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Long-term behavioral and morphological consequences of nonconvulsive status epilepticus in rats
Epilepsy & Behavior Epilepsy & Behavior 5 (2004) 180–191 www.elsevier.com/locate/yebeh
Long-term behavioral and morphological consequences of nonconvulsive status epilepticus in rats Pavel Krsek, Anna Mikuleck a, Rastislav Druga, Hana Kubov a, Zdenek Hli nak, * Lucie Suchomelov a, and Pavel Mares Institute of Physiology, Academy of Sciences of the Czech Republic, Vıdenska 1083, CZ 142 20 Prague 4, Czech Republic Received 23 July 2003; revised 24 November 2003; accepted 25 November 2003
Abstract The aims of the present study were to ascertain whether nonconvulsive status epilepticus (NCSE) could give rise to long-term behavioral deficits and permanent brain damage. Two months after NCSE was elicited with pilocarpine (15 mg/kg ip) in LiCl-pretreated adult male rats, animals were assigned to either behavioral (spontaneous behavior, social interaction, elevated plus-maze, rotorod, and bar-holding tests) or EEG studies. Another group of animals was sacrificed and their brains were processed for Nissl and Timm staining as well as for parvalbumin and calbindin immunohistochemistry. Behavioral analysis revealed motor deficits (shorter latencies to fall from rotorod as well as from bar) and disturbances in the social behavior of experimental animals (decreased interest in juvenile conspecific). EEGs showed no apparent abnormalities. Quantification of immunohistochemically stained sections revealed decreased amounts of parvalbumin- and calbindin-immunoreactive neurons in the motor cortex and of parvalbumin-positive neurons in the dentate gyrus. Despite relatively inconspicuous manifestations, NCSE may represent a risk for long-term deficits. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Lithium–pilocarpine status epilepticus; Epileptic brain damage; Motor performance; Behavior; Calcium-binding proteins
1. Introduction It has been proved that convulsive status epilepticus causes serious brain damage via excitotoxic mechanisms [e.g., 1]. A complex neurotoxic cascade consisting of multiple serial and parallel processes leading to both necrotic and apoptotic cell death has been described [2,3]. It is now evident that ‘‘epileptic‘‘ brain damage results from excessive neuronal activity during status epilepticus (SE), with complicating systemic factors playing an additional role. Consequently, there is also a conceptual framework for brain damage resulting from nonconvulsive status epilepticus (NCSE). However, although a number of clinical reports support the view that convulsive SE represents a serious risk for the human brain, possible harmful effects of NCSE are still a source of controversy.
* Corresponding author. Fax: +420-2-41062488. E-mail address: [email protected] (P. Maresˇ).
1525-5050/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2003.11.032
It has repeatedly been demonstrated that complex partial status epilepticus, one of the two main types of NCSE in humans, can be accompanied by considerable neurological morbidity, as well as mortality [4–8]. These clinical observations are supported by MRI findings [9] as well as by studies on serum neuron-specific enolase, an accepted marker of acute brain injury [10,11]. However, most deficits are completely reversible [12,13]. It is also stressed that the poor outcome of patients may be related to the course of a systemic disease and not to complex partial SE itself [14,15]. It is therefore doubtful whether clinical studies can definitively answer questions concerning harmful effects of complex partial SE on the brain. Some electrically and pharmacologically induced animal models of NCSE have been introduced. Hosford recently asserted that the following models satisfy the criteria for an animal model for complex partial SE: kainic acid model, pilocarpine model, prolonged kindling, and LothmanÕs self-sustaining limbic SE [16]. All these models cause extensive brain damage. However, a
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marked discrepancy between the dire consequences of artificially induced SE in animals and the relatively good outcome of patients after complex partial SE has been reported [15]. We suggest that one possible explanation of this disagreement is that these experimental models represent much more severe insults compared with human complex partial SE. Similarly, Drislane recently suggested that EEG discharges recorded in the majority of animal models are better suited to convulsive SE than to complex partial SE, with its usually less dramatic electroencephalographic patterns [17]. Short-term and self-limited NCSE induced by the administration of low doses of pilocarpine in lithium chloride-pretreated rats was developed in our laboratory [18]. NCSE consisted of approximately 90 minutes of abnormal behavior characterized by the occurrence of various epileptic automatisms, e.g., chewing, nodding, face washing, and exploratory behavior as if in a new environment. Both cortical and hippocampal spikes, isolated or in bursts, as well as other types of epileptic EEG activity, accompanied these behavioral features. Profound impairment of responsiveness to exteroceptive stimuli correlating with the occurrence of epileptic EEG activity was observed [19]. One and two weeks after NCSE, seizure-related brain damage consisting of dark and shrunken neurons in Nissl-stained brain sections was observed mainly in the motor neocortical fields [18,19]. On the basis of these observations, we were able to define a new pharmacologically induced model of human complex partial SE. However, it remained to be ascertained whether this relatively brief NCSE could cause long-term functional and morphological consequences. This presumption was supported by the study of Cook and Persinger, who described a deficit in longterm memory using the radial maze paradigm in rats injected with a subconvulsive dose of pilocarpine 5 months before testing [20]. To determine the possible delayed consequences of NCSE, video EEG monitoring, behavioral testing, and morphological examination were conducted 2 months later. Histological examination of brain sections was performed to clarify whether morphological damage observed 1 and 2 weeks after NCSE was permanent. Moreover, brain slices were immunostained using antibodies to the calcium-binding proteins calbindin D-28k and parvalbumin to specifically evaluate affected cell populations in both the neocortex and limbic structures.
2. Methods 2.1. Animals Adult (90-day-old) male albino Wistar rats (N ¼ 114) weighing 250–350 g were used. They were housed in
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standard plastic cages in a temperature-controlled room (22 1 °C) with a 12-hour light/dark cycle (lights on at 6:00 A M ). Food and water were available ad libitum in the home cage. In addition, 32 juvenile rats 21 days old were used in the social interaction test. All experiments were conducted under approval of the Animal Care and Use Committee of the Institute of Physiology of the Academy of Sciences in agreement with the Animal Protection Law of the Czech Republic (fully compatible with the guidelines of European Community Council Directives 88/609/EEC). 2.2. Experimental procedure NCSE was elicited with pilocarpine (15 mg/kg ip) 24 hours after lithium chloride pretreatment (3 mEq/kg ip). The rats were placed in separate plastic boxes and their behavior was observed for 120 minutes. EEG recording was not performed in this study because of data from the previous study [18]. Experimental groups were formed only by animals exhibiting clear-cut features of NCSE (N ¼ 63). Control rats (N ¼ 51) received an equal volume of saline instead of pilocarpine; other conditions were the same as for the pilocarpine-treated animals. These controls were put together from all ongoing studies on nonconvulsive status. Two rats included in the control group received pilocarpine also but they did not develop any signs of NCSE. Animals exhibiting short-lasting behavioral signs of nonconvulsive seizures (N ¼ 3) were not included in the present study; it would be necessary to have more rats to make an additional group. Animals exhibiting motor seizures were included in other studies focused on convulsive status epilepticus. All experiments were performed 2 months after NCSE. Three groups of animals were established. First, video EEG monitoring was conducted in 6 pilocarpinetreated and 2 control animals. The second group, consisting of 46 pilocarpine-treated and 44 control rats, was started on behavioral testing. Rats in the third group (11 pilocarpine-treated rats and 5 controls) were sacrificed and their brains processed for morphological techniques. Animals from the first and the third groups were not tested behaviorally to ensure that possible changes are due to their previous nonconvulsive status. 2.3. Video EEG monitoring Animals were anesthetized with ketamine (100 mg/kg ip) and xylazine (20 mg/kg im). Two flat silver electrodes were placed over the sensorimotor areas of both hemispheres (AP ¼ 0, L ¼ 2 mm), two Teflon-coated stainless-steel electrodes were inserted into the dorsal hippocampus (AP ¼ 3.5, L ¼ 2, H ¼ 3:8 mm), and an indifferent electrode was inserted into the nasal bone. The electrodes were fixed to the skull with dental acrylic.
