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Different types of status epilepticus lead to different levels of brain damage in rats
Epilepsy & Behavior 7 (2005) 401–410 www.elsevier.com/locate/yebeh
Different types of status epilepticus lead to different levels of brain damage in rats Cristiane Queixa Tilelli, Flavio Del Vecchio, Artur Fernandes, Norberto Garcia-Cairasco * Departamento de Fisiologia, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil Received 16 December 2004; revised 8 June 2005; accepted 10 June 2005 Available online 2 September 2005
Abstract We investigated a possible correlation between behavior during status epilepticus (SE) and underlying brain damage. Adult rats were electrically stimulated in the left amygdala to induce SE, which was stopped 2 hours later. We observed two different types of SE: (1) typical SE (TSE), with facial automatisms, neck and forelimb myoclonus, rearing and falling, and tonic–clonic seizures; (2) ambulatory SE (ASE), with facial automatisms, neck myoclonus, and concomitant ambulatory behavior. TSE was behaviorally more severe than ASE (P < 0.05). Histology revealed neuronal loss in several brain areas. There was a positive correlation between SE type and amount of injured areas 24 hours and 14 days after SE (P < 0.01). The areas more affected were piriform cortex and hippocampal formation. We suggest quality of seizures during SE may be considered in further SE studies, as our results indicate its influence on the severity of brain damage following this paradigm. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Temporal lobe epilepsy; Hippocampus; Piriform cortex; Status epilepticus; Neuronal loss; Brain injury; Plasticity; Behavior; Seizure; Electrical stimulation of amygdala
1. Introduction Epilepsy affects about 1–2% of the worldÕs population [1]. Thirty percent of patients with epilepsy are resistant to drug treatment; among them, the majority have socalled temporal lobe epilepsy (TLE), which is generally associated with an initial insult, followed by a latent period that can last several years and, then, spontaneous recurrent seizures [2–4]. In TLE associated with hippocampal sclerosis, the chance of controlling seizures with conventional drug treatment is decreased [5]. To better understand the process of epileptogenesis and eventually discover new tools for the prevention and treatment of epilepsy, many research groups have spent decades studying the factors that may be involved
*
Corresponding author. Fax : +55 16 602 0017. E-mail address: [email protected] (N. Garcia-Cairasco).
1525-5050/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2005.06.013
in the pathological alterations underlying TLE [4,6–14]. Several animal models for epilepsy research use the status epilepticus (SE) paradigm [15–21] to induce epileptogenesis, claiming that it mimics behavioral, electroencephalographic, and morphological phenomena such as those observed in humans [2,22,23]. As a consequence of all these efforts, some key factors in the process of the development of epilepsy have been identified. First, the duration of SE seems to be very important with respect to subsequent epileptogenesis and morphological alterations of the limbic structures involved [23–27]. Animals that experience a short SE may not develop epilepsy and/or may not present with the equivalent of human hippocampal sclerosis. Second, it is hypothesized that both the gene cascades that follow SE and their consequent limbic system morphological modifications [2,22,23,28–36] constitute the basis for the appearance of the spontaneous recurrent seizures that define the epilepsy condition.
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Rats subjected to SE manifest diverse behaviors that can be categorized according to their severity [37–40]. We hypothesized that the behavior presented by the animals during SE, because it is a complex consequence of regional or wider abnormal brain activity, may reflect the pathogenesis of epilepsy. In other words, behavioral seizures might be a direct indication of the correspondent activated circuitry. To verify this hypothesis, we induced SE in rats by electrical stimulation of the amygdaloid complex (AmyC) according to the method developed by Nissinen et al. [20], controlling for the duration of the SE, and compared behavior observed during SE with its neuroanatomical consequences.
2. Methods 2.1. Animals Adult, male albino Wistar rats (n = 36) from the Central Breeding Stock of the University of Sa˜o Paulo, Ribeira˜o Preto, 240–320 g, were used. During all procedures, animals received food and water ad libitum, with a light/dark cycle of 12 hours (lights on: 7 AM to 7 PM), maintained under optimal temperature and humidity conditions. Experiments with laboratory animals were conducted in accordance with the rules of the Brazilian Society for Neuroscience and Behavior, which are based on the Society for Neuroscience guidelines for animal experimentation. All efforts were made to avoid any suffering or pain of the animals. 2.2. Surgery and SE induction Animals were stereotaxically implanted, under anesthesia (ketamine, 0.7 mg/kg, Bayer S.A., and xylazine, 1.0 mg/kg, Agener LTDA), with Teflon-coated bipolar stainless-steel electrodes (A-M Systems, Inc). One stimulation/recording electrode was implanted into the AmyC in the left hemisphere and the other recording electrode into the ipsilateral posterior hippocampus (HIP). A stainless-steel screw, attached to the frontal skull, was used as the ground and reference electrode, according to Romcy-Pereira and Garcia-Cairasco [41]. Twenty-two animals received monopolar electrical stimulation of the left AmyC and developed self-sustained generalized SE (GSE), and 14 animals were subjected to the same procedures, except for electrical stimulation. Animals that were stimulated but did not develop GSE were excluded from this study. The stimulus characteristics were: biphasic square waves, 300 lA of amplitude, frequency 60 Hz, duration 100 ms, each half-a-second, for 30 minutes. Animals were monitored by video/EEG as described by Dutra-Moraes et al. [42]. Briefly, video/EEG recordings were made using a video blaster board (VBSE, Creative Labs) and an A/
