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Estrogen effects on pilocarpine-induced temporal lobe epilepsy in rats
Maturitas 62 (2009) 190–196
Contents lists available at ScienceDirect
Maturitas journal homepage: www.elsevier.com/locate/maturitas
Estrogen effects on pilocarpine-induced temporal lobe epilepsy in rats夽 Melquíades Pereira Jr. a , José M. Soares Jr. a,∗ , Sandra G. Valente b , Patricia B. Oliveira a , Esper A. Cavalheiro b , Débora Amado b , Edmund C. Baracat a,c a
Departamento de Ginecologia, Escola Paulista de Medicina/Universidade Federal de São Paulo, Rua Sena Madureira 1245 apt 11, 04021-051 São Paulo, SP, Brazil Disciplina de Neurologia Experimental, Escola Paulista de Medicina/Universidade Federal de São Paulo, São Paulo, SP, Brazil c Departamento de Obstetrícia e Ginecologia da Universidade de São Paulo, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 11 August 2008 Received in revised form 24 October 2008 Accepted 30 October 2008 Keywords: Experimental models Epilepsy Pilocarpine Menopause Estrogen
a b s t r a c t Objetive: To evaluate the effects of conjugated equine estrogens (CEE) on the pilocarpine-induced epilepsy in rats. Study design: 40 female rats were divided into: GPC (positive control) presented “status epilepticus” (SE) induced by pilocarpine; GOC (ovariectomized control) only castrated; GNC (negative control) received only saline solution; GPE received pilocarpine, presented SE, castrated and received 50 g/kg CEE treatment; GPV received pilocarpine, castrated and received propylene glycol (vehicle). The animals were monitored by a video system. At the end of observation, the brains removed for later histologic analysis using NeoTimm and Nissl methods. Results: The GPE presented a reduction in number of seizures compared to GPV. The Neo-Timm analysis showed that GPV had greater sprouting of mossy fibers, with a denser band in the area of the dentate gyrus hilum compared to GPE. On Nissl staining, GPE showed evident neuronal loss in the CA3 area. GPV presented loss in CA1 and dentate gyrus. Conclusion: Estrogen may have a protecting effect on the central nervous system. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Postmenopausal women present deleterious phenomena related to the hypoestrogenism status [1]. Innumerable studies demonstrated the benefits of hormone therapy in these women, resulting in their better quality of life and improving symptomatology In addition, estrogen seems to have an effect on cognitive function [2]. Hormone privation may interfere with the course of epilepsy in postmenopausal women [3]. The course of epilepsy in women at the beginning of menopause is still unknown, the pattern of epileptic seizures being liable to decrease or increase, as related to the presence or not of hormonal deficiency. Steroid hormones interfere with neuronal excitability through specific mechanisms which change cell metabolism and nerve transmission [4,5]. Endocrine and reproductive alterations are frequently reported in women with temporal lobe epilepsy and studies show that gonadal steroids modify dendritic spine density in hippocampal pyramidal cells
夽 Sources of financial support: FAPESP, São Paulo, Brazil CNPq; CINAPCE; CAPESBrasilia, Brazil. ∗ Corresponding author. Tel.: +55 11 50813685; fax: +55 11 50813685. E-mail address: [email protected] (J.M. Soares Jr.). 0378-5122/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2008.10.014
and in neurons of the hypothalamic ventromedial region in adult rats, suggesting an afferent synaptic modulation for these cells [6–8]. Some studies demonstrated the existence of neurons containing nuclear receptors for estrogen in certain areas of the brain, predominantly in the hypophysis, hypothalamus, limbic system and cerebral cortex. Clinical and experimental studies showed that ovarian steroids could alter susceptibility to epileptic activity, with estradiol decreasing the threshold of convulsive seizures, an effect that would be reversed by progesterone [9–14]. This symptomatology may be observed in women with anovulatory cycles as polycystic ovary syndrome. Some studies show that 50% of epileptic women have associated chronic anovulation [15]. However, the actual estrogen action on the course of epilepsy is still unknown. Conjugated equine estrogens (CEE) are commonly used in postmenopausal hormone replacement therapy. These hormones are a combination of soluble estrogens from the urine of pregnant mares, and they include several biologically active components such as estrone, estradiol, and its respective sulphates, and sulphates of equine estrogens (equilin and equilenin). In a previous study with rats we showed that, following treatment with 50 g/kg of CEE, estrogen levels are close to 40 pg/ml [16]. These values are similar to those found in postmenopausal women receiving estrogen therapy [17].
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were confined at random in appropriate metal cages with water and food ad libitum, and maintained at a temperature of 22 ◦ C under a 12-h light-dark cycle. The IRB of Universidade Federal de Sao Paulo – Escola Paulista de Medicina approved this study and the animals were carried according to the Standard Animal Manual Care of the same institution. 2.2. Pilocarpine model
Fig. 1. Number of epileptic seizures in GPC, GPE, GPV during the treatment period. *p < 0.05 compared to GPC and GPE.
In order to evaluate the influence of estrogen on epilepsy evolution in castrated rats, the pilocarpine-induced epilepsy model was used. The cholinergic muscarinic agonist is administrated intraperitoneally reproducing the main characteristics of temporal lobe epilepsy in humans [18]. Thus, the behavior and frequency of seizures were observed, as well neuronal alterations (by light microscopy) of the hippocampus of adult castrated rats, with pharmacologically induced epilepsy using pilocarpine and treated with estrogen. Therefore this model allows the evaluation of estrogen effects on epilepsy. 2. Material and methods 2.1. Animals Eighty virgin and intact, approximately 120-day-old albino Wistar-EPM1 rats weighing about 200 g were used. The animals
To prevent peripheral cholinergic pilocarpine effects, subcutaneous methylscopolamine (Sigma, 1 mg/kg) was previously administered to the animals; 30 min thereafter 4% pilocarpine was intraperitoneal administered to 64 animals at a dose of 350 mg/kg for induction of “status epilepticus”. All animals that survived to SE and presented spontaneous seizures after the silent period were considered to made epilepsy (chronic period, [21,22]). During pilocapine administration, 30 animals were lost due to tonic seizures leading to instantaneous death. 2.3. Negative control 16 animals received only physiological saline (0.9%) (vehicle) and constituted the control group. No deaths occurred in the animals receiving only physiological saline. 2.4. Seizure control After approximately thirty days the animals were placed in cylindrical cones, maintained under controlled conditions with a 12/12 h light/dark cycle, 21 ◦ C temperature, with water and food ad libitum and monitored by filming during 24 h/day using a videoEEG system. Seizures were accompanied during 30 days. In this phase, another five animals were lost. At the end of the experiment
Fig. 2. Photomicrograph of coronal brain section, at the level of the hippocampus, stained by Nissl technique. (A) Animals submitted to pilocarpine; (B) castrated animals; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (25×).