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One week after surgery, animals were individually placed in plastic boxes (30 40 45 cm) and attached to the EEG apparatus (BrainScope) with flexible wires and swivels so that their movement was not restricted. The EEG was continuously recorded for 5 hours for six experimental and two control rats. Behavior was simultaneously videotaped. Localization of hippocampal electrodes was checked histologically after the end of the experiment. Again, all animals included in EEG study had tips of electrodes placed in the CA3 field of the dorsal hippocampus. 2.4. Behavioral procedure Animals (N ¼ 90) were tested between 9:00 A M and 3:00 P M . All the experimental devices used were thoroughly cleaned before a new animal was tested. In both the spontaneous behavior test and the social interaction test, rat behavior was monitored using a video camera located 100 cm away from the arena. The videotape was scored by an experienced observer (intrarater reliability > 0.9) using the Observer program (Noldus Information Technology). Rats were never used for two behavioral tests; only some animals were exposed to one behavioral and one motor (rotorod or bar holding) test. 2.5. Spontaneous behavior test Animals (14 pilocarpine-treated rats and 14 controls) were placed individually in the square arena (plexiglas cage, 45 45 30 cm) and their behavior was monitored for 5 minutes. The following categories of spontaneous behavior were distinguished: walking (movement around the arena); rearing (upright posture both against and away from the wall); sniffing (investigation of the floor, walls, and space); face washing (brief period of vibrating movements of the forepaws in front of the snout followed by nose or head washing and paw licking); grooming (face washing with a subsequent cephalocaudal progression); and immobility (lying or sitting). The total number, total duration (in seconds), and mean duration (a derived measure: total duration divided by total number) of individual categories were evaluated. 2.6. Social interaction test On the day of the experiment, both pilocarpinetreated (N ¼ 16) and control (N ¼ 16) adults and juveniles (21 days old, N ¼ 32) were housed individually. After 1 hour of adaptation to the experimental room, an adult animal was placed in the experimental arena (identical to that described above) to explore the new environment. Two minutes later, a juvenile conspecific was placed in the arena. Each interaction lasted 5 minutes. Reexposure to the same juvenile occurred 30 minutes after the first interaction. Between successive
exposures, both adults and juveniles were maintained individually. The total number and total duration of social investigations directed toward the juvenile (approaching, nosing, and body sniffing) were calculated. 2.7. Elevated plus-maze test Pilocarpine-treated (N ¼ 16) and control (N ¼ 14) animals were placed individually on the open arm of the plus-maze, and the transfer latency (time required for an animal to move from the open arm to the enclosed arm) was recorded. The second session was carried out 24 hours later. If the rat did not enter the enclosed arm within 120 seconds, it was excluded from the evaluation (2 of the original group of 30 rats). 2.8. Motor performance 2.8.1. Rotorod test The rotorod apparatus was set to rotate at a constant 5 rpm. The rats (25 pilocarpine-treated and 22 control rats; these groups were formed by animals exposed to the open field and social interaction test) were placed on the rod with the body axis perpendicular to the rodÕs long axis and with the head directed against the direction of rotation. The maximal score in maintaining equilibrium was arbitrarily fixed at 60 seconds. To observe the behavioral strategy used by an animal to maintain equilibrium, rats were placed on the rod individually. Three strategies were observed: grasping (motionless grasping of the rod), walking (walking on the rod), and orientation (described as scanning relative to the rod and attempting to stand upright). 2.8.2. Bar-holding test Rats (14 pilocarpine-treated and 14 control rats, the same animals as in the spontaneous behavior test) were hung by both forepaws on a wooden rod (25 cm long, 1 cm in diameter) located 1 m above a landing platform covered with a thick sheet of soft plastic to cushion falls. The duration of grasping was evaluated. 2.9. Morphology 2.9.1. Fixation Two months after treatment, an overdose of urethane (2 g/kg ip) was administered and animals were perfused with 0.37% sulfide solution (1 mL/g) for 10 minutes followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (1 mL/g), for 10 minutes. The brains were removed from the skull and postfixed in buffered 4% paraformaldehyde for 3 hours and then cryoprotected in a solution containing 20% glycerol in 0.02 M potassiumbuffered saline for 24 hours. The brains were frozen in dry ice and stored at )70 °C until cut. Brains were sectioned in the coronal plane (50 lm, 1-in-5 series) with a
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sliding microtome and sections were stored in a cryoprotectant tissue-collecting solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium phosphate buffer) at )20 °C until processed. Adjacent series of sections were used for Nissl and Timm staining. 2.9.2. Nissl and Timm staining The first series of 1-in-5 sections were mounted onto gelatin-coated slides and stained with cresyl violet. For the Timm sulfide/silver method, all sections (1-in-5 series) in which hippocampus was present were mounted on gelatin-coated slides, air-dried, and stained in the dark according to the following protocol: A working solution containing arabic gum (300 g/L), sodium citrate buffer (25.5 g/L citric acid monohydrate and 23.4 g/L sodium citrate), hydroquinone (16.9 g/L), and silver nitrate (84.5 mg/kg) was poured into the staining dish, which contained the slides. The sections were developed until an appropriate staining intensity was attained (approximately 60 minutes). Then the slides were rinsed with tap water for 30 minutes and placed in 5% sodium thiosulfate for 12 minutes. Finally, sections were dehydrated through an ascending series of ethanol, cleared in xylene, and coverslipped. Nissl-stained slices were examined to identify the cytoarchitectonic boundaries as well as the possible distribution of neuronal damage and to localize hippocampal electrodes in animals tested electrophysiologically. 2.9.3. Immunohistochemistry Sections were sequentially incubated in 0.15% hydrogen peroxide in PBS (0.01 M, pH 7.4) for 10 minutes, rinsed with PBS four times, and then incubated with blocking solution containing 2% normal horse serum (Vector Laboratories, Burlinghame, CA, USA) and 0.1% Triton X-100 in PBS at room temperature. The sections were incubated with the primary antibody (antiparvalbumin and/or anti-calbindin monoclonal antibody, Sigma, dilution; 1:5000 in PBS containing 1.5% normal horse serum and 0.1% Triton X-100) for 48 hours at 4 °C and then rinsed four times in PBS. Sections were incubated for 1 hour at room temperature with secondary antibody, biotinylated anti-mouse antibody made in horse serum (Vector); dilution 1:50 in PBS containing 1.5% normal horse serum and 0.1% Triton X-100. After this step, the tissue was rinsed with PBS four times and covered with the ABC reagent (Vectastain Kit, Vector) for 1 hour at room temperature. After being rinsed, sections were incubated for 5–7 minutes with a mixture of 0.02% diaminobenzidine and 0.05% hydrogen peroxide in PBS. Control sections were prepared with the same procedures, but omitting the primary antibody. All sections were mounted onto gelatin-coated slides, coverslipped, and examined by light microscopy (Olympus AX 70 microscope with brightfield optics).
