation coefficients between seizure frequency and Nissl staining showed a strong correlation between seizure frequency
Group
Grade
Saline SE SE + NAS SE + MEL
0 4 4 3
and number of surviving cells in the CA3 region (− 0.846, P = 0.002), an average correlation between seizure frequency and number of surviving cells in the CA1 region (0.738, P = 0.015), and a weak correlation between seizure frequency and number of surviving cells in the hilus (0.235, P = 0.51). The results are summarized in Table 2. Analysis of neo-Timm-stained sections showed different patterns for each group according to a scale developed by Cavazos et al. [34]. Neo-Timm-stained sections obtained from animals treated with melatonin after SE were grade 3, in contrast to those of animals that were treated only with pilocarpine and exhibited SE and animals that were treated with N-acetylserotonin after SE, which were grade 4. Sections from saline-treated animals were grade 0; that is, mossy fiber
Fig. 2. Photomicrographs of neo-Timm-stained mossy fibers of the dorsal hippocampus. (A) Saline group. (B) Animals that received pilocarpine and exhibited SE. (C) Animals that exhibited SE and were treated with melatonin. (D) Animals that exhibited SE and were treated with N-acetylserotonin. Magnification = 200×, bar = 150 μm. Note the supragranular mossy fiber sprouting (arrow).
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sprouting could not be detected in animals that were not treated with systemic pilocarpine. Grades of neo-Timm-stained cells were expressed as means ± SD, and are provided in Fig. 2 and Table 3.
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Acknowledgments The authors thank FAPESP, CAPES, CNPq, CInAPCe, and FAPESP/ CNPq/MCT—Instituto Nacional de Neurociência Tranlacional for supporting this study.
4. Discussion Our results indicate that the animals that received pilocarpine, developed SE, and were treated with melatonin during SE had a decreased number of seizures in the chronic period when compared with the SE group (not treated with melatonin or NAS). In contrast, we did not observe any difference in the animals that received pilocarpine, developed SE, and were treated with N-acetylserotonin. In addition, no changes in mortality rate were observed in the animals of the SE + MEL and SE + NAS groups when compared with the SE group. Another interesting point to be considered is that the animals treated with melatonin exhibited neuronal preservation in the CA3 subfield of the hippocampus associated with a minor grade of mossy fiber sprouting when compared with the other groups. In light of these data and considering the process of neuronal death and mossy fiber sprouting, which are markers of mesial temporal sclerosis, these results indicate an important role for melatonin in the epileptogenic process as well as in the control of seizures. Moreover, these results could have been due to the anticonvulsant effect of melatonin. There exists the possibility that melatonin diminishes subconvulsive seizures induced by pilocarpine, and therefore, its main action would be anticonvulsant. However, there was no difference in the severity or intensity of convulsive status epilepticus among the groups, suggesting that the effect of melatonin was antiepileptogenic. In this context, although we did not observe significant statistical alterations in the animals treated with N-acetylserotonin with respect to morphology and behavior, in the literature there are many studies demonstrating the anti-inflammatory and antioxidant properties of N-acetylserotonin [4,10]. Other studies have demonstrated the anticonvulsant effects of melatonin on different convulsive stimuli, including seizures induced with pentylenetretazole [10,16,17,35], quinolate, kainate, glutamate, N-methyl-D-aspartate [17], L-cysteine, cyanide [18], maximal electroshock [36], and electrically induced stimulation of the amygdala [19]. Our results clearly indicate that melatonin interferes with the natural course of epileptogenesis in the pilocarpine model of epilepsy. Our data indicate that melatonin has an important role in diminishing the excitotoxity promoted by SE, probably because the melatonin could be decreasing the inflammation and oxidative stress induced by SE. Along these lines, Chung and Han [37] suggested that melatonin is a potentially useful hormone in the treatment of acute brain pathologies associated with oxidative stress induced by neuronal damage such as epilepsy, stroke, and traumatic brain injury. In general, the important role of melatonin as a neuroprotective agent has been observed in several pathologies. Status epilepticus is a life-threatening and poorly understood neurological condition with potentially devastating consequences [38] and several neuropathological studies have implied that it causes brain damage. This is a major problem both medically and socially. Although most seizures do not require emergency medical treatment, a patient with a prolonged seizure lasting more than 5 minutes may be in status epilepticus and should be taken to an emergency room immediately. It is important to treat a person with SE as soon as possible. Our results suggest that treatment with melatonin after the onset of SE may be an important way to decrease excitability. We believe that melatonin could be used as a co-adjuvant in the treatment of SE, reducing the inflammation and excitotoxicity induced by SE, preventing or even minimizing the possible development of temporal lobe epilepsy. Nevertheless, other molecular studies are needed in this area to gain a full understanding of the mechanisms underlying melatonin's action during epileptogenesis and status epilepticus.
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