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Fig. 3. Photomicrograph of coronal brain section, at the level the dentate gyrus, stained by Nissl technique. (A) Animals submitted to pilocarpine; (B) animals only castrated; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (100×).
29 rats with epilepsy remained. After this period, surgical castration was performed.
2.5. Epilepsy control group A group of eight animals was randomly separated from the 29 epileptic rats, constituting the epilepsy control (with ovaries).
2.6. Castration procedure 21 animals were submitted to anesthesia and their abdomen was shaved; they were submitted to sterile preparation, and opened with a 1.5 cm lower midline incision. After that bilateral ovariectomy (castration) was performed. The procedure was carried out under anesthesia with sodium pentobarbital, at a 60 mg/kg intraperitoneal dose. During this procedure, five animals of the epileptic group were lost due to complications after surgical procedure. Also, half of negative control (n = 8) was ovariectomized.
2.8. Groups of animals The 40 animals were classified into five groups of 8 animals each, following previous procedures: GPC: rats which received only pilocarpine and entered “status epilepticus”—positive control. GOC: rats which received physiological saline (0.9%) and were castrated—negative control. GNC: non-castrated rats which received physiological saline (0.9%)—negative control. GPE (PILO/CT/EC): rats which received pilocarpine, entered “status epilepticus”, became epileptic, were castrated (CT) and received conjugated equine estrogens at a dose of 50 g/animal/day [19] by gavage. GPV (PILO/CT/vehicle): rats which received pilocarpine, entered “status epilepticus”, became epileptic, were castrated (CT), and received propylene glycol vehicle by gavage. 2.9. Control of seizures during the treatment
2.7. Treatment groups In all castrated animals a colpocytologic examination was performed to confirm genital atrophy. After confirming castration, 16 castrated animals were randomly divided into groups: one with eight rats that received only vehicle and another with eight animals that received estrogen [19].
All animals of GPE and GPV were placed in the individual cylindrical cones and were submitted to 24 h/day filming. After four days, administration of drugs started (conjugated equine estrogens and propylene glycol) by gavage using a metal probe, for thirty consecutive days. At the end of 60 days of observation, the animals were perfused and the brains removed for later histologic analysis using Neo-Timm and Nissl methods.
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Fig. 4. Photomicrograph of coronal brain section, at the level of the hippocampus stained by Neo-Timm technique. (A) Animals submitted to pilocarpine; (B) animals only castrated; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (25×).
2.10. Qualitative histological analysis
3. Results
After histological routine, the tissue sections stained with NeoTimm were classified according to the scale by Tauck and Nadler [20] into:
3.1. Effects of pilocarpine on the female rats
Grade 0: no or occasional Neo-Timm staining of mossy fibers; Grade 1: sparse staining over all parts of the granular layer; Grade 2: isolated, discontinuous intensely stained mossy fibers or a continuous band of supragranular staining of intermediate intensity, tween classification degrees 1 and 3; Grade 3: with a dense and a continuous stained band of supragranular mossy fibers. Nissl stain was used to evidence neuronal cytoplasm, as an indicator of neuronal viability since these corpuscles may disappear if neuronal injury occurs. This phenomenon is known as “chromatolysis”.
2.11. Statistical analysis The following parameters were analyzed by “t” Student test for parametric analysis to compare before and after treatment: Frequency and time of seizures. Analysis of variance (ANOVA) test and Tukey and Kremer tests were performed to compare the differences among the treatment groups and controls. A p-value < 0.05 was considered to be significant and the results are presented as the mean ± S.E.
All animals submitted to pilocarpine (GPC, GPE and GPV) presented salivation, tremors and repetitive movements progressing to motor limbic seizures and then presenting clonus of anterior paws supported by the posterior paws and finally falling. These seizures were recurrent, culminating with the animal’s death or “status epilepticus”. After a period without seizures (silent phase), the surviving animals presented spontaneous and recurrent seizures, characterizing the chronic period which continued throughout their whole life [21–23]. GPC, GPE and GPV animals did not present any behavioral alteration different from that expected in the chronic phase of the pilocarpine model. GOC and GNC animals were not submitted to pilocarpine; therefore they did not present any behavioral alteration (seizures). Of the animals which received pilocarpine, 30 died after the first clonic seizures (46.8 ± 24). Of the animals surviving after the first seizures and which remained in “status epilepticus” after 30 days, 5 died (19.5 ± 20.5). The mean latency time until the first convulsive seizure in the animals was 29.55 ± 17.11 min. Latency until “status epilepticus” was 55.93 ± 23.66 min. Mean time interval for establishment of the chronic period was 50 ± 10.36 days. 3.2. Estrogen therapy effect Frequency of seizures in the chronic phase before and after castration GPC animals (PILO/control) did not present any change in their seizure pattern (17.79 ± 3.12 seizures/month). GOC
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Fig. 5. Photomicrograph of coronal brain section at the level of the dentate gyrus stained by the Neo-Timm technique. (A) Animals submitted to pilocarpine; (B) animals only castrated; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (100×).