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2.9.4. Analysis of the sections Synaptic reorganization (mossy fiber sprouting) was analyzed in sections stained the using the Timm method. Sprouting was analyzed along the septotemporal axis of the hippocampus; method was described in detail by Nissinen et al. [21]. The septal end included the coronal sections between AP levels 2.3 and 6.0 mm posterior from bregma [22]. The dorsal and ventral midportions of the dentate gyrus included dorsal and ventral parts of hippocampus where the granule cell layer of the septal and temporal ends becomes fused and forms a standardized ‘‘oval-shaped’’ layer (AP level 6.1–6.7 mm posterior to bregma). Mossy fiber sprouting was scored according to Cavazos et al. [23]. The distribution of Timm granules in the supragranular layer was rated from 0 to 5. While the lowest levels (0 and 1) indicate negative finding or sparse granules, respectively, more numerous granules are scored as 2, occasional patches of granules as 3, presence of a dense laminar band of granules as 4, and extension into the inner molecular layer as 5. Parvalbumin-immunoreactive (PV-IR) neurons were counted throughout layers II–VI of motor neocortical fields Fr 1, Fr 2, and Fr 3 (where the most prominent effect was observed 1 and 2 weeks after NCSE in the previous studies [18,19]). For each animal 12 sections in the range of stereotaxic planes AP 2.2 and AP )0.3 were counted [22]. Calbindin-immunoreactive (CB-IR) neurons were counted in the same anteroposterior range of motor fields using 0.3 0.3-mm squares localized randomly in neocortical layers V and VI. Sixteen squares per each of 12 sections from each rat were evaluated. Classification of the neocortical areas was based on the cytoarchitectonic criteria of Zilles [24]. PV-IR neurons in the dentate gyrus were counted in the whole mediolateral extent of both blades in 12 sections per each animal in the range of stereotaxic planes AP )2.8 and AP 4.3. PV-IR neurons in CA1 and CA3 hippocampal fields were counted in 0.3 0.3-mm squares (12 squares per each animal). PV-IR neurons in the CA1 field were restricted to the segment between sagittal planes 1 and 2 or 1 and 3 in more caudal sections, respectively. Terminology used in description of the limbic structures is according to Paxinos and Watson [22]. All analyses were conducted blind to treatment group. 2.10. Statistics Behavioral data were analyzed nonparametrically. To compare the differences between independent groups (pilocarpine-treated animals vs controls) within a given session, the Mann–Whitney U test was used. To compare the differences within matched pairs (subsequent sessions for both pilocarpine-treated and control rats) in the social interaction and elevated plus-maze tests, the
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Wilcoxon matched-pairs signed-ranks test was used. Morphological data (numbers of PV-IR and CB-IR neurons) were analyzed parametrically with the t test. Statistical significance was accepted at P < 0:05 (twotailed).
3. Results NCSE was elicited in all 63 experimental animals included in this study. It was characterized by hypersalivation, piloerection, licking, swallowing, and chewing starting immediately after pilocarpine administration. These phenomena could not be taken as epileptic; rather, they represented the peripheral action of pilocarpine. Then automatisms like face washing, head nodding, and creep walking (ataxia-like movements) were observed. Fully developed ictal behavior was characterized by immobility (with chewing and head nodding) interrupted by periods of creep walking. Gradual disappearance of the above-mentioned abnormal behavior was accompanied by repeated periods of exploration/searching activity (in a well-known cage) and self-grooming. These activities also subsided so that there were no apparent behavioral changes in the experimental animals 2 hours after pilocarpine treatment. Rats were returned to the animal facility and examined 2 months later.
Fig. 1. Relative representation of the occurrence (left) and duration (right) of individual behavioral patterns. C, control group (n ¼ 14); P, pilocarpine group (n ¼ 14). Statistical significance, P < 0:05 (twotailed): Pilocarpine versus control group (only duration of face washing exhibited a significant difference).
3.1. Video EEG monitoring Evaluation of video EEG recordings revealed no apparent EEG as well as behavioral abnormalities in experimental as well as control rats. 3.2. Behavioral data 3.2.1. Spontaneous behavior test There was no difference in the total frequency of all recorded behavioral patterns between pilocarpine-treated animals (48.1 5.6, mean SEM) and controls (42.5 5.7). Analysis of particular patterns revealed no change except for face washing (Fig. 1). The total time spent and the mean duration of face washing were significantly increased in pilocarpine-treated animals. 3.2.2. Social interaction test When compared with the controls (Fig. 2), a significant reduction in the total duration of social investigation was observed for pilocarpine-treated animals during the initial exposure. No difference in social investigation was found between pilocarpine-treated and control rats during the second exposure. Intersession comparisons of social investigation revealed significantly lower levels during the second exposure for both experimental and control rats.
Fig. 2. Total time spent by adult males in investigation of juveniles in the social interaction test (means + SEM). The second exposure to the same juveniles was performed 30 minutes after the first one. Open bars: control group (n ¼ 16); crosshatched bars: pilocarpine group (n ¼ 16). Statistical significance, P < 0:05 (two tailed): Pilocarpine versus control group, d second session versus first session.
3.2.3. Elevated plus-maze test No statistical difference in transfer latency between pilocarpine-treated and control animals was noted during both the initial session and the second session. As compared with the initial session, a significant
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shortening of the transfer latency during reexposure was noted in both groups of animals. 3.3. Motor performance: rotorod and bar-holding tests Maintenance of equilibrium on the rotorod without falling (Fig. 3) was significantly decreased in pilocarpine-treated animals. Moreover, the strategy of the pilocarpine-treated animals was different from that of the controls. While control rats walked on the rod and maintained equilibrium, pilocarpine-treated rats attempted to rear, scan, and turn in the direction of the rotation and to sit; 16 of 25 pilocarpine-treated animals fell. In the bar-holding test, the duration of grasping was significantly decreased in the pilocarpine-treated animals. Similarly to controls, pilocarpine-treated rats attempted to climb the bar; however, they were less skillful (Fig. 3). 3.4. Morphology 3.4.1. Nissl and Timm staining Whole brain was always checked in Nissl-stained sections. There were dark and shrunken neurons in layers V and VI of the motor neocortical areas (fields Fr 1–3) in pilocarpine-treated rats (Figs. 4A,B). Small numbers of dark neurons were inconsistently found in the corpus striatum and CA3 hippocampal field. Exact quantification of the changes was precluded by the thickness of the slices; however, the number of affected neurons appeared to be smaller in comparison with the numbers 1 and 2 weeks after NCSE [18,19]. No obvious changes were observed in any other brain structure.