D converter interfacing board (MPW100, Biopac) to present both the animalÕs image and EEG data on the computer screen. The EEG data were digitally recorded for further analysis, after passage through a signal conditioner (Cyberamp 320, Axon Instruments), and the computerÕs screen image was recorded in VHS. A source follower circuit was used to reduce movement artifacts, and an electric swivel avoided cable twisting during SE. EEG data were acquired with a 1-kHz low-pass filter, 0.1-Hz high-pass filter, and Notch filter in 60 Hz. Data were sampled at 500 Hz in both the amygdala and hippocampus channels. The experiment comprised four periods: (1) control period—5 minutes of basal behavior and EEG recording; (2) induction period—30 minutes of electrical stimulation (not in control group) with concomitant behavior and EEG recording; (3) maintenance period—90 minutes of behavior and EEG recording, when animals were expected to develop GSE; and (4) recovery period—rescue of animals from GSE with a diazepam injection (5 mg/kg) and observation for 30–120 minutes. Whenever necessary, additional diazepam was injected. All animals were treated with diazepam independent of the presence of GSE. 2.3. Behavioral analysis During the four periods described above, a behavioral study was conducted, using RacineÕs scale [37], as modified by Pinel and Rovner [38]: (1) facial myoclonus; (2) facial and neck myoclonus; (3) facial, neck, and forelimb myoclonus; (4) class 3 with rearing; (5) class 4 with falling; (6) more then three times class 5; (7) running and jumping; (8) generalized tonic–clonic seizure. The four periods were subdivided into 5-minute intervals, animal behavior was observed, and the maximum score in each interval was recorded. The animals were divided into groups based on observations of SE behavior; the curves obtained by averaging the data of all animals in each group for each interval were compared to verify the consistency of our observations. 2.4. Tissue analysis After diazepam treatment, animals were maintained in the Physiology Department Vivarium, under the same conditions previously described. They were killed at 3 hours, 24 hours, or 14 days after stimulus onset. These times were selected to verify the possible acute and semichronic anatomical injury consequent to GSE. Animals were killed under profound anesthesia (sodium thiopental, 60 mg/kg) by transcardiac perfusion with (1) 100 mL of MillonigÕs buffer (sodium hydroxide 0.39% w/v, sodium phosphate monobasic monohydrate 1.66% w/v; sodium chloride 0.002% w/v); (2) 300 mL of
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0.1% sodium sulfide in MillonigÕs buffer; and (3) 300 mL of 4% paraformaldehyde (PFA) in MillonigÕs buffer. Brains were removed from skulls and immersed in PFA for 2–4 hours and then in 20% sucrose for 24–48 hours. Brains were frozen in isopentane on dry ice and cut into 40-lm slices. The slices were maintained in antifreezing medium (50% phosphate buffer 50 mM, 30% ethylene glycol, and 20% glycerine) until processed for Nissl and Neo-Timm histochemistry and neuronal nucleus protein (Neu-N) immunohistochemistry. Immunohistochemistry was done in a free-floating system. Tissue was washed with 10 mM phosphate buffer, pH 7.4 (PB), incubated for 45 minutes in 3% hydrogen peroxide in methanol, and washed in PB with 0.9% saline (PBS). Slices were then transferred to a buffer containing 100 mM glycine in PBS, and washed again in PBS and in PBS containing 0.1% Triton X-100 (Sigma)
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(PBS-T). Nonspecific binding was blocked by incubating the sections in PBS-T containing 3% bovine serum albumin (BSA, Sigma). Tissue was incubated with primary antibody (Neu-N, MAB 377, Chemicon Internationals, Inc; 1:700) diluted in BSA for 18 hours, at room temperature. Slices were then washed in PBS and incubated in secondary antibody (Chemicon International, Inc; 1:1000) diluted in BSA for 1–2 hours at room temperature. They were washed again in PBS and incubated in the biotin–avidin–peroxidase ABC kit (Vector Laboratories) for 30 minutes, and then the immunoreaction was made with 1 mg/mL diaminobenzidine (DAB, Sigma) diluted in PBS with 0.03% hydrogen peroxide for 2 minutes. The reaction was stopped with distilled water and slices were mounted on gelatin-coated slides (0.5% gelatin and 0.05% sulfate–chrome–aluminum w/v), dehydrated, and coverslipped.
Fig. 1. Comparison of lesions in piriform cortices. (A) normal piriform cortex with Neu-N staining, (B) normal piriform cortex with Nissl histochemistry, (C) lesioned piriform cortex with Neu-N staining, (D) lesioned piriform cortex with Nissl histochemistry. Note that the lesion is more evident with Neu-N staining, but is confirmed with Nissl histochemistry. Bar = 500 lm.
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The tissues were observed under a light microscope (Olympus BX-60), and images were acquired with a digital camera (Cool-CCD, Optronics). We observed several anteroposterior areas per brain, ranging from the most anterior part of the hippocampus to its most posterior part, including the amygdala and entorhinal and piriform cortices. Two blinded observers counted the number of areas with evident cell loss in each animal. Only brain areas with extensive damage were added, making quantitative cell counting unnecessary (Fig. 1 shows examples of what we call ‘‘evident cell loss’’). Each of the following areas was counted as a separate structure: in hippocampus, dentate gyrus, hilus, CA4, CA3, CA2, CA1; subiculum; entorhinal cortex; pyriform cortex; and lateral/basolateral amygdala. Substantial neuronal loss observed unilaterally in one of those areas was scored as 1. Damage to the same area in both hemispheres was scored 2. Damage of the same area, ipsilaterally, in different sections of the same animal, was scored 1. Damage immediately surrounding the electrodes tips or their tract was ignored.
3. Results 3.1. Electrode positioning Electrodes occupied the AmyC and functionally related areas. Fig. 2 illustrates electrode positioning in the stimulated animals (based on Paxinos and WatsonÕs atlas of the rat brain [43]). The nuclei stimulated were lateral amygdala (n = 6), basolateral amygdala (n = 8),
basomedial amygdala (n = 2), medial amygdala (n = 1), bed nucleus of stria terminalis (n = 1), caudate putamen (n = 1), deep endopiriform nucleus (n = 2), and piriform cortex (n = 1). Because the main purpose of this work was to analyze the behavior and consequent neuroanatomical injury caused by GSE, we decided not to exclude animals that developed GSE but in which the electrodes were positioned outside the AmyC. Because areas outside the AmyC included in this study are intimately related to the AmyC [44], they are included when we refer to AmyC stimulation. 3.2. Behavior analysis We observed two different types of GSE induced by electrical stimulation of the AmyC. ‘‘Typical GSE’’ (TSE, 50% of animals) precludes the presence of all behaviors described by Pinel and Rovner [38]: facial automatisms, neck and forelimb myoclonus, rearing, falling, running, jumping, and generalized tonic–clonic seizures. On average, animals continuously exhibit heavy forelimb myoclous. The second type of GSE, so-called ‘‘ambulatory GSE’’ (ASE, 50% of animals) includes only the lowest scores, such as facial automatisms and neck myoclonus. Concomitantly, animals with ASE manifested continuous ambulatory behavior in the observation cage. TSE scores were higher scores than ASE scores during the entire maintenance period, when self-sustained SE occurred but not concomitant with electrical stimulation (P < 0.05, ANOVA on ranks with Tukey test) (Fig. 3). Once GSE was established (maintenance period), there was no relationship between the
Fig. 2. Distribution of electrode tips from the stimulated rats: black dots indicate electrodes inside the AmyC and gray squares indicate electrodes in AmyC-related areas.