(CT/control) and GNC animals (negative control) did not present seizures. GPE animals (PILO/CT/EC) showed a significant reduction in seizure number after castration and estrogen replacement (21.62 ± 2.53 seizures/month) when compared to baseline (before castration, 33.51 ± 3.14 seizures/month, p < 0.01). GPV animals (PILO/CT/PG) presented a significant increase in seizure number after castration and propylene glycol administration (32.37 ± 2.94 seizures/month) when compared to baseline (18.36 ± 3.54 seizures/month, p < 0.01). When compared the GPC, GPE and GPV (after estrogen treatment), the highest number of seizures was detected in GPV (p < 0.05) compared to GPC and GPE (Fig. 1).
fibers in the dentate gyrus hilum. GOC (CT/control) and GNC (control): grade 0 staining with no or only an occasional staining of mossy fibers. GPE (PILO/CT/EC): grade 2 staining, with sparse staining over all parts of the granular layer. GPV (PILO/CT/PG): grade 3 staining with a dense and continuous band of intense staining of supragranular mossy fibers. Based on these results, an intense sprouting of mossy fibers was observed in the animals which received pilocarpine, were castrated and did not receive estrogen replacement (GEV), while sprouting was less in epileptic non-castrated animals (GPC) and in those with estrogen replacement (GPE). In the castrated only animals (GOC) and in the controls (GNC) a sparse marking of mossy fibers was observed.
3.3. Predominant time of seizures
4.2. Nissl staining
Regarding the predominant time of seizures during the day, there were no significant differences among the groups (GPC, GPE and GPV). The animals, in general, presented more seizures during the light period (7:00–18:59 h) than in the dark (19:00–6:59) (p < 0.24). 4. Histological analysis
GPC and GPV histologic sections showed neuronal loss in the CA3, CA1 and dentate gyrus pyramidal layer, with breakdown of the structure of cell architecture of the hippocampus. In GOC and GNC animals, the sections did not present morphologic alterations. In GPE animals, the sections showed a greater neuronal loss in the CA3 layer and breakdown of the structure of the hippocampal cell architecture.
4.1. Neo-Timm staining
5. Discussion
Intensity of Neo-Timm staining of brain sections, in the dentate gyrus was visually assessed using the morphologic grading scale described by Tauck and Nadler [20]. Different patterns were found between the five animal groups (Figs. 2–5): GPC (PILO/control): grade 2 staining with a continuous band of supragranular mossy
It is known that estrogens act on the central nervous system by mechanisms which include a greater ability of neuronal repair, increase in the number of dendritic terminals, reduction in oxidative stress and, due to vascular effect, they promote a greater blood flow [23]. Development of experimental models in animals which
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mimic human epilepsy was of fundamental importance in the elucidation and study of epileptic phenomena. The pilocarpine-induced epilepsy model has been widely studied, both in males [22] and females [24] (reason for using it in our study). Based on these facts, the animals were castrated, provoking a hypoestrogenic state and estrogen replacement was used to evaluate its effect on epilepsy. Our results suggested that estrogen treatment may decrease the seizures in our animal model. During the preparation of the animals, the observed mortality rate after administration of pilocarpine (mortality after the first tonic seizure and after “status epilepticus”), latency to first seizure, “status epilepticus”, and the chronic period were similar to those described in the literature [25]. The mortality of rats is a limitation of this model. On analyzing the effect of estrogen replacement, it was observed that frequency of seizures among animals receiving estrogen replacement was lower than that of the animals receiving the vehicle. Such result shows that somehow estrogen would act as a moderator of susceptibility to seizures. The strength of this study is objective form to analyze the estrogen treatment on seizures and histological findings that may support our results. Thus, it was observed that estrogen seems to have a protecting effect on susceptibility to seizures induced by pilocarpine. However, some authors report that estrogen would act as seizure promoter while others relate that its absence would not offer protection against them. Also, Valente et al. [26] found a higher mortality rate in castrated animals which received pilocarpine, suggesting that female steroid hormones would protect the brain against cell death induced by “status epilepticus”. The authors demonstrated also that castrated females which received pilocarpine presented short latency period to first seizure, showing that estrogen has a protecting effect on susceptibility to pilocarpine-induced seizures. Our study showed that estrogen treatment may decrease the number of seizures in ovariectomized after pilocarpine-induced epileptic stabilized. This point was not evaluate in Valente et al. [26]. It can also be observed that seizure frequency was higher in the diurnal than in the nocturnal period. These data confirm the observations by Arida et al. [27] who observed that the animals have a higher frequency of seizures in the diurnal period (7:00–18:59 h) than in the nocturnal (19:00–6:59 h). The estrogen treatment may not change this pattern. Brains of the animals used in the study were submitted to Nissl stain for localization of the region where neuronal death could have occurred. The pattern found in epileptic rats and mice is of intense neuronal loss in the pyramidal layers of the CA1 and CA3 regions and in the granular layer of the hilum, areas of glial cell proliferation and breakdown of hippocampal cell architecture [28]. However, analyzing the staining pattern of GPE animals (estrogen treatment), the sections presented neuronal loss in the CA3 layer and breakdown of structure of hippocampal cell architecture, while in GPV animals (without estrogen), besides breakdown of structure and loss in CA3, there was also loss in CA1 and dentate gyrus. This result may suggest that estrogen may induce less brain damage than vehicle in castrated rats. Neuronal loss pattern in humans with epilepsy is frequently associated with supragranular mossy fiber sprouting. These fibers, displaying a great zinc concentration, were identified bye NeoTimm staining, thus making identification of axonal reorganization found in epileptic tissues easy. Consistent increase in the projections of the supragranular layer of the dentate gyrus has been identified by this staining after induction of pilocarpine-induced epileptic seizures [29]. Regarding the studied animals it was observed that GPE presented positive grade 2 mark, while GPV had positive grade 3 mark. Thus it was observed that in animals not treated with estrogen there was intense mossy fiber sprouting, which would indicate that estrogen plays a fundamental role in
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epileptogenesis. The anomalous synaptic reorganization, characterized by mossy fiber innervation, would thus form a monosynaptic excitatory feedback for dendrites of the internal molecular layer of the dentate gyrus [30]. Based on these results, it is suggested that estrogen replacement would avoid more neuronal cell architecture damage in the hippocampus. Future studies along this research line could produce more subsidies in order to better understand the role of estrogen in the central nervous system, mainly in cell signaling and gene action.