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Evaluation of Timm-stained sections showed no sprouting of mossy fibers in the hippocampus of pilocarpine-treated animals, i.e., level 0 according to Cavazos et al. [23]. 3.4.2. Immunohistochemistry In control animals, PV-IR cells were found in all cortical layers except layer I, and exhibited a variety of somatic and dendritic morphologies (Fig. 4C). Multipolar cell bodies predominated in the upper layers while small pyramidal and bipolar neurons were less frequent. In layers V and VI, some bipolar and triangular (pyramidal-like) neurons were distinguished in addition to the prevailing multipolar neurons. Most PV-IR cells were moderately to heavily labeled. The highest density of PV-IR neurons was located in the supragranular layers of the motor area. Two populations of CB-IR cells could be distinguished. First, weakly to moderately positive neurons predominated in layers II and III and could also be found in layer V. Many weakly and moderately CB-IR cells were obscured by dense diffuse staining of the neuropil in layers I–III. Second, heavily labeled CB-IR neurons were found scattered in layers II–VI but most of them were localized in two belts corresponding to layers II–III and V–VI. Both bipolar and multipolar perikarya were present in the supragranular layers, while the majority of heavily labeled cells in layer V–VI were multipolar (Fig. 4E). In pilocarpine-treated animals, a significant reduction in both PV-IR and CB-IR neurons in the motor neocortical area was evident in comparison with controls (Figs. 4D,F). The number of PV-IR neurons throughout layers II–VI was 47.7 2.88 (mean SEM)
Fig. 3. Motor performance in the rotorod test (left, expressed as the time spent on the rod without falling down) and the bar-holding test (right, expressed as the time of grasping). Data are expressed as means + SEM. Open bars: control group (n ¼ 22 and 14, respectively); crosshatched bars: pilocarpine group (n ¼ 25 and 14, respectively). Statistical significance, P < 0:05 (two-tailed): Pilocarpine versus control group.
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Fig. 4. Photomicrographs demonstrating parvalbumin-containing cells in the sensorimotor cortex (A,B), calbindin-containing cells in the motor cortex (C,D), and parvalbumin-containing cells in the dentate gyrus (E,F). Control rats: A, C, E; animals after nonconvulsive status: B, D, F. Bars ¼ 500 lm. Roman numerals denote cortical layers. WM, white matter. CA3, hippocampal field CA3; sm, stratum moleculare; sg, stratum granulare. Arrowheads in (E) and (F) denote parvalbumin-positive cells on the borderline between the granular layer and hilus of the dentate gyrus.
in pilocarpine-treated and 94.3 4.72 in control animals. A decline in PV-IR cells was particularly prominent in layers II and III. Some of the persisting neurons exhibited weaker immunopositivity of perikarya and dendrites when compared with controls. The average number of CB-IR neurons in a 0.3 0.3-mm area localized randomly in layers V and VI was 4.57 0.13 in pilocarpine-treated rats and 6.33 0.26 in the control group (Fig. 5). In control animals, PV-IR neurons predominated in the stratum pyramidale and oriens of the hippocampus and in the stratum granulosum and hilus of the dentate gyrus (Fig. 4G). A decline in the number of PV-IR neurons was demonstrated in both blades of the dentate gyrus in pilocarpine-treated rats (Fig. 4H). The average
number of PV-IR cells in the dentate gyrus was 6.75 0.49 in pilocarpine-treated and 10.27 0.82 in control animals. On the other hand, no significant differences in the numbers of PV-IR neurons were found in the CA1 (17.6 1.78 pilocarpine-treated vs 20.7 1.0 controls) and CA3 (12.2 0.72 pilocarpine-treated vs 12.4 0.89 controls) hippocampal fields (Fig. 6).
4. Discussion Classic pilocarpine-induced SE represents a model of secondary generalized convulsive status epilepticus [25,26]. Studies dealing with subconvulsive doses of pilocarpine are rare. Two original studies described
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Fig. 5. Average number of calbindin-positive neurons counted in the square of 0.3 0.3 mm localized randomly in layers V and VI of the motor neocortex (left); average number of parvalbumin-positive neurons counted in the whole range of layers V and VI of the motor neocortical fields Fr 1– 3 of both hemispheres (right). Open bars: control group (n ¼ 5); crosshatched bars: pilocarpine group (n ¼ 11). Data are expressed as means + SEM. Statistical significance, P < 0:05 (two-tailed): pilocarpine versus control group.
Fig. 6. Average number of parvalbumin-positive neurons in the dentate gyrus (counted in the whole extent of both blades) and in the CA1 and CA3 hippocampal fields (counted in a square of 0.3 0.3 mm). Open bars: control group (n ¼ 5); crosshatched bars: pilocarpine group (n ¼ 11). Data are expressed as mean + SEM. Statistical significance, P < 0:05 (two-tailed): Pilocarpine group versus control group.
effects of nonconvulsant pilocarpine doses in rats not pretreated with lithium chloride [27,28]. Sixty to one hundred twenty minutes of abnormal behavior concurrently with EEG changes (theta rhythm and spiking in
the hippocampus and low-voltage fast activity in the cortex) were observed. However, we found only one subsequent study that focused on these phenomena and reported expression of the c-fos protein in certain limbic areas induced by a subconvulsant dose of pilocarpine [29]. Our previous results demonstrated that the model of NCSE induced by a subconvulsant dose of pilocarpine in lithium chloride-pretreated rats, characterized by behavioral automatisms and spikes (isolated as well as in series) in hippocampal and cortical EEGs, is equivalent to human CPSE [18,19]. The striking result of the analysis of this model was the finding of morphological damage in the motor neocortex 1 and 2 weeks after NCSE. As one of the most critical questions regarding NCSE in humans concerns the risk of long-term harmful sequelae to the brain, we turned our attention to analyzing both behavioral and morphological consequences of nonconvulsive seizures in our model. The possibility of behavioral sequelae was suggested by the study of Cook and Persinger [20], a pioneering morphological study. Video EEG monitoring did not reveal any epileptic activity in our animals. This contrasts sharply with data for convulsive status epilepticus (lithium–pilocarpine model with pilocarpine doses of about 40 mg/kg and/or high-dose pilocarpine model) where life-long spontaneous seizures were recorded under conditions similar to those for our monitoring [30]. Behavioral tests revealed some deficits 2 months after NCSE. Conversely to the prominent disturbances observed in rats during pilocarpine-induced NCSE [18,19], discernible long-term behavioral consequences were only subtle. No long-term structural disintegration
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of spontaneous behavior was observed. However, the animals experiencing NCSE displayed more face washing. In intact animals, this behavioral component occupies the second position in the sequential organization of grooming behavior [31,32]. An increased display of face washing in animals experiencing NCSE probably reflects different levels of responsiveness following exposure to a stressor when placed into a new environment. It has been proved that the grooming response is related to age [33,34] and, among others, also to neurohormonal modulation [35,36]. The social interaction test revealed decreased investigation by both pilocarpine-treated and control animals reexposed to the same juvenile 30 minutes after the initial interaction, suggesting that the animals are able to recognize juvenile conspecifics [37–40]. However, duration of investigation during the initial exposure was markedly decreased in animals after NCSE. One possible explanation is that the decreased interest in juveniles by pilocarpine-treated animals might reflect deficits in sensitivity as well as in responsiveness of animals exposed for the first time to social novelty. On the other hand, pilocarpine-treated rats remembered their juvenile conspecifics; there was a significant reduction in duration of investigation during the second exposure. The reduction in the transfer latency in the elevated plus-maze test during the second session showed that spatial orientation and/or short-term memory of pilocarpine-treated animals were not impaired. This, to a certain extent, contrasts with the finding of Cook and Persinger [20], who reported long-term memory disturbance in animals 5 months after treatment with a subconvulsive dose of pilocarpine. However, it is necessary to note that animals tested on the elevated plus-maze were apparently submitted to a less difficult memory task in comparison with the eight-arm radial maze used by Cook and Persinger. Long-term motor deficits were observed in animals that experienced NCSE. These may be caused by either a muscular tone deficiency or impaired skillfulness of the pilocarpine-treated animals. The static component of the equilibrium, in particular, seemed to be impaired. Moreover, pilocarpine-treated animals attempted to turn the body in the direction of the rotation on the rotorod and therefore fell. Equilibrium behavior depends on a number of sensory cues and on training [41,42]. The different behavioral strategy of the pilocarpine-treated animals could be related to deficits in perception, for example, of visual, proprioceptive, vestibular, and tactile stimuli. It has been demonstrated that the olivocerebellar pathway is involved in the temporal organization of motor learning and skills [43,44]. However, no discernible damage was observed in the brainstem (including oliva inferior) in our experimental rats. Therefore, a more plausible explanation is that the motor deficits reflect damage to the motor neocortical areas.