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Fig. 3. There are two different types of GSE consequent to AmyC electrical stimulation. It is shown that these GSE types are established during the maintenance phase and that TSE (triangles) is more severe than ASE (squares). ANOVA on ranks followed by Tukey test, P < 0.05.
severity of behavior and the area stimulated (data not shown).
EEGs (Fig. 4), marked by the presence of high-amplitude and -frequency discharges intermixed with flattened periods.
3.3. EEG 3.4. Histology In this work, we used the model of SE proposed by Nissinen et al. [20]. In addition to behavioral observation, we also recorded EEG patterns during seizures (Fig. 4). Instead of performing the extensive EEG analysis already done by that research group with EEG data, we compared patterns qualitatively. Comparison of our observations with AmyC stimulation and their description of the EEGs of rats exhibiting SE in response to lateral amygdaloid nucleus stimulation indicates that our animals with SE had EEGs very similar to their animalsÕ
Fig. 4. EEG recordings from amygdala (AMY) and hippocampus (HIP) during SE, demonstrating the so-called ‘‘high-amplitude and frequency discharge’’ (third EEG line, between arrows) and the ‘‘flat periods’’ of 1 to 3 seconds following it (third EEG line, asterisk). There is shown an approximately 120-second period of EEG, divided into three successive epochs (upper, middle, and bottom). Bars = (horizontal, time) 3 seconds and (vertical, voltage) 2 mV.
Histological analysis (Nissl staining confirmed with selective Neu-N immunostaining) revealed that, at 3 hours (n = 2 ambulatory GSE, n = 5 TSE, and n = 4 controls), no temporal lobe-related area showed evident cell loss in any of the groups studied. In contrast, 24 hours after stimulus onset (n = 4 ambulatory GSE, n = 2 TSE, and n = 5 controls), piriform cortex was intact or injured unilaterally in all ASE animals and bilaterally in all TSE animals. Fourteen days after GSE (n = 5 ambulatory GSE, n = 4 TSE, and n = 3 controls), ASE animals had unilateral or bilateral cell loss in piriform cortex and TSE animals had bilateral cell loss in piriform cortex and in the hippocampal formation, including the subiculum. Examples of what we considered evident lesions are compared with control tissues in Fig. 5A (Neu-N immunohistochemistry). The damage was also evident by Nissl staining, indicating that it was not a problem of impaired Neu-N expression, but real neuronal loss. Statistical analysis of these data (Pearson correlation) revealed a statistically significant positive correlation between the median seizure score of each rat (considering all 5-minute intervals of the SE protocol) and the total number of injured areas both 24 hours (R = 0.873, P < 0.001) and 14 days (R = 0.798, P < 0.01) after GSE (Fig. 5B). Neo-Timm staining showed that only one animal, at 14 days after TSE, exhibited light mossy fiber sprouting. All other animals, in all the groups studied, did not exhibit mossy fiber sprouting (Fig. 6).
4. Discussion To our knowledge, this is the first time that the behavioral severity of SE is shown to be related to the
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Fig. 5. (A) Images of hippocampal formation (top) and piriform cortex (bottom) of rats killed 14 days after GSE induction. From left to right are tissues from rats with control, ambulatory and typical GSE. Asterisks and arrows indicate what we considered areas with evident lesions. Neu-N immunohistochemistry. Bars = 500 lm. (B) Severity of behavior during the maintenance phase of GSE correlates with the number of areas with evident lesions, at both 24 hours and 14 days after GSE induction (bottom). Pearson correlation, P < 0.05.
pathological alterations subjacent to TLE in animals. Our results demonstrate that stimulation of the AmyC and related areas for 30 minutes, with the parameters used in this work, is effective in inducing a self-sustained SE that can be maintained at least for 2 hours. Nissinen et al. [20] had proposed this protocol of SE induction with stimulation of the lateral nucleus of the amygdala. In this work, we also stimulated other AmyC subnuclei, as well as other nuclei closely related to the AmyC. We found that, once the self-sustained SE is established, it does not matter what nucleus was stimulated. However, we also observed that stimulation of the lateral and basolateral nuclei of the amygdala is more effective in
inducing self-sustained SE than stimulation of other nuclei (about 80% of the animals stimulated in the lateral and basolateral nuclei develop GSE, as compared with about 0–50% of the animals stimulated in other nuclei; data not shown). The EEG data obtained by our group also resemble those of Nissinen et al. [20], and constitute another parameter that confirms the success of this model using the stimulation of other nuclei related to the amygdala. It may also suggest that the circuitries involved in the maintenance of SE are similar, if not the same. During the maintenance phase of our experiments (self-sustained SE) we observed that the rats could be
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Fig. 6. Animals with GSE did not manifest sprouting (center, compare with control in top), with the exception of one with a very light sprouting (bottom, arrows). Bar = 500 lm.