References [1] Galhardo CL, Soares Jr JM, Simoes RS, Haidar MA, Rodrigues de Lima G, Baracat EC. Estrogen effects on the vaginal pH, flora and cytology in late postmenopause after a long period without hormone therapy. Clin Exp Obstet Gynecol 2006;33:85–9. [2] Marinho RM, Soares Jr JM, Santiago RC, et al. Effects of estradiol on the cognitive function of postmenopausal women. Maturitas 2008;60:230–4. [3] Harden CL. Issues for mature women with epilepsy. Int Rev Neurobiol 2008;83:385–95. [4] Smith SS. Estradiol administration increases neuronal responses to excitatory amino acids as a long-term effect. Brain res 1989;503:354–7. [5] Herzog AG. Progesterone therapy in women with complex partial and secondary generalized seizures. Neurology 1995;45:1660–2. [6] Valente SG, Marques RH, Baracat EC, Cavalheiro EA, Naffah-Mazzacoratti MG, Amado D. Effect of hormonal replacement therapy in the hippocampus of ovariectomized epileptic female rats using the pilocarpine experimental model. Epilepsy Res 2008;82:48–58. [7] Gould E, Wooley CS, Frankfurt M, McEwen BS. Gonadal steroids regulate dentritic spine density in hipocampal pyramidal cells in adulthood. J Neurosci 1990;10:1286–91. [8] Segarra AC, McEwen BS. Estrogen increases spine density in ventromedial hypothalamic neuron of peripuberal rats. Neuroendocrinology 1991;54:365–72. [9] Verrotti A, Latini G, Manco R, De Simone M, Chiarelli F. Influence of sex hormones on brain excitability and epilepsy. J Endocrinol Invest 2007;30:797– 803. [10] Buterbaugh GG. Postictal events in amygdala-kindled female rats: with and without estradiol replacement. Exp Neurol 1987;95:697–713. [11] Buterbaugh GG. Estradiol replacement facilitates the acquisition of seizures kindled from the anterior neocortex in female rats. Epilepsy Res 1989;4:207–15. [12] Tolmacheva EA, van Luijtelaar G. The role of ovarian steroid hormones in the regulation of basal and stress induced absence seizures. J Steroid Biochem Mol Biol 2007;104:281–8. [13] Nicoletti F, Speciale C, Sortino MA, et al. Comparative effects of estradiol benzoate, the antiestrogen clomiphene citrate and the progestin medroxiprogesterone acetate on kainic acid-induced seizures in male and female rats. Epilepsia 1985;26:252–7. [14] Harden CL, Herzog AG, Nikolov BG, Koppel BS, Christos PJ, Fowler K, et al. Hormone replacement therapy in women with epilepsy: a randomized, doubleblind, placebo-controlled study. Epilepsia 2006;47:1447–51. [15] Herzog AG, Seibel MM, Schomer DL, Vaitukatis JL, Geschwind N. Reproductive endocrine disorders in women with partial seizures of temporal lobe origin. Arch Neurol 1986;43:341–6. [16] de Araujo LF, Soares Jr JM, Simões RS, et al. Effect of conjugated equine estrogens and tamoxifen administration on thyroid gland histomorphology of the rat. Clinics 2006;61:321–6. [17] Penteado SR, Fonseca AM, Bagnoli VR, Abdo CH, Júnior JM, Baracat EC. Effects of the addition of methyltestosterone to combined hormone therapy with estrogens and progestogens on sexual energy and on orgasm in postmenopausal women. Climacteric 2008;11:17–25. [18] Cavalheiro EA. The pilocarpine model of epilepsy. Ital J Neurol Sci 1995;16:33–7. [19] Mosquette R, de Jesus Simões M, da Silva ID, et al. The effects of soy extract on the uterus of castrated adult rats. Maturitas 2007;56(2):173–83. [20] Tauck DL, Nadler JV. Evidence of functional mossy fiber sprouting in hipocampal formation of kainic acid-treated rats. J Neurosci 1985;5:1016–22. [21] Leite JP, Bortolotto ZA, Turski L, Cavalheiro EA. Spontaneous recurrent seizures in rats: an experimental model of partial epilepsy. Neurosci Biobehav Rev 1990;14:511–7. [22] Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L. Longterm effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures. Epilesia 1991;32:778–82. [23] Cooke BM, Woolley CS. Gonadal hormone modulation of dendrites in the mammalian CNS. J Neurobiol 2005;64:34–46. [24] Amado D, Cavalheiro EA, Bentivoglio M. Epilepsy and hormonal regulation: the patterns of GnRH and galanin immunoreactivity in the hypothalamus of epileptic female rats. Epilepsy Res 1993;14:149–59. [25] Cavalheiro EA, Fernandes JM, Turski L, Mazzacoratti MGN. Spontaneous recurrent seizures in rats: amino acid and monoamine determination in the hippocampus. Epilepsia 1994;35:1–11.
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[26] Valente SG, Naffah-Mazzacoratti MG, Pereira M, et al. Castration in female rats modifies the development of the pilocarpine model of epilepsy. Epilepsy Res 2002;49:181–8. [27] Arida RM, Sacorza FA, Peres CA, Cavalheiro EA. The course of untreated in the pilocarpine model of epilepsy. Epilepsy Res 1999;34:99–107. [28] Santos NF, Marques RH, Correia L, et al. Multiple pilocarpine-induced “status epilepticus” in developing rats: a long-term behavioral and electrophysiological study. Epilepsia 2000;41:S57–63.
[29] Mello LEAM, Cavalheiro EA, Tan AM, et al. Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: cell loss and mossy fiber sprouting. Epilepsia 1993;34:985–95. [30] Babb TL, Kupfer WR, Pretorius JK, Crandall PH, Evesque MF. Synaptic reorganization by mossy fiber in human epileptic fascia dentate. Neurosciense 1991;42:351–63.