Both histological and immunohistochemical techniques demonstrated damage in different brain structures 2 months after NCSE, strongly supporting the view that short-term NCSE is able to damage the brain permanently. Damage to motor neocortical fields corresponding to motor deficits was the most conspicuous morphological finding. This is in accordance with our former observations [18]. It was suggested that the motor neocortex might be damaged by projection of the epileptic activity from the limbic structures via the amygdala [19]. Calcium-binding proteins like parvalbumin and calbindin play an important role as calcium transporters, a buffering system for intracellular calcium ions, and represent one of the most important calcium compartments in the brain [45]. The decrease in immunoreactivity (and probably in the concentrations of PV and CB) may thus increase neuronal susceptibility to calcium fluctuations, leading to structural and functional impairment. Analysis of tissue obtained from patients undergoing epilepsy surgery indicated reduced numbers of PV-IR and CB-IR neurons in human neocortical epileptic foci [46]. Loss of PV-IR neurons was also detected in the neocortex of the temporal pole in patients with intractable temporal epilepsy [47]. A decrease in glutamate decarboxylase and PV immunostaining was observed in the high-dose pilocarpine model in rats surviving 60 days after the onset of chronic seizures [48]. These findings support an involvement of neocortex in temporal lobe epilepsy with secondary generalization. The distribution of PV-IR and CB-IR neurons in the motor cortical areas (fields Fr1, Fr2, and Fr3) of control animals was similar to that reported in the studies of Celio [49] and van Brederode et al. [50]. In our pilocarpine-treated rats, a decline in both PV-IR and CB-IR neurons was demonstrated in layers II–VI and V–VI of the motor neocortical fields, respectively. This finding indicates that NCSE influenced neuronal expression of the calcium-binding proteins in the same direction as convulsive SE. PV-IR and CB-IR cells represent a subpopulation of cortical GABAergic inhibitory neurons [49]. PV is localized in fast-spiking, metabolically active large basket cells and chandelier cells in the neocortex. On the contrary, CB-IR cells are typically regular-spiking double-bouquet cells (some are fast-spiking ones) [51–53]. A reduction in neocortical PV and CB immunoreactivity thus suggests an impairment of GABAergic inhibition as a consequence of nonconvulsive SE. Our prior work failed to demonstrate an apparent effect on limbic structures, where the most prominent epileptic brain damage has repeatedly been described in humans [1,54], as well as in the majority of animal models of SE [2,16]. For that reason, discovery of the dentate gyrus lesion demonstrable by PV immunohistochemistry could be regarded as a crucial result of the
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present work. Although dark neurons were found in some pilocarpine-treated animals, examination of Nisslstained brain sections failed to reveal discernible damage to limbic structures 2 months after NCSE. However, it was suggested that conventional cresyl violet staining of the hippocampus might not be able to demonstrate cell loss because of a lack of sensitivity [55]. The pattern of PV-IR cells in the hippocampus in control rats was similar to that known from previous studies [56,57]. PV is present exclusively in interneurons of all hippocampal subregions [54]. These neurons can be classified as basket or chandelier cells [57]. Similarly to the neocortex, these cells are regarded as GABAergic interneurons [58] and constitute 25–31% of the GABAergic cell population in the normal hippocampus [56]. These cells innervate the somata, proximal dendrites, and axon initial segments of principal neurons. They may thus control the pattern and timing of output of principal cells and synchronize their action potential discharges [57,59]. Concerning the fascia dentata, the existence of two almost separate populations of GABAergic nongranular cells—somatostatin- and PV-containing neurons—has been reported. It was demonstrated that PV-IR neurons are less sensitive to seizure-induced calcium overload than somatostatin-containing cells [60]. Granular cell pathology was observed only in animals that exhibited a loss of adjacent hilar neurons [61]. A severe decrease in PV-IR cells was reported in the hippocampal formation of rats after convulsive pilocarpine-induced SE [62]. Partial loss of PV-IR neurons and markedly reduced pericellular innervation of dentate granular cells by PV-positive axons was recently described in the lithium–pilocarpine model as well as after intrahippocampal injection of kainic acid [63,64]. Similarly, there was a significant reduction in PV-IR and somatostatin-IR neurons in the hilar region accompanied by extensive mossy fiber sprouting after tetanic stimulation of the angular bundle [65]. A decrease in PV-IR neurons was detected in the dentate gyrus not only after stimulation of the angular bundle but also in systemic kainic acid-induced SE [66]. In contrast to the above-mentioned findings, a loss of PV-IR neurons occurred in the kainic acid model in areas CA1–CA3 of the ipsilateral hippocampus following a unilateral lesion, but not in the dentate gyrus [67,68]. A decrease in PV immunoreactivity does not necessarily mean cell death; it might be due to decreased PV expression. Anyway, it was suggested that a decrease in PV CB immunorectivity is associated with decreased phosphorylation and subsequent degradation of neurofilaments, leading eventually to neuronal degeneration [69]. On the other hand, a decrease in the number of PV-IR cells in the stratum oriens of region CA1 in pilocarpine-induced chronic seizures was ascribed to the death of these neurons [70]. It was, however, proven that PV- and CB-IR neurons are not spared in pilocarpine-induced brain damage.
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In contrast to a severe decrease in PV-IR cells reported in the hippocampal formation of rats experiencing convulsive pilocarpine-induced SE [62], the changes caused by NCSE were understandably less pronounced. Nevertheless, the loss of the specific and important population of nonpyramidal interneurons in the dentate gyrus might be responsible for a chronic alteration of inhibition. Our results support the view that no direct evidence had been obtained linking these calcium-binding proteins to specific protection against calcium-mediated cell injury [49]. It should be emphasized again that PV and CB immunonegativity can mean either that the cells have died or that neurons may be spared but do not express PV and CB [67]. No matter which of these is true, because calcium-binding proteins are present mainly in GABAergic interneurons, their disturbance could thus refer to an alteration of inhibitory mechanisms as the consequence of NCSE. Such an alteration was demonstrated in the dentate gyrus of rats exposed previously to convulsive pilocarpine-induced SE. Dentate granule cells exhibited prolonged EPSPs and discharged more action potentials in comparison with controls. In addition, IPSP conductances as well as frequency of GABA-A spontaneous and miniature IPSCs were decreased, thus confirming a loss of inhibition of granule cells [71]. This alteration might be responsible for the long-term deficits in equilibrium behavior and grasping observed in the rotorod and barholding tests. It is not easy to answer the question whether this effect on the dentate gyrus is directly responsible for the behavioral changes revealed by the social interaction test. As part of the hippocampal formation, the dentate gyrus participates in the processes of learning, memory, motivation, integration of cognitive functions, alerting responses, and awareness as well as in cardiovascular, endocrine, and reproductive functions [72]. Via the entorhinal cortex, the dentate gyrus integrates inputs from a variety of cortical regions [73]. The loss of inhibition in the dentate gyrus may cause hyperexcitability of the CA3 and CA1 subregions, and this changed excitability state in the whole hippocampal formation might cause diverse behavioral abnormalities. In conclusion, pilocarpine-induced NCSE represents a suitable model for studying possible consequences of complex partial SE (without secondary generalization) in the brain. It is necessary to study other well-defined neuronal populations (e.g., somatostatin-positive neurons), changes in excitability of at least neocortex and hippocampal formation, and memory functions in our model. The present finding of seizure-related brain damage associated with long-term behavioral deficits following this relatively subtle epileptic condition warns against underestimating the harmful effects of complex partial SE in patients.