subdivided into two groups: one exhibiting very strong SE, the so-called typical SE, where the behaviors observed corresponded to high categories of limbic seizures (based on RacineÕs scale [37], as modified by Pinel and Rovner [38]), such as forelimb myoclonus and rearing; and the other exhibiting a much lower severity of SE, corresponding to scores 1–2 on the scale, such as face and neck myoclonus. As several articles that cite the behavior observed during SE describe a SE similar to the high-score SE observed in this work, we decided to name it ‘‘typical GSE.’’ On the other hand, rats exhibiting low-score SE concomitantly walked around the observation box, with intermixed myoclonic jerks, which is why we called it ‘‘ambulatory SE.’’ Ambulatory SE is described elsewhere, but we do not know if those descriptions correspond exactly to the GSE we described here [45,46]. During the preparation of this article, we observed that other SE that could also be called ‘‘ambulatory’’ occurred in our rats, but the hippocampal electrodes were silent indicating a localized more than a generalized SE, and the animals did not exhibit concomitant face and neck myoclonus (unpublished data). We sacrificed the animals at different intervals after the initial stimulation to verify the possible neuropatho-
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logical consequences of SE. We observed neuronal loss as soon as 24 hours after SE, and this neuronal loss was aggravated after 14 days of SE. We did not count cells because, at this point, we consider only the neuronal loss that was evident, as can be seen in Fig. 1and 5A. A test for the relationship between number of areas with cell loss and severity of SE showed a strong positive correlation both 3 hours and 14 days after SE. Interestingly, 24 hours after GSE, lesions were restricted to piriform cortex, unilaterally or bilaterally, with no distinction between typical and ambulatory SE. But at 14 days after GSE, only the animals with TSE had hippocampal cell loss. No cell loss was observed in the hippocampal formation in the absence of cell loss in the piriform cortex. The piriform cortex was the first and most severely injured area in all rat brains studied in this work. The most affected area of the piriform cortex was layer III. Although the piriform cortex is not usually a main focus when TLE is discussed, some data indicate its relation to the pathology of epilepsy. Ebert et al. [47] verified that female rats injected with kainic acid (potent excitatory drug for induction of SE) manifested cell loss in the piriform cortex 24 to 28 hours after SE. Absence of the piriform cortex (when destroyed by ibotenic acid) causes a delay in the development of the kindling process [48]. Activation of piriform cortex by bicuculline also induces severe seizures in rats [49]. More interestingly, Librizzi and de Curtis [50] suggested that interictal activity of the piriform cortex could have a blocking effect over itself afterward, when epileptic activity occurred in the hippocampus or entorhinal cortex. The lesions observed in the hippocampal formation were more expected, because cell loss in this structure is extensively described [51,52]. The pattern of cell loss observed in the HIP in this work is in agreement with the patterns previously described: loss of neurons in the hilus of the dentate gyrus, in CA4, CA3, and CA1, with preservation of CA2. Some hypotheses try to explain the epileptogenic process by the pathology of the hippocampal formation observed in this and other studies. According to those theories, the sprouting observed in hippocampi from animals with chronic epilepsy occurs partly because of the death of neurons in the hilus of the dentate gyrus and in the CA4–CA3 regions. Interestingly, we did not find sprouting in our animals, with the exception of really light sprouting, unilaterally, in a brain from an animal with TSE sacrificed 14 days after SE induction (see Fig. 6). The cause/consequence relationship of the sprouting with epilepsy in animals subjected to SE induction is very controversial [21,53–59]. Unfortunately, we did not have the opportunity to observe if our animals exhibited spontaneous seizures by the time they were sacrificed, which could help to clarify the role of sprouting in the establishment of epilepsy in animal models.
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One obvious criticism that can be made of our results is the possibility of continuation of electrographic epileptiform activity despite the arrest of behavioral seizures. If stronger behavioral SE leads to longer-lasting electrographic SE, then the presence of more areas with neuron loss would be more a consequence of longer SE than of the severity of SE itself. Our data suggest that this may not be the case: 9 animals manifested EEG activity down to less than baseline 5 to 45 minutes after diazepam injection (4 typical SE, 5 ambulatory SE), and in the other 11, the duration of epileptiform activity on the EEG was not known (still above the baseline when we finished the EEG recording, up to 1 hour after diazepam injection; 6 typical SE and 5 ambulatory SE). The even distribution of short and unknown EEG epileptiform activity durations between typical and ambulatory SE is already evidence that this is not the factor leading to differences in the quantity of severely damaged areas between groups. To confirm this suspicion, we compared data from TSE animals whose epileptiform EEG activity, together with behavioral epileptiform arrest activity, ended within 30 minutes of diazepam injection (n = 2; one killed 24 hours and one killed 14 days after SE induction) with data from ASE animals that still exhibited EEG epileptiform activity after behavioral epileptiform arrest and termination of EEG recording (unknown duration of electrographic seizure on EEG; n = 4; 2 killed 24 hours and 2 killed 14 days after SE induction). TSE animals had 7 and 11 damaged areas
and ASE had 0, 1, 2, and 3 damaged areas, confirming that the number of areas with severe neuronal loss depends on the type of behavior exhibited during SE more than the EEG duration of the SE. Fig. 7 shows some examples of the analysis just described. Interestingly, in a recent publication, Mohapel et al. [46] also reported that different severities of SE lead to different pathologies in rat brains. They found that rats with the so-called ‘‘fully convulsive SE’’ (more severe, probably corresponding to TSE) exhibit more neurogenesis in the dentate gyrus of the hippocampus, although those new neurons died after 4 weeks of SE. On the other hand, what they called ‘‘partial convulsive SE’’ (less severe, probably corresponding to ASE) exhibited less neurogenesis, but those new neurons survived after 4 weeks of SE. Our work complements that of Mohapel et al., corroborating their findings and adding some further data on the time-related events of the injury consequent to different severities of SE. In conclusion, this work showed that electrical stimulation of the amygdala and related areas induces SE of different severities. The severity of the SE observed in our animals induced different levels of injury in brain areas that participate in the seizures. Based on the results, we suggest that the behavior of animals submitted to SE may be an important factor in analysis of the pathophysiology of epileptogenesis in future studies using SE as a paradigm for the investigation of epilepsy.
Fig. 7. Lack of relationship between electrographic seizure duration after diazepam injection and area of brain damage. Data from three representative cases: TSE (upper row); ASE (middle row), and control (bottom row). The first column shows the compacted EEG from each animal (gray: hippocampus channel; black: amygdala channel). The second and third columns show left and right hippocampi (respectively), and the fourth and fifth columns, left and right amygdala (respectively) of each animal. Top row: animal with TSE with electrographic epileptiform activity completed within 5–15 minutes of diazepam (DZP) injection. Middle row: animal with ASE with electrographic epileptiform activity not completed within recording protocol (time of EEG seizures unknown). EEG recordings: black, HIP; gray, AmyC; /, stimulation window; #, electrode tract. The control rat received no stimulation to induce SE induction or a brain lesion. Note that, although the rat with TSE experienced SE for a shorter time than the animal with ASE, the former manifested more areas with neuronal loss (asterisks) than the latter. Bar = 250 lm.
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Acknowledgments The authors thank the following Brazilian agencies for funding: Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Programa de Apoio a Nu´cleos de Exceleˆncia do Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (PRONEX-CNPq), Programa de Apoio a Po´s-Graduac¸a˜o da Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (PROAP-Capes) and Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assinteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜o Paulo (FAEPA-HC/FMRP-USP). C.Q.T. and N.G.-C. held FAPESP and CNPq Research Fellowships, respectively, during the execution of this work.