Contents lists available at ScienceDirect
Maturitas journal homepage: www.elsevier.com/locate/maturitas
Estrogen effects on pilocarpine-induced temporal lobe epilepsy in rats夽 Melquíades Pereira Jr. a , José M. Soares Jr. a,∗ , Sandra G. Valente b , Patricia B. Oliveira a , Esper A. Cavalheiro b , Débora Amado b , Edmund C. Baracat a,c a
Departamento de Ginecologia, Escola Paulista de Medicina/Universidade Federal de São Paulo, Rua Sena Madureira 1245 apt 11, 04021-051 São Paulo, SP, Brazil Disciplina de Neurologia Experimental, Escola Paulista de Medicina/Universidade Federal de São Paulo, São Paulo, SP, Brazil c Departamento de Obstetrícia e Ginecologia da Universidade de São Paulo, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 11 August 2008 Received in revised form 24 October 2008 Accepted 30 October 2008 Keywords: Experimental models Epilepsy Pilocarpine Menopause Estrogen
a b s t r a c t Objetive: To evaluate the effects of conjugated equine estrogens (CEE) on the pilocarpine-induced epilepsy in rats. Study design: 40 female rats were divided into: GPC (positive control) presented “status epilepticus” (SE) induced by pilocarpine; GOC (ovariectomized control) only castrated; GNC (negative control) received only saline solution; GPE received pilocarpine, presented SE, castrated and received 50 g/kg CEE treatment; GPV received pilocarpine, castrated and received propylene glycol (vehicle). The animals were monitored by a video system. At the end of observation, the brains removed for later histologic analysis using NeoTimm and Nissl methods. Results: The GPE presented a reduction in number of seizures compared to GPV. The Neo-Timm analysis showed that GPV had greater sprouting of mossy fibers, with a denser band in the area of the dentate gyrus hilum compared to GPE. On Nissl staining, GPE showed evident neuronal loss in the CA3 area. GPV presented loss in CA1 and dentate gyrus. Conclusion: Estrogen may have a protecting effect on the central nervous system. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Postmenopausal women present deleterious phenomena related to the hypoestrogenism status [1]. Innumerable studies demonstrated the benefits of hormone therapy in these women, resulting in their better quality of life and improving symptomatology In addition, estrogen seems to have an effect on cognitive function [2]. Hormone privation may interfere with the course of epilepsy in postmenopausal women [3]. The course of epilepsy in women at the beginning of menopause is still unknown, the pattern of epileptic seizures being liable to decrease or increase, as related to the presence or not of hormonal deficiency. Steroid hormones interfere with neuronal excitability through specific mechanisms which change cell metabolism and nerve transmission [4,5]. Endocrine and reproductive alterations are frequently reported in women with temporal lobe epilepsy and studies show that gonadal steroids modify dendritic spine density in hippocampal pyramidal cells
夽 Sources of financial support: FAPESP, São Paulo, Brazil CNPq; CINAPCE; CAPESBrasilia, Brazil. ∗ Corresponding author. Tel.: +55 11 50813685; fax: +55 11 50813685. E-mail address: [email protected] (J.M. Soares Jr.). 0378-5122/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.maturitas.2008.10.014
and in neurons of the hypothalamic ventromedial region in adult rats, suggesting an afferent synaptic modulation for these cells [6–8]. Some studies demonstrated the existence of neurons containing nuclear receptors for estrogen in certain areas of the brain, predominantly in the hypophysis, hypothalamus, limbic system and cerebral cortex. Clinical and experimental studies showed that ovarian steroids could alter susceptibility to epileptic activity, with estradiol decreasing the threshold of convulsive seizures, an effect that would be reversed by progesterone [9–14]. This symptomatology may be observed in women with anovulatory cycles as polycystic ovary syndrome. Some studies show that 50% of epileptic women have associated chronic anovulation [15]. However, the actual estrogen action on the course of epilepsy is still unknown. Conjugated equine estrogens (CEE) are commonly used in postmenopausal hormone replacement therapy. These hormones are a combination of soluble estrogens from the urine of pregnant mares, and they include several biologically active components such as estrone, estradiol, and its respective sulphates, and sulphates of equine estrogens (equilin and equilenin). In a previous study with rats we showed that, following treatment with 50 g/kg of CEE, estrogen levels are close to 40 pg/ml [16]. These values are similar to those found in postmenopausal women receiving estrogen therapy [17].
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were confined at random in appropriate metal cages with water and food ad libitum, and maintained at a temperature of 22 ◦ C under a 12-h light-dark cycle. The IRB of Universidade Federal de Sao Paulo – Escola Paulista de Medicina approved this study and the animals were carried according to the Standard Animal Manual Care of the same institution. 2.2. Pilocarpine model
Fig. 1. Number of epileptic seizures in GPC, GPE, GPV during the treatment period. *p < 0.05 compared to GPC and GPE.
In order to evaluate the influence of estrogen on epilepsy evolution in castrated rats, the pilocarpine-induced epilepsy model was used. The cholinergic muscarinic agonist is administrated intraperitoneally reproducing the main characteristics of temporal lobe epilepsy in humans [18]. Thus, the behavior and frequency of seizures were observed, as well neuronal alterations (by light microscopy) of the hippocampus of adult castrated rats, with pharmacologically induced epilepsy using pilocarpine and treated with estrogen. Therefore this model allows the evaluation of estrogen effects on epilepsy. 2. Material and methods 2.1. Animals Eighty virgin and intact, approximately 120-day-old albino Wistar-EPM1 rats weighing about 200 g were used. The animals
To prevent peripheral cholinergic pilocarpine effects, subcutaneous methylscopolamine (Sigma, 1 mg/kg) was previously administered to the animals; 30 min thereafter 4% pilocarpine was intraperitoneal administered to 64 animals at a dose of 350 mg/kg for induction of “status epilepticus”. All animals that survived to SE and presented spontaneous seizures after the silent period were considered to made epilepsy (chronic period, [21,22]). During pilocapine administration, 30 animals were lost due to tonic seizures leading to instantaneous death. 2.3. Negative control 16 animals received only physiological saline (0.9%) (vehicle) and constituted the control group. No deaths occurred in the animals receiving only physiological saline. 2.4. Seizure control After approximately thirty days the animals were placed in cylindrical cones, maintained under controlled conditions with a 12/12 h light/dark cycle, 21 ◦ C temperature, with water and food ad libitum and monitored by filming during 24 h/day using a videoEEG system. Seizures were accompanied during 30 days. In this phase, another five animals were lost. At the end of the experiment
Fig. 2. Photomicrograph of coronal brain section, at the level of the hippocampus, stained by Nissl technique. (A) Animals submitted to pilocarpine; (B) castrated animals; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (25×).
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Fig. 3. Photomicrograph of coronal brain section, at the level the dentate gyrus, stained by Nissl technique. (A) Animals submitted to pilocarpine; (B) animals only castrated; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (100×).
29 rats with epilepsy remained. After this period, surgical castration was performed.
2.5. Epilepsy control group A group of eight animals was randomly separated from the 29 epileptic rats, constituting the epilepsy control (with ovaries).
2.6. Castration procedure 21 animals were submitted to anesthesia and their abdomen was shaved; they were submitted to sterile preparation, and opened with a 1.5 cm lower midline incision. After that bilateral ovariectomy (castration) was performed. The procedure was carried out under anesthesia with sodium pentobarbital, at a 60 mg/kg intraperitoneal dose. During this procedure, five animals of the epileptic group were lost due to complications after surgical procedure. Also, half of negative control (n = 8) was ovariectomized.
2.8. Groups of animals The 40 animals were classified into five groups of 8 animals each, following previous procedures: GPC: rats which received only pilocarpine and entered “status epilepticus”—positive control. GOC: rats which received physiological saline (0.9%) and were castrated—negative control. GNC: non-castrated rats which received physiological saline (0.9%)—negative control. GPE (PILO/CT/EC): rats which received pilocarpine, entered “status epilepticus”, became epileptic, were castrated (CT) and received conjugated equine estrogens at a dose of 50 g/animal/day [19] by gavage. GPV (PILO/CT/vehicle): rats which received pilocarpine, entered “status epilepticus”, became epileptic, were castrated (CT), and received propylene glycol vehicle by gavage. 2.9. Control of seizures during the treatment
2.7. Treatment groups In all castrated animals a colpocytologic examination was performed to confirm genital atrophy. After confirming castration, 16 castrated animals were randomly divided into groups: one with eight rats that received only vehicle and another with eight animals that received estrogen [19].
All animals of GPE and GPV were placed in the individual cylindrical cones and were submitted to 24 h/day filming. After four days, administration of drugs started (conjugated equine estrogens and propylene glycol) by gavage using a metal probe, for thirty consecutive days. At the end of 60 days of observation, the animals were perfused and the brains removed for later histologic analysis using Neo-Timm and Nissl methods.
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Fig. 4. Photomicrograph of coronal brain section, at the level of the hippocampus stained by Neo-Timm technique. (A) Animals submitted to pilocarpine; (B) animals only castrated; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (25×).
2.10. Qualitative histological analysis
3. Results
After histological routine, the tissue sections stained with NeoTimm were classified according to the scale by Tauck and Nadler [20] into:
3.1. Effects of pilocarpine on the female rats
Grade 0: no or occasional Neo-Timm staining of mossy fibers; Grade 1: sparse staining over all parts of the granular layer; Grade 2: isolated, discontinuous intensely stained mossy fibers or a continuous band of supragranular staining of intermediate intensity, tween classification degrees 1 and 3; Grade 3: with a dense and a continuous stained band of supragranular mossy fibers. Nissl stain was used to evidence neuronal cytoplasm, as an indicator of neuronal viability since these corpuscles may disappear if neuronal injury occurs. This phenomenon is known as “chromatolysis”.
2.11. Statistical analysis The following parameters were analyzed by “t” Student test for parametric analysis to compare before and after treatment: Frequency and time of seizures. Analysis of variance (ANOVA) test and Tukey and Kremer tests were performed to compare the differences among the treatment groups and controls. A p-value < 0.05 was considered to be significant and the results are presented as the mean ± S.E.
All animals submitted to pilocarpine (GPC, GPE and GPV) presented salivation, tremors and repetitive movements progressing to motor limbic seizures and then presenting clonus of anterior paws supported by the posterior paws and finally falling. These seizures were recurrent, culminating with the animal’s death or “status epilepticus”. After a period without seizures (silent phase), the surviving animals presented spontaneous and recurrent seizures, characterizing the chronic period which continued throughout their whole life [21–23]. GPC, GPE and GPV animals did not present any behavioral alteration different from that expected in the chronic phase of the pilocarpine model. GOC and GNC animals were not submitted to pilocarpine; therefore they did not present any behavioral alteration (seizures). Of the animals which received pilocarpine, 30 died after the first clonic seizures (46.8 ± 24). Of the animals surviving after the first seizures and which remained in “status epilepticus” after 30 days, 5 died (19.5 ± 20.5). The mean latency time until the first convulsive seizure in the animals was 29.55 ± 17.11 min. Latency until “status epilepticus” was 55.93 ± 23.66 min. Mean time interval for establishment of the chronic period was 50 ± 10.36 days. 3.2. Estrogen therapy effect Frequency of seizures in the chronic phase before and after castration GPC animals (PILO/control) did not present any change in their seizure pattern (17.79 ± 3.12 seizures/month). GOC
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Fig. 5. Photomicrograph of coronal brain section at the level of the dentate gyrus stained by the Neo-Timm technique. (A) Animals submitted to pilocarpine; (B) animals only castrated; (C) control animals; (D) animals submitted to pilocarpine, castrated and treated with propylene glycol and (E) animals submitted to pilocarpine, castrated and treated with estrogen (100×).
(CT/control) and GNC animals (negative control) did not present seizures. GPE animals (PILO/CT/EC) showed a significant reduction in seizure number after castration and estrogen replacement (21.62 ± 2.53 seizures/month) when compared to baseline (before castration, 33.51 ± 3.14 seizures/month, p < 0.01). GPV animals (PILO/CT/PG) presented a significant increase in seizure number after castration and propylene glycol administration (32.37 ± 2.94 seizures/month) when compared to baseline (18.36 ± 3.54 seizures/month, p < 0.01). When compared the GPC, GPE and GPV (after estrogen treatment), the highest number of seizures was detected in GPV (p < 0.05) compared to GPC and GPE (Fig. 1).
fibers in the dentate gyrus hilum. GOC (CT/control) and GNC (control): grade 0 staining with no or only an occasional staining of mossy fibers. GPE (PILO/CT/EC): grade 2 staining, with sparse staining over all parts of the granular layer. GPV (PILO/CT/PG): grade 3 staining with a dense and continuous band of intense staining of supragranular mossy fibers. Based on these results, an intense sprouting of mossy fibers was observed in the animals which received pilocarpine, were castrated and did not receive estrogen replacement (GEV), while sprouting was less in epileptic non-castrated animals (GPC) and in those with estrogen replacement (GPE). In the castrated only animals (GOC) and in the controls (GNC) a sparse marking of mossy fibers was observed.
3.3. Predominant time of seizures
4.2. Nissl staining
Regarding the predominant time of seizures during the day, there were no significant differences among the groups (GPC, GPE and GPV). The animals, in general, presented more seizures during the light period (7:00–18:59 h) than in the dark (19:00–6:59) (p < 0.24). 4. Histological analysis
GPC and GPV histologic sections showed neuronal loss in the CA3, CA1 and dentate gyrus pyramidal layer, with breakdown of the structure of cell architecture of the hippocampus. In GOC and GNC animals, the sections did not present morphologic alterations. In GPE animals, the sections showed a greater neuronal loss in the CA3 layer and breakdown of the structure of the hippocampal cell architecture.
4.1. Neo-Timm staining
5. Discussion
Intensity of Neo-Timm staining of brain sections, in the dentate gyrus was visually assessed using the morphologic grading scale described by Tauck and Nadler [20]. Different patterns were found between the five animal groups (Figs. 2–5): GPC (PILO/control): grade 2 staining with a continuous band of supragranular mossy
It is known that estrogens act on the central nervous system by mechanisms which include a greater ability of neuronal repair, increase in the number of dendritic terminals, reduction in oxidative stress and, due to vascular effect, they promote a greater blood flow [23]. Development of experimental models in animals which
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mimic human epilepsy was of fundamental importance in the elucidation and study of epileptic phenomena. The pilocarpine-induced epilepsy model has been widely studied, both in males [22] and females [24] (reason for using it in our study). Based on these facts, the animals were castrated, provoking a hypoestrogenic state and estrogen replacement was used to evaluate its effect on epilepsy. Our results suggested that estrogen treatment may decrease the seizures in our animal model. During the preparation of the animals, the observed mortality rate after administration of pilocarpine (mortality after the first tonic seizure and after “status epilepticus”), latency to first seizure, “status epilepticus”, and the chronic period were similar to those described in the literature [25]. The mortality of rats is a limitation of this model. On analyzing the effect of estrogen replacement, it was observed that frequency of seizures among animals receiving estrogen replacement was lower than that of the animals receiving the vehicle. Such result shows that somehow estrogen would act as a moderator of susceptibility to seizures. The strength of this study is objective form to analyze the estrogen treatment on seizures and histological findings that may support our results. Thus, it was observed that estrogen seems to have a protecting effect on susceptibility to seizures induced by pilocarpine. However, some authors report that estrogen would act as seizure promoter while others relate that its absence would not offer protection against them. Also, Valente et al. [26] found a higher mortality rate in castrated animals which received pilocarpine, suggesting that female steroid hormones would protect the brain against cell death induced by “status epilepticus”. The authors demonstrated also that castrated females which received pilocarpine presented short latency period to first seizure, showing that estrogen has a protecting effect on susceptibility to pilocarpine-induced seizures. Our study showed that estrogen treatment may decrease the number of seizures in ovariectomized after pilocarpine-induced epileptic stabilized. This point was not evaluate in Valente et al. [26]. It can also be observed that seizure frequency was higher in the diurnal than in the nocturnal period. These data confirm the observations by Arida et al. [27] who observed that the animals have a higher frequency of seizures in the diurnal period (7:00–18:59 h) than in the nocturnal (19:00–6:59 h). The estrogen treatment may not change this pattern. Brains of the animals used in the study were submitted to Nissl stain for localization of the region where neuronal death could have occurred. The pattern found in epileptic rats and mice is of intense neuronal loss in the pyramidal layers of the CA1 and CA3 regions and in the granular layer of the hilum, areas of glial cell proliferation and breakdown of hippocampal cell architecture [28]. However, analyzing the staining pattern of GPE animals (estrogen treatment), the sections presented neuronal loss in the CA3 layer and breakdown of structure of hippocampal cell architecture, while in GPV animals (without estrogen), besides breakdown of structure and loss in CA3, there was also loss in CA1 and dentate gyrus. This result may suggest that estrogen may induce less brain damage than vehicle in castrated rats. Neuronal loss pattern in humans with epilepsy is frequently associated with supragranular mossy fiber sprouting. These fibers, displaying a great zinc concentration, were identified bye NeoTimm staining, thus making identification of axonal reorganization found in epileptic tissues easy. Consistent increase in the projections of the supragranular layer of the dentate gyrus has been identified by this staining after induction of pilocarpine-induced epileptic seizures [29]. Regarding the studied animals it was observed that GPE presented positive grade 2 mark, while GPV had positive grade 3 mark. Thus it was observed that in animals not treated with estrogen there was intense mossy fiber sprouting, which would indicate that estrogen plays a fundamental role in
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epileptogenesis. The anomalous synaptic reorganization, characterized by mossy fiber innervation, would thus form a monosynaptic excitatory feedback for dendrites of the internal molecular layer of the dentate gyrus [30]. Based on these results, it is suggested that estrogen replacement would avoid more neuronal cell architecture damage in the hippocampus. Future studies along this research line could produce more subsidies in order to better understand the role of estrogen in the central nervous system, mainly in cell signaling and gene action.
References [1] Galhardo CL, Soares Jr JM, Simoes RS, Haidar MA, Rodrigues de Lima G, Baracat EC. Estrogen effects on the vaginal pH, flora and cytology in late postmenopause after a long period without hormone therapy. Clin Exp Obstet Gynecol 2006;33:85–9. [2] Marinho RM, Soares Jr JM, Santiago RC, et al. Effects of estradiol on the cognitive function of postmenopausal women. Maturitas 2008;60:230–4. [3] Harden CL. Issues for mature women with epilepsy. Int Rev Neurobiol 2008;83:385–95. [4] Smith SS. Estradiol administration increases neuronal responses to excitatory amino acids as a long-term effect. Brain res 1989;503:354–7. [5] Herzog AG. Progesterone therapy in women with complex partial and secondary generalized seizures. Neurology 1995;45:1660–2. [6] Valente SG, Marques RH, Baracat EC, Cavalheiro EA, Naffah-Mazzacoratti MG, Amado D. Effect of hormonal replacement therapy in the hippocampus of ovariectomized epileptic female rats using the pilocarpine experimental model. Epilepsy Res 2008;82:48–58. [7] Gould E, Wooley CS, Frankfurt M, McEwen BS. Gonadal steroids regulate dentritic spine density in hipocampal pyramidal cells in adulthood. J Neurosci 1990;10:1286–91. [8] Segarra AC, McEwen BS. Estrogen increases spine density in ventromedial hypothalamic neuron of peripuberal rats. Neuroendocrinology 1991;54:365–72. [9] Verrotti A, Latini G, Manco R, De Simone M, Chiarelli F. Influence of sex hormones on brain excitability and epilepsy. J Endocrinol Invest 2007;30:797– 803. [10] Buterbaugh GG. Postictal events in amygdala-kindled female rats: with and without estradiol replacement. Exp Neurol 1987;95:697–713. [11] Buterbaugh GG. Estradiol replacement facilitates the acquisition of seizures kindled from the anterior neocortex in female rats. Epilepsy Res 1989;4:207–15. [12] Tolmacheva EA, van Luijtelaar G. The role of ovarian steroid hormones in the regulation of basal and stress induced absence seizures. J Steroid Biochem Mol Biol 2007;104:281–8. [13] Nicoletti F, Speciale C, Sortino MA, et al. Comparative effects of estradiol benzoate, the antiestrogen clomiphene citrate and the progestin medroxiprogesterone acetate on kainic acid-induced seizures in male and female rats. Epilepsia 1985;26:252–7. [14] Harden CL, Herzog AG, Nikolov BG, Koppel BS, Christos PJ, Fowler K, et al. Hormone replacement therapy in women with epilepsy: a randomized, doubleblind, placebo-controlled study. Epilepsia 2006;47:1447–51. [15] Herzog AG, Seibel MM, Schomer DL, Vaitukatis JL, Geschwind N. Reproductive endocrine disorders in women with partial seizures of temporal lobe origin. Arch Neurol 1986;43:341–6. [16] de Araujo LF, Soares Jr JM, Simões RS, et al. Effect of conjugated equine estrogens and tamoxifen administration on thyroid gland histomorphology of the rat. Clinics 2006;61:321–6. [17] Penteado SR, Fonseca AM, Bagnoli VR, Abdo CH, Júnior JM, Baracat EC. Effects of the addition of methyltestosterone to combined hormone therapy with estrogens and progestogens on sexual energy and on orgasm in postmenopausal women. Climacteric 2008;11:17–25. [18] Cavalheiro EA. The pilocarpine model of epilepsy. Ital J Neurol Sci 1995;16:33–7. [19] Mosquette R, de Jesus Simões M, da Silva ID, et al. The effects of soy extract on the uterus of castrated adult rats. Maturitas 2007;56(2):173–83. [20] Tauck DL, Nadler JV. Evidence of functional mossy fiber sprouting in hipocampal formation of kainic acid-treated rats. J Neurosci 1985;5:1016–22. [21] Leite JP, Bortolotto ZA, Turski L, Cavalheiro EA. Spontaneous recurrent seizures in rats: an experimental model of partial epilepsy. Neurosci Biobehav Rev 1990;14:511–7. [22] Cavalheiro EA, Leite JP, Bortolotto ZA, Turski WA, Ikonomidou C, Turski L. Longterm effects of pilocarpine in rats: structural damage of the brain triggers kindling and spontaneous recurrent seizures. Epilesia 1991;32:778–82. [23] Cooke BM, Woolley CS. Gonadal hormone modulation of dendrites in the mammalian CNS. J Neurobiol 2005;64:34–46. [24] Amado D, Cavalheiro EA, Bentivoglio M. Epilepsy and hormonal regulation: the patterns of GnRH and galanin immunoreactivity in the hypothalamus of epileptic female rats. Epilepsy Res 1993;14:149–59. [25] Cavalheiro EA, Fernandes JM, Turski L, Mazzacoratti MGN. Spontaneous recurrent seizures in rats: amino acid and monoamine determination in the hippocampus. Epilepsia 1994;35:1–11.
196
M. Pereira Jr. et al. / Maturitas 62 (2009) 190–196
[26] Valente SG, Naffah-Mazzacoratti MG, Pereira M, et al. Castration in female rats modifies the development of the pilocarpine model of epilepsy. Epilepsy Res 2002;49:181–8. [27] Arida RM, Sacorza FA, Peres CA, Cavalheiro EA. The course of untreated in the pilocarpine model of epilepsy. Epilepsy Res 1999;34:99–107. [28] Santos NF, Marques RH, Correia L, et al. Multiple pilocarpine-induced “status epilepticus” in developing rats: a long-term behavioral and electrophysiological study. Epilepsia 2000;41:S57–63.
[29] Mello LEAM, Cavalheiro EA, Tan AM, et al. Circuit mechanisms of seizures in the pilocarpine model of chronic epilepsy: cell loss and mossy fiber sprouting. Epilepsia 1993;34:985–95. [30] Babb TL, Kupfer WR, Pretorius JK, Crandall PH, Evesque MF. Synaptic reorganization by mossy fiber in human epileptic fascia dentate. Neurosciense 1991;42:351–63.