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Acknowledgments This study was supported by Grants 309/00/1643 and 309/03/0770 of the Grant Agency of the Czech Republic and by Research Project AVOZ 5011922.
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Long-term behavioral and morphological consequences of nonconvulsive status epilepticus in rats Pavel Krsek, Anna Mikuleck a, Rastislav Druga, Hana Kubov a, Zdenek Hli nak, * Lucie Suchomelov a, and Pavel Mares Institute of Physiology, Academy of Sciences of the Czech Republic, Vıdenska 1083, CZ 142 20 Prague 4, Czech Republic Received 23 July 2003; revised 24 November 2003; accepted 25 November 2003
Abstract The aims of the present study were to ascertain whether nonconvulsive status epilepticus (NCSE) could give rise to long-term behavioral deficits and permanent brain damage. Two months after NCSE was elicited with pilocarpine (15 mg/kg ip) in LiCl-pretreated adult male rats, animals were assigned to either behavioral (spontaneous behavior, social interaction, elevated plus-maze, rotorod, and bar-holding tests) or EEG studies. Another group of animals was sacrificed and their brains were processed for Nissl and Timm staining as well as for parvalbumin and calbindin immunohistochemistry. Behavioral analysis revealed motor deficits (shorter latencies to fall from rotorod as well as from bar) and disturbances in the social behavior of experimental animals (decreased interest in juvenile conspecific). EEGs showed no apparent abnormalities. Quantification of immunohistochemically stained sections revealed decreased amounts of parvalbumin- and calbindin-immunoreactive neurons in the motor cortex and of parvalbumin-positive neurons in the dentate gyrus. Despite relatively inconspicuous manifestations, NCSE may represent a risk for long-term deficits. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Lithium–pilocarpine status epilepticus; Epileptic brain damage; Motor performance; Behavior; Calcium-binding proteins
1. Introduction It has been proved that convulsive status epilepticus causes serious brain damage via excitotoxic mechanisms [e.g., 1]. A complex neurotoxic cascade consisting of multiple serial and parallel processes leading to both necrotic and apoptotic cell death has been described [2,3]. It is now evident that ‘‘epileptic‘‘ brain damage results from excessive neuronal activity during status epilepticus (SE), with complicating systemic factors playing an additional role. Consequently, there is also a conceptual framework for brain damage resulting from nonconvulsive status epilepticus (NCSE). However, although a number of clinical reports support the view that convulsive SE represents a serious risk for the human brain, possible harmful effects of NCSE are still a source of controversy.
* Corresponding author. Fax: +420-2-41062488. E-mail address: [email protected] (P. Maresˇ).
1525-5050/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2003.11.032
It has repeatedly been demonstrated that complex partial status epilepticus, one of the two main types of NCSE in humans, can be accompanied by considerable neurological morbidity, as well as mortality [4–8]. These clinical observations are supported by MRI findings [9] as well as by studies on serum neuron-specific enolase, an accepted marker of acute brain injury [10,11]. However, most deficits are completely reversible [12,13]. It is also stressed that the poor outcome of patients may be related to the course of a systemic disease and not to complex partial SE itself [14,15]. It is therefore doubtful whether clinical studies can definitively answer questions concerning harmful effects of complex partial SE on the brain. Some electrically and pharmacologically induced animal models of NCSE have been introduced. Hosford recently asserted that the following models satisfy the criteria for an animal model for complex partial SE: kainic acid model, pilocarpine model, prolonged kindling, and LothmanÕs self-sustaining limbic SE [16]. All these models cause extensive brain damage. However, a
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marked discrepancy between the dire consequences of artificially induced SE in animals and the relatively good outcome of patients after complex partial SE has been reported [15]. We suggest that one possible explanation of this disagreement is that these experimental models represent much more severe insults compared with human complex partial SE. Similarly, Drislane recently suggested that EEG discharges recorded in the majority of animal models are better suited to convulsive SE than to complex partial SE, with its usually less dramatic electroencephalographic patterns [17]. Short-term and self-limited NCSE induced by the administration of low doses of pilocarpine in lithium chloride-pretreated rats was developed in our laboratory [18]. NCSE consisted of approximately 90 minutes of abnormal behavior characterized by the occurrence of various epileptic automatisms, e.g., chewing, nodding, face washing, and exploratory behavior as if in a new environment. Both cortical and hippocampal spikes, isolated or in bursts, as well as other types of epileptic EEG activity, accompanied these behavioral features. Profound impairment of responsiveness to exteroceptive stimuli correlating with the occurrence of epileptic EEG activity was observed [19]. One and two weeks after NCSE, seizure-related brain damage consisting of dark and shrunken neurons in Nissl-stained brain sections was observed mainly in the motor neocortical fields [18,19]. On the basis of these observations, we were able to define a new pharmacologically induced model of human complex partial SE. However, it remained to be ascertained whether this relatively brief NCSE could cause long-term functional and morphological consequences. This presumption was supported by the study of Cook and Persinger, who described a deficit in longterm memory using the radial maze paradigm in rats injected with a subconvulsive dose of pilocarpine 5 months before testing [20]. To determine the possible delayed consequences of NCSE, video EEG monitoring, behavioral testing, and morphological examination were conducted 2 months later. Histological examination of brain sections was performed to clarify whether morphological damage observed 1 and 2 weeks after NCSE was permanent. Moreover, brain slices were immunostained using antibodies to the calcium-binding proteins calbindin D-28k and parvalbumin to specifically evaluate affected cell populations in both the neocortex and limbic structures.