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Different types of status epilepticus lead to different levels of brain damage in rats Cristiane Queixa Tilelli, Flavio Del Vecchio, Artur Fernandes, Norberto Garcia-Cairasco * Departamento de Fisiologia, Faculdade de Medicina de Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil Received 16 December 2004; revised 8 June 2005; accepted 10 June 2005 Available online 2 September 2005
Abstract We investigated a possible correlation between behavior during status epilepticus (SE) and underlying brain damage. Adult rats were electrically stimulated in the left amygdala to induce SE, which was stopped 2 hours later. We observed two different types of SE: (1) typical SE (TSE), with facial automatisms, neck and forelimb myoclonus, rearing and falling, and tonic–clonic seizures; (2) ambulatory SE (ASE), with facial automatisms, neck myoclonus, and concomitant ambulatory behavior. TSE was behaviorally more severe than ASE (P < 0.05). Histology revealed neuronal loss in several brain areas. There was a positive correlation between SE type and amount of injured areas 24 hours and 14 days after SE (P < 0.01). The areas more affected were piriform cortex and hippocampal formation. We suggest quality of seizures during SE may be considered in further SE studies, as our results indicate its influence on the severity of brain damage following this paradigm. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Temporal lobe epilepsy; Hippocampus; Piriform cortex; Status epilepticus; Neuronal loss; Brain injury; Plasticity; Behavior; Seizure; Electrical stimulation of amygdala
1. Introduction Epilepsy affects about 1–2% of the worldÕs population [1]. Thirty percent of patients with epilepsy are resistant to drug treatment; among them, the majority have socalled temporal lobe epilepsy (TLE), which is generally associated with an initial insult, followed by a latent period that can last several years and, then, spontaneous recurrent seizures [2–4]. In TLE associated with hippocampal sclerosis, the chance of controlling seizures with conventional drug treatment is decreased [5]. To better understand the process of epileptogenesis and eventually discover new tools for the prevention and treatment of epilepsy, many research groups have spent decades studying the factors that may be involved
*
Corresponding author. Fax : +55 16 602 0017. E-mail address: [email protected] (N. Garcia-Cairasco).
1525-5050/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2005.06.013
in the pathological alterations underlying TLE [4,6–14]. Several animal models for epilepsy research use the status epilepticus (SE) paradigm [15–21] to induce epileptogenesis, claiming that it mimics behavioral, electroencephalographic, and morphological phenomena such as those observed in humans [2,22,23]. As a consequence of all these efforts, some key factors in the process of the development of epilepsy have been identified. First, the duration of SE seems to be very important with respect to subsequent epileptogenesis and morphological alterations of the limbic structures involved [23–27]. Animals that experience a short SE may not develop epilepsy and/or may not present with the equivalent of human hippocampal sclerosis. Second, it is hypothesized that both the gene cascades that follow SE and their consequent limbic system morphological modifications [2,22,23,28–36] constitute the basis for the appearance of the spontaneous recurrent seizures that define the epilepsy condition.
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Rats subjected to SE manifest diverse behaviors that can be categorized according to their severity [37–40]. We hypothesized that the behavior presented by the animals during SE, because it is a complex consequence of regional or wider abnormal brain activity, may reflect the pathogenesis of epilepsy. In other words, behavioral seizures might be a direct indication of the correspondent activated circuitry. To verify this hypothesis, we induced SE in rats by electrical stimulation of the amygdaloid complex (AmyC) according to the method developed by Nissinen et al. [20], controlling for the duration of the SE, and compared behavior observed during SE with its neuroanatomical consequences.
2. Methods 2.1. Animals Adult, male albino Wistar rats (n = 36) from the Central Breeding Stock of the University of Sa˜o Paulo, Ribeira˜o Preto, 240–320 g, were used. During all procedures, animals received food and water ad libitum, with a light/dark cycle of 12 hours (lights on: 7 AM to 7 PM), maintained under optimal temperature and humidity conditions. Experiments with laboratory animals were conducted in accordance with the rules of the Brazilian Society for Neuroscience and Behavior, which are based on the Society for Neuroscience guidelines for animal experimentation. All efforts were made to avoid any suffering or pain of the animals. 2.2. Surgery and SE induction Animals were stereotaxically implanted, under anesthesia (ketamine, 0.7 mg/kg, Bayer S.A., and xylazine, 1.0 mg/kg, Agener LTDA), with Teflon-coated bipolar stainless-steel electrodes (A-M Systems, Inc). One stimulation/recording electrode was implanted into the AmyC in the left hemisphere and the other recording electrode into the ipsilateral posterior hippocampus (HIP). A stainless-steel screw, attached to the frontal skull, was used as the ground and reference electrode, according to Romcy-Pereira and Garcia-Cairasco [41]. Twenty-two animals received monopolar electrical stimulation of the left AmyC and developed self-sustained generalized SE (GSE), and 14 animals were subjected to the same procedures, except for electrical stimulation. Animals that were stimulated but did not develop GSE were excluded from this study. The stimulus characteristics were: biphasic square waves, 300 lA of amplitude, frequency 60 Hz, duration 100 ms, each half-a-second, for 30 minutes. Animals were monitored by video/EEG as described by Dutra-Moraes et al. [42]. Briefly, video/EEG recordings were made using a video blaster board (VBSE, Creative Labs) and an A/
D converter interfacing board (MPW100, Biopac) to present both the animalÕs image and EEG data on the computer screen. The EEG data were digitally recorded for further analysis, after passage through a signal conditioner (Cyberamp 320, Axon Instruments), and the computerÕs screen image was recorded in VHS. A source follower circuit was used to reduce movement artifacts, and an electric swivel avoided cable twisting during SE. EEG data were acquired with a 1-kHz low-pass filter, 0.1-Hz high-pass filter, and Notch filter in 60 Hz. Data were sampled at 500 Hz in both the amygdala and hippocampus channels. The experiment comprised four periods: (1) control period—5 minutes of basal behavior and EEG recording; (2) induction period—30 minutes of electrical stimulation (not in control group) with concomitant behavior and EEG recording; (3) maintenance period—90 minutes of behavior and EEG recording, when animals were expected to develop GSE; and (4) recovery period—rescue of animals from GSE with a diazepam injection (5 mg/kg) and observation for 30–120 minutes. Whenever necessary, additional diazepam was injected. All animals were treated with diazepam independent of the presence of GSE. 2.3. Behavioral analysis During the four periods described above, a behavioral study was conducted, using RacineÕs scale [37], as modified by Pinel and Rovner [38]: (1) facial myoclonus; (2) facial and neck myoclonus; (3) facial, neck, and forelimb myoclonus; (4) class 3 with rearing; (5) class 4 with falling; (6) more then three times class 5; (7) running and jumping; (8) generalized tonic–clonic seizure. The four periods were subdivided into 5-minute intervals, animal behavior was observed, and the maximum score in each interval was recorded. The animals were divided into groups based on observations of SE behavior; the curves obtained by averaging the data of all animals in each group for each interval were compared to verify the consistency of our observations. 2.4. Tissue analysis After diazepam treatment, animals were maintained in the Physiology Department Vivarium, under the same conditions previously described. They were killed at 3 hours, 24 hours, or 14 days after stimulus onset. These times were selected to verify the possible acute and semichronic anatomical injury consequent to GSE. Animals were killed under profound anesthesia (sodium thiopental, 60 mg/kg) by transcardiac perfusion with (1) 100 mL of MillonigÕs buffer (sodium hydroxide 0.39% w/v, sodium phosphate monobasic monohydrate 1.66% w/v; sodium chloride 0.002% w/v); (2) 300 mL of
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0.1% sodium sulfide in MillonigÕs buffer; and (3) 300 mL of 4% paraformaldehyde (PFA) in MillonigÕs buffer. Brains were removed from skulls and immersed in PFA for 2–4 hours and then in 20% sucrose for 24–48 hours. Brains were frozen in isopentane on dry ice and cut into 40-lm slices. The slices were maintained in antifreezing medium (50% phosphate buffer 50 mM, 30% ethylene glycol, and 20% glycerine) until processed for Nissl and Neo-Timm histochemistry and neuronal nucleus protein (Neu-N) immunohistochemistry. Immunohistochemistry was done in a free-floating system. Tissue was washed with 10 mM phosphate buffer, pH 7.4 (PB), incubated for 45 minutes in 3% hydrogen peroxide in methanol, and washed in PB with 0.9% saline (PBS). Slices were then transferred to a buffer containing 100 mM glycine in PBS, and washed again in PBS and in PBS containing 0.1% Triton X-100 (Sigma)
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(PBS-T). Nonspecific binding was blocked by incubating the sections in PBS-T containing 3% bovine serum albumin (BSA, Sigma). Tissue was incubated with primary antibody (Neu-N, MAB 377, Chemicon Internationals, Inc; 1:700) diluted in BSA for 18 hours, at room temperature. Slices were then washed in PBS and incubated in secondary antibody (Chemicon International, Inc; 1:1000) diluted in BSA for 1–2 hours at room temperature. They were washed again in PBS and incubated in the biotin–avidin–peroxidase ABC kit (Vector Laboratories) for 30 minutes, and then the immunoreaction was made with 1 mg/mL diaminobenzidine (DAB, Sigma) diluted in PBS with 0.03% hydrogen peroxide for 2 minutes. The reaction was stopped with distilled water and slices were mounted on gelatin-coated slides (0.5% gelatin and 0.05% sulfate–chrome–aluminum w/v), dehydrated, and coverslipped.
Fig. 1. Comparison of lesions in piriform cortices. (A) normal piriform cortex with Neu-N staining, (B) normal piriform cortex with Nissl histochemistry, (C) lesioned piriform cortex with Neu-N staining, (D) lesioned piriform cortex with Nissl histochemistry. Note that the lesion is more evident with Neu-N staining, but is confirmed with Nissl histochemistry. Bar = 500 lm.
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The tissues were observed under a light microscope (Olympus BX-60), and images were acquired with a digital camera (Cool-CCD, Optronics). We observed several anteroposterior areas per brain, ranging from the most anterior part of the hippocampus to its most posterior part, including the amygdala and entorhinal and piriform cortices. Two blinded observers counted the number of areas with evident cell loss in each animal. Only brain areas with extensive damage were added, making quantitative cell counting unnecessary (Fig. 1 shows examples of what we call ‘‘evident cell loss’’). Each of the following areas was counted as a separate structure: in hippocampus, dentate gyrus, hilus, CA4, CA3, CA2, CA1; subiculum; entorhinal cortex; pyriform cortex; and lateral/basolateral amygdala. Substantial neuronal loss observed unilaterally in one of those areas was scored as 1. Damage to the same area in both hemispheres was scored 2. Damage of the same area, ipsilaterally, in different sections of the same animal, was scored 1. Damage immediately surrounding the electrodes tips or their tract was ignored.
3. Results 3.1. Electrode positioning Electrodes occupied the AmyC and functionally related areas. Fig. 2 illustrates electrode positioning in the stimulated animals (based on Paxinos and WatsonÕs atlas of the rat brain [43]). The nuclei stimulated were lateral amygdala (n = 6), basolateral amygdala (n = 8),
basomedial amygdala (n = 2), medial amygdala (n = 1), bed nucleus of stria terminalis (n = 1), caudate putamen (n = 1), deep endopiriform nucleus (n = 2), and piriform cortex (n = 1). Because the main purpose of this work was to analyze the behavior and consequent neuroanatomical injury caused by GSE, we decided not to exclude animals that developed GSE but in which the electrodes were positioned outside the AmyC. Because areas outside the AmyC included in this study are intimately related to the AmyC [44], they are included when we refer to AmyC stimulation. 3.2. Behavior analysis We observed two different types of GSE induced by electrical stimulation of the AmyC. ‘‘Typical GSE’’ (TSE, 50% of animals) precludes the presence of all behaviors described by Pinel and Rovner [38]: facial automatisms, neck and forelimb myoclonus, rearing, falling, running, jumping, and generalized tonic–clonic seizures. On average, animals continuously exhibit heavy forelimb myoclous. The second type of GSE, so-called ‘‘ambulatory GSE’’ (ASE, 50% of animals) includes only the lowest scores, such as facial automatisms and neck myoclonus. Concomitantly, animals with ASE manifested continuous ambulatory behavior in the observation cage. TSE scores were higher scores than ASE scores during the entire maintenance period, when self-sustained SE occurred but not concomitant with electrical stimulation (P < 0.05, ANOVA on ranks with Tukey test) (Fig. 3). Once GSE was established (maintenance period), there was no relationship between the
Fig. 2. Distribution of electrode tips from the stimulated rats: black dots indicate electrodes inside the AmyC and gray squares indicate electrodes in AmyC-related areas.
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Fig. 3. There are two different types of GSE consequent to AmyC electrical stimulation. It is shown that these GSE types are established during the maintenance phase and that TSE (triangles) is more severe than ASE (squares). ANOVA on ranks followed by Tukey test, P < 0.05.
severity of behavior and the area stimulated (data not shown).
EEGs (Fig. 4), marked by the presence of high-amplitude and -frequency discharges intermixed with flattened periods.
3.3. EEG 3.4. Histology In this work, we used the model of SE proposed by Nissinen et al. [20]. In addition to behavioral observation, we also recorded EEG patterns during seizures (Fig. 4). Instead of performing the extensive EEG analysis already done by that research group with EEG data, we compared patterns qualitatively. Comparison of our observations with AmyC stimulation and their description of the EEGs of rats exhibiting SE in response to lateral amygdaloid nucleus stimulation indicates that our animals with SE had EEGs very similar to their animalsÕ
Fig. 4. EEG recordings from amygdala (AMY) and hippocampus (HIP) during SE, demonstrating the so-called ‘‘high-amplitude and frequency discharge’’ (third EEG line, between arrows) and the ‘‘flat periods’’ of 1 to 3 seconds following it (third EEG line, asterisk). There is shown an approximately 120-second period of EEG, divided into three successive epochs (upper, middle, and bottom). Bars = (horizontal, time) 3 seconds and (vertical, voltage) 2 mV.
Histological analysis (Nissl staining confirmed with selective Neu-N immunostaining) revealed that, at 3 hours (n = 2 ambulatory GSE, n = 5 TSE, and n = 4 controls), no temporal lobe-related area showed evident cell loss in any of the groups studied. In contrast, 24 hours after stimulus onset (n = 4 ambulatory GSE, n = 2 TSE, and n = 5 controls), piriform cortex was intact or injured unilaterally in all ASE animals and bilaterally in all TSE animals. Fourteen days after GSE (n = 5 ambulatory GSE, n = 4 TSE, and n = 3 controls), ASE animals had unilateral or bilateral cell loss in piriform cortex and TSE animals had bilateral cell loss in piriform cortex and in the hippocampal formation, including the subiculum. Examples of what we considered evident lesions are compared with control tissues in Fig. 5A (Neu-N immunohistochemistry). The damage was also evident by Nissl staining, indicating that it was not a problem of impaired Neu-N expression, but real neuronal loss. Statistical analysis of these data (Pearson correlation) revealed a statistically significant positive correlation between the median seizure score of each rat (considering all 5-minute intervals of the SE protocol) and the total number of injured areas both 24 hours (R = 0.873, P < 0.001) and 14 days (R = 0.798, P < 0.01) after GSE (Fig. 5B). Neo-Timm staining showed that only one animal, at 14 days after TSE, exhibited light mossy fiber sprouting. All other animals, in all the groups studied, did not exhibit mossy fiber sprouting (Fig. 6).
4. Discussion To our knowledge, this is the first time that the behavioral severity of SE is shown to be related to the
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Fig. 5. (A) Images of hippocampal formation (top) and piriform cortex (bottom) of rats killed 14 days after GSE induction. From left to right are tissues from rats with control, ambulatory and typical GSE. Asterisks and arrows indicate what we considered areas with evident lesions. Neu-N immunohistochemistry. Bars = 500 lm. (B) Severity of behavior during the maintenance phase of GSE correlates with the number of areas with evident lesions, at both 24 hours and 14 days after GSE induction (bottom). Pearson correlation, P < 0.05.
pathological alterations subjacent to TLE in animals. Our results demonstrate that stimulation of the AmyC and related areas for 30 minutes, with the parameters used in this work, is effective in inducing a self-sustained SE that can be maintained at least for 2 hours. Nissinen et al. [20] had proposed this protocol of SE induction with stimulation of the lateral nucleus of the amygdala. In this work, we also stimulated other AmyC subnuclei, as well as other nuclei closely related to the AmyC. We found that, once the self-sustained SE is established, it does not matter what nucleus was stimulated. However, we also observed that stimulation of the lateral and basolateral nuclei of the amygdala is more effective in
inducing self-sustained SE than stimulation of other nuclei (about 80% of the animals stimulated in the lateral and basolateral nuclei develop GSE, as compared with about 0–50% of the animals stimulated in other nuclei; data not shown). The EEG data obtained by our group also resemble those of Nissinen et al. [20], and constitute another parameter that confirms the success of this model using the stimulation of other nuclei related to the amygdala. It may also suggest that the circuitries involved in the maintenance of SE are similar, if not the same. During the maintenance phase of our experiments (self-sustained SE) we observed that the rats could be
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Fig. 6. Animals with GSE did not manifest sprouting (center, compare with control in top), with the exception of one with a very light sprouting (bottom, arrows). Bar = 500 lm.
subdivided into two groups: one exhibiting very strong SE, the so-called typical SE, where the behaviors observed corresponded to high categories of limbic seizures (based on RacineÕs scale [37], as modified by Pinel and Rovner [38]), such as forelimb myoclonus and rearing; and the other exhibiting a much lower severity of SE, corresponding to scores 1–2 on the scale, such as face and neck myoclonus. As several articles that cite the behavior observed during SE describe a SE similar to the high-score SE observed in this work, we decided to name it ‘‘typical GSE.’’ On the other hand, rats exhibiting low-score SE concomitantly walked around the observation box, with intermixed myoclonic jerks, which is why we called it ‘‘ambulatory SE.’’ Ambulatory SE is described elsewhere, but we do not know if those descriptions correspond exactly to the GSE we described here [45,46]. During the preparation of this article, we observed that other SE that could also be called ‘‘ambulatory’’ occurred in our rats, but the hippocampal electrodes were silent indicating a localized more than a generalized SE, and the animals did not exhibit concomitant face and neck myoclonus (unpublished data). We sacrificed the animals at different intervals after the initial stimulation to verify the possible neuropatho-
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logical consequences of SE. We observed neuronal loss as soon as 24 hours after SE, and this neuronal loss was aggravated after 14 days of SE. We did not count cells because, at this point, we consider only the neuronal loss that was evident, as can be seen in Fig. 1and 5A. A test for the relationship between number of areas with cell loss and severity of SE showed a strong positive correlation both 3 hours and 14 days after SE. Interestingly, 24 hours after GSE, lesions were restricted to piriform cortex, unilaterally or bilaterally, with no distinction between typical and ambulatory SE. But at 14 days after GSE, only the animals with TSE had hippocampal cell loss. No cell loss was observed in the hippocampal formation in the absence of cell loss in the piriform cortex. The piriform cortex was the first and most severely injured area in all rat brains studied in this work. The most affected area of the piriform cortex was layer III. Although the piriform cortex is not usually a main focus when TLE is discussed, some data indicate its relation to the pathology of epilepsy. Ebert et al. [47] verified that female rats injected with kainic acid (potent excitatory drug for induction of SE) manifested cell loss in the piriform cortex 24 to 28 hours after SE. Absence of the piriform cortex (when destroyed by ibotenic acid) causes a delay in the development of the kindling process [48]. Activation of piriform cortex by bicuculline also induces severe seizures in rats [49]. More interestingly, Librizzi and de Curtis [50] suggested that interictal activity of the piriform cortex could have a blocking effect over itself afterward, when epileptic activity occurred in the hippocampus or entorhinal cortex. The lesions observed in the hippocampal formation were more expected, because cell loss in this structure is extensively described [51,52]. The pattern of cell loss observed in the HIP in this work is in agreement with the patterns previously described: loss of neurons in the hilus of the dentate gyrus, in CA4, CA3, and CA1, with preservation of CA2. Some hypotheses try to explain the epileptogenic process by the pathology of the hippocampal formation observed in this and other studies. According to those theories, the sprouting observed in hippocampi from animals with chronic epilepsy occurs partly because of the death of neurons in the hilus of the dentate gyrus and in the CA4–CA3 regions. Interestingly, we did not find sprouting in our animals, with the exception of really light sprouting, unilaterally, in a brain from an animal with TSE sacrificed 14 days after SE induction (see Fig. 6). The cause/consequence relationship of the sprouting with epilepsy in animals subjected to SE induction is very controversial [21,53–59]. Unfortunately, we did not have the opportunity to observe if our animals exhibited spontaneous seizures by the time they were sacrificed, which could help to clarify the role of sprouting in the establishment of epilepsy in animal models.
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One obvious criticism that can be made of our results is the possibility of continuation of electrographic epileptiform activity despite the arrest of behavioral seizures. If stronger behavioral SE leads to longer-lasting electrographic SE, then the presence of more areas with neuron loss would be more a consequence of longer SE than of the severity of SE itself. Our data suggest that this may not be the case: 9 animals manifested EEG activity down to less than baseline 5 to 45 minutes after diazepam injection (4 typical SE, 5 ambulatory SE), and in the other 11, the duration of epileptiform activity on the EEG was not known (still above the baseline when we finished the EEG recording, up to 1 hour after diazepam injection; 6 typical SE and 5 ambulatory SE). The even distribution of short and unknown EEG epileptiform activity durations between typical and ambulatory SE is already evidence that this is not the factor leading to differences in the quantity of severely damaged areas between groups. To confirm this suspicion, we compared data from TSE animals whose epileptiform EEG activity, together with behavioral epileptiform arrest activity, ended within 30 minutes of diazepam injection (n = 2; one killed 24 hours and one killed 14 days after SE induction) with data from ASE animals that still exhibited EEG epileptiform activity after behavioral epileptiform arrest and termination of EEG recording (unknown duration of electrographic seizure on EEG; n = 4; 2 killed 24 hours and 2 killed 14 days after SE induction). TSE animals had 7 and 11 damaged areas
and ASE had 0, 1, 2, and 3 damaged areas, confirming that the number of areas with severe neuronal loss depends on the type of behavior exhibited during SE more than the EEG duration of the SE. Fig. 7 shows some examples of the analysis just described. Interestingly, in a recent publication, Mohapel et al. [46] also reported that different severities of SE lead to different pathologies in rat brains. They found that rats with the so-called ‘‘fully convulsive SE’’ (more severe, probably corresponding to TSE) exhibit more neurogenesis in the dentate gyrus of the hippocampus, although those new neurons died after 4 weeks of SE. On the other hand, what they called ‘‘partial convulsive SE’’ (less severe, probably corresponding to ASE) exhibited less neurogenesis, but those new neurons survived after 4 weeks of SE. Our work complements that of Mohapel et al., corroborating their findings and adding some further data on the time-related events of the injury consequent to different severities of SE. In conclusion, this work showed that electrical stimulation of the amygdala and related areas induces SE of different severities. The severity of the SE observed in our animals induced different levels of injury in brain areas that participate in the seizures. Based on the results, we suggest that the behavior of animals submitted to SE may be an important factor in analysis of the pathophysiology of epileptogenesis in future studies using SE as a paradigm for the investigation of epilepsy.
Fig. 7. Lack of relationship between electrographic seizure duration after diazepam injection and area of brain damage. Data from three representative cases: TSE (upper row); ASE (middle row), and control (bottom row). The first column shows the compacted EEG from each animal (gray: hippocampus channel; black: amygdala channel). The second and third columns show left and right hippocampi (respectively), and the fourth and fifth columns, left and right amygdala (respectively) of each animal. Top row: animal with TSE with electrographic epileptiform activity completed within 5–15 minutes of diazepam (DZP) injection. Middle row: animal with ASE with electrographic epileptiform activity not completed within recording protocol (time of EEG seizures unknown). EEG recordings: black, HIP; gray, AmyC; /, stimulation window; #, electrode tract. The control rat received no stimulation to induce SE induction or a brain lesion. Note that, although the rat with TSE experienced SE for a shorter time than the animal with ASE, the former manifested more areas with neuronal loss (asterisks) than the latter. Bar = 250 lm.
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Acknowledgments The authors thank the following Brazilian agencies for funding: Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP), Programa de Apoio a Nu´cleos de Exceleˆncia do Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (PRONEX-CNPq), Programa de Apoio a Po´s-Graduac¸a˜o da Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (PROAP-Capes) and Fundac¸a˜o de Apoio ao Ensino, Pesquisa e Assinteˆncia do Hospital das Clı´nicas da Faculdade de Medicina de Ribeira˜o Preto da Universidade de Sa˜o Paulo (FAEPA-HC/FMRP-USP). C.Q.T. and N.G.-C. held FAPESP and CNPq Research Fellowships, respectively, during the execution of this work.
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