2. Methods 2.1. Animals Adult (90-day-old) male albino Wistar rats (N ¼ 114) weighing 250–350 g were used. They were housed in
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standard plastic cages in a temperature-controlled room (22 1 °C) with a 12-hour light/dark cycle (lights on at 6:00 A M ). Food and water were available ad libitum in the home cage. In addition, 32 juvenile rats 21 days old were used in the social interaction test. All experiments were conducted under approval of the Animal Care and Use Committee of the Institute of Physiology of the Academy of Sciences in agreement with the Animal Protection Law of the Czech Republic (fully compatible with the guidelines of European Community Council Directives 88/609/EEC). 2.2. Experimental procedure NCSE was elicited with pilocarpine (15 mg/kg ip) 24 hours after lithium chloride pretreatment (3 mEq/kg ip). The rats were placed in separate plastic boxes and their behavior was observed for 120 minutes. EEG recording was not performed in this study because of data from the previous study [18]. Experimental groups were formed only by animals exhibiting clear-cut features of NCSE (N ¼ 63). Control rats (N ¼ 51) received an equal volume of saline instead of pilocarpine; other conditions were the same as for the pilocarpine-treated animals. These controls were put together from all ongoing studies on nonconvulsive status. Two rats included in the control group received pilocarpine also but they did not develop any signs of NCSE. Animals exhibiting short-lasting behavioral signs of nonconvulsive seizures (N ¼ 3) were not included in the present study; it would be necessary to have more rats to make an additional group. Animals exhibiting motor seizures were included in other studies focused on convulsive status epilepticus. All experiments were performed 2 months after NCSE. Three groups of animals were established. First, video EEG monitoring was conducted in 6 pilocarpinetreated and 2 control animals. The second group, consisting of 46 pilocarpine-treated and 44 control rats, was started on behavioral testing. Rats in the third group (11 pilocarpine-treated rats and 5 controls) were sacrificed and their brains processed for morphological techniques. Animals from the first and the third groups were not tested behaviorally to ensure that possible changes are due to their previous nonconvulsive status. 2.3. Video EEG monitoring Animals were anesthetized with ketamine (100 mg/kg ip) and xylazine (20 mg/kg im). Two flat silver electrodes were placed over the sensorimotor areas of both hemispheres (AP ¼ 0, L ¼ 2 mm), two Teflon-coated stainless-steel electrodes were inserted into the dorsal hippocampus (AP ¼ 3.5, L ¼ 2, H ¼ 3:8 mm), and an indifferent electrode was inserted into the nasal bone. The electrodes were fixed to the skull with dental acrylic.
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One week after surgery, animals were individually placed in plastic boxes (30 40 45 cm) and attached to the EEG apparatus (BrainScope) with flexible wires and swivels so that their movement was not restricted. The EEG was continuously recorded for 5 hours for six experimental and two control rats. Behavior was simultaneously videotaped. Localization of hippocampal electrodes was checked histologically after the end of the experiment. Again, all animals included in EEG study had tips of electrodes placed in the CA3 field of the dorsal hippocampus. 2.4. Behavioral procedure Animals (N ¼ 90) were tested between 9:00 A M and 3:00 P M . All the experimental devices used were thoroughly cleaned before a new animal was tested. In both the spontaneous behavior test and the social interaction test, rat behavior was monitored using a video camera located 100 cm away from the arena. The videotape was scored by an experienced observer (intrarater reliability > 0.9) using the Observer program (Noldus Information Technology). Rats were never used for two behavioral tests; only some animals were exposed to one behavioral and one motor (rotorod or bar holding) test. 2.5. Spontaneous behavior test Animals (14 pilocarpine-treated rats and 14 controls) were placed individually in the square arena (plexiglas cage, 45 45 30 cm) and their behavior was monitored for 5 minutes. The following categories of spontaneous behavior were distinguished: walking (movement around the arena); rearing (upright posture both against and away from the wall); sniffing (investigation of the floor, walls, and space); face washing (brief period of vibrating movements of the forepaws in front of the snout followed by nose or head washing and paw licking); grooming (face washing with a subsequent cephalocaudal progression); and immobility (lying or sitting). The total number, total duration (in seconds), and mean duration (a derived measure: total duration divided by total number) of individual categories were evaluated. 2.6. Social interaction test On the day of the experiment, both pilocarpinetreated (N ¼ 16) and control (N ¼ 16) adults and juveniles (21 days old, N ¼ 32) were housed individually. After 1 hour of adaptation to the experimental room, an adult animal was placed in the experimental arena (identical to that described above) to explore the new environment. Two minutes later, a juvenile conspecific was placed in the arena. Each interaction lasted 5 minutes. Reexposure to the same juvenile occurred 30 minutes after the first interaction. Between successive
exposures, both adults and juveniles were maintained individually. The total number and total duration of social investigations directed toward the juvenile (approaching, nosing, and body sniffing) were calculated. 2.7. Elevated plus-maze test Pilocarpine-treated (N ¼ 16) and control (N ¼ 14) animals were placed individually on the open arm of the plus-maze, and the transfer latency (time required for an animal to move from the open arm to the enclosed arm) was recorded. The second session was carried out 24 hours later. If the rat did not enter the enclosed arm within 120 seconds, it was excluded from the evaluation (2 of the original group of 30 rats). 2.8. Motor performance 2.8.1. Rotorod test The rotorod apparatus was set to rotate at a constant 5 rpm. The rats (25 pilocarpine-treated and 22 control rats; these groups were formed by animals exposed to the open field and social interaction test) were placed on the rod with the body axis perpendicular to the rodÕs long axis and with the head directed against the direction of rotation. The maximal score in maintaining equilibrium was arbitrarily fixed at 60 seconds. To observe the behavioral strategy used by an animal to maintain equilibrium, rats were placed on the rod individually. Three strategies were observed: grasping (motionless grasping of the rod), walking (walking on the rod), and orientation (described as scanning relative to the rod and attempting to stand upright). 2.8.2. Bar-holding test Rats (14 pilocarpine-treated and 14 control rats, the same animals as in the spontaneous behavior test) were hung by both forepaws on a wooden rod (25 cm long, 1 cm in diameter) located 1 m above a landing platform covered with a thick sheet of soft plastic to cushion falls. The duration of grasping was evaluated. 2.9. Morphology 2.9.1. Fixation Two months after treatment, an overdose of urethane (2 g/kg ip) was administered and animals were perfused with 0.37% sulfide solution (1 mL/g) for 10 minutes followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (1 mL/g), for 10 minutes. The brains were removed from the skull and postfixed in buffered 4% paraformaldehyde for 3 hours and then cryoprotected in a solution containing 20% glycerol in 0.02 M potassiumbuffered saline for 24 hours. The brains were frozen in dry ice and stored at )70 °C until cut. Brains were sectioned in the coronal plane (50 lm, 1-in-5 series) with a
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sliding microtome and sections were stored in a cryoprotectant tissue-collecting solution (30% ethylene glycol, 25% glycerol in 0.05 M sodium phosphate buffer) at )20 °C until processed. Adjacent series of sections were used for Nissl and Timm staining. 2.9.2. Nissl and Timm staining The first series of 1-in-5 sections were mounted onto gelatin-coated slides and stained with cresyl violet. For the Timm sulfide/silver method, all sections (1-in-5 series) in which hippocampus was present were mounted on gelatin-coated slides, air-dried, and stained in the dark according to the following protocol: A working solution containing arabic gum (300 g/L), sodium citrate buffer (25.5 g/L citric acid monohydrate and 23.4 g/L sodium citrate), hydroquinone (16.9 g/L), and silver nitrate (84.5 mg/kg) was poured into the staining dish, which contained the slides. The sections were developed until an appropriate staining intensity was attained (approximately 60 minutes). Then the slides were rinsed with tap water for 30 minutes and placed in 5% sodium thiosulfate for 12 minutes. Finally, sections were dehydrated through an ascending series of ethanol, cleared in xylene, and coverslipped. Nissl-stained slices were examined to identify the cytoarchitectonic boundaries as well as the possible distribution of neuronal damage and to localize hippocampal electrodes in animals tested electrophysiologically. 2.9.3. Immunohistochemistry Sections were sequentially incubated in 0.15% hydrogen peroxide in PBS (0.01 M, pH 7.4) for 10 minutes, rinsed with PBS four times, and then incubated with blocking solution containing 2% normal horse serum (Vector Laboratories, Burlinghame, CA, USA) and 0.1% Triton X-100 in PBS at room temperature. The sections were incubated with the primary antibody (antiparvalbumin and/or anti-calbindin monoclonal antibody, Sigma, dilution; 1:5000 in PBS containing 1.5% normal horse serum and 0.1% Triton X-100) for 48 hours at 4 °C and then rinsed four times in PBS. Sections were incubated for 1 hour at room temperature with secondary antibody, biotinylated anti-mouse antibody made in horse serum (Vector); dilution 1:50 in PBS containing 1.5% normal horse serum and 0.1% Triton X-100. After this step, the tissue was rinsed with PBS four times and covered with the ABC reagent (Vectastain Kit, Vector) for 1 hour at room temperature. After being rinsed, sections were incubated for 5–7 minutes with a mixture of 0.02% diaminobenzidine and 0.05% hydrogen peroxide in PBS. Control sections were prepared with the same procedures, but omitting the primary antibody. All sections were mounted onto gelatin-coated slides, coverslipped, and examined by light microscopy (Olympus AX 70 microscope with brightfield optics).
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2.9.4. Analysis of the sections Synaptic reorganization (mossy fiber sprouting) was analyzed in sections stained the using the Timm method. Sprouting was analyzed along the septotemporal axis of the hippocampus; method was described in detail by Nissinen et al. [21]. The septal end included the coronal sections between AP levels 2.3 and 6.0 mm posterior from bregma [22]. The dorsal and ventral midportions of the dentate gyrus included dorsal and ventral parts of hippocampus where the granule cell layer of the septal and temporal ends becomes fused and forms a standardized ‘‘oval-shaped’’ layer (AP level 6.1–6.7 mm posterior to bregma). Mossy fiber sprouting was scored according to Cavazos et al. [23]. The distribution of Timm granules in the supragranular layer was rated from 0 to 5. While the lowest levels (0 and 1) indicate negative finding or sparse granules, respectively, more numerous granules are scored as 2, occasional patches of granules as 3, presence of a dense laminar band of granules as 4, and extension into the inner molecular layer as 5. Parvalbumin-immunoreactive (PV-IR) neurons were counted throughout layers II–VI of motor neocortical fields Fr 1, Fr 2, and Fr 3 (where the most prominent effect was observed 1 and 2 weeks after NCSE in the previous studies [18,19]). For each animal 12 sections in the range of stereotaxic planes AP 2.2 and AP )0.3 were counted [22]. Calbindin-immunoreactive (CB-IR) neurons were counted in the same anteroposterior range of motor fields using 0.3 0.3-mm squares localized randomly in neocortical layers V and VI. Sixteen squares per each of 12 sections from each rat were evaluated. Classification of the neocortical areas was based on the cytoarchitectonic criteria of Zilles [24]. PV-IR neurons in the dentate gyrus were counted in the whole mediolateral extent of both blades in 12 sections per each animal in the range of stereotaxic planes AP )2.8 and AP 4.3. PV-IR neurons in CA1 and CA3 hippocampal fields were counted in 0.3 0.3-mm squares (12 squares per each animal). PV-IR neurons in the CA1 field were restricted to the segment between sagittal planes 1 and 2 or 1 and 3 in more caudal sections, respectively. Terminology used in description of the limbic structures is according to Paxinos and Watson [22]. All analyses were conducted blind to treatment group. 2.10. Statistics Behavioral data were analyzed nonparametrically. To compare the differences between independent groups (pilocarpine-treated animals vs controls) within a given session, the Mann–Whitney U test was used. To compare the differences within matched pairs (subsequent sessions for both pilocarpine-treated and control rats) in the social interaction and elevated plus-maze tests, the
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Wilcoxon matched-pairs signed-ranks test was used. Morphological data (numbers of PV-IR and CB-IR neurons) were analyzed parametrically with the t test. Statistical significance was accepted at P < 0:05 (twotailed).
3. Results NCSE was elicited in all 63 experimental animals included in this study. It was characterized by hypersalivation, piloerection, licking, swallowing, and chewing starting immediately after pilocarpine administration. These phenomena could not be taken as epileptic; rather, they represented the peripheral action of pilocarpine. Then automatisms like face washing, head nodding, and creep walking (ataxia-like movements) were observed. Fully developed ictal behavior was characterized by immobility (with chewing and head nodding) interrupted by periods of creep walking. Gradual disappearance of the above-mentioned abnormal behavior was accompanied by repeated periods of exploration/searching activity (in a well-known cage) and self-grooming. These activities also subsided so that there were no apparent behavioral changes in the experimental animals 2 hours after pilocarpine treatment. Rats were returned to the animal facility and examined 2 months later.
Fig. 1. Relative representation of the occurrence (left) and duration (right) of individual behavioral patterns. C, control group (n ¼ 14); P, pilocarpine group (n ¼ 14). Statistical significance, P < 0:05 (twotailed): Pilocarpine versus control group (only duration of face washing exhibited a significant difference).
3.1. Video EEG monitoring Evaluation of video EEG recordings revealed no apparent EEG as well as behavioral abnormalities in experimental as well as control rats. 3.2. Behavioral data 3.2.1. Spontaneous behavior test There was no difference in the total frequency of all recorded behavioral patterns between pilocarpine-treated animals (48.1 5.6, mean SEM) and controls (42.5 5.7). Analysis of particular patterns revealed no change except for face washing (Fig. 1). The total time spent and the mean duration of face washing were significantly increased in pilocarpine-treated animals. 3.2.2. Social interaction test When compared with the controls (Fig. 2), a significant reduction in the total duration of social investigation was observed for pilocarpine-treated animals during the initial exposure. No difference in social investigation was found between pilocarpine-treated and control rats during the second exposure. Intersession comparisons of social investigation revealed significantly lower levels during the second exposure for both experimental and control rats.
Fig. 2. Total time spent by adult males in investigation of juveniles in the social interaction test (means + SEM). The second exposure to the same juveniles was performed 30 minutes after the first one. Open bars: control group (n ¼ 16); crosshatched bars: pilocarpine group (n ¼ 16). Statistical significance, P < 0:05 (two tailed): Pilocarpine versus control group, d second session versus first session.
3.2.3. Elevated plus-maze test No statistical difference in transfer latency between pilocarpine-treated and control animals was noted during both the initial session and the second session. As compared with the initial session, a significant
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shortening of the transfer latency during reexposure was noted in both groups of animals. 3.3. Motor performance: rotorod and bar-holding tests Maintenance of equilibrium on the rotorod without falling (Fig. 3) was significantly decreased in pilocarpine-treated animals. Moreover, the strategy of the pilocarpine-treated animals was different from that of the controls. While control rats walked on the rod and maintained equilibrium, pilocarpine-treated rats attempted to rear, scan, and turn in the direction of the rotation and to sit; 16 of 25 pilocarpine-treated animals fell. In the bar-holding test, the duration of grasping was significantly decreased in the pilocarpine-treated animals. Similarly to controls, pilocarpine-treated rats attempted to climb the bar; however, they were less skillful (Fig. 3). 3.4. Morphology 3.4.1. Nissl and Timm staining Whole brain was always checked in Nissl-stained sections. There were dark and shrunken neurons in layers V and VI of the motor neocortical areas (fields Fr 1–3) in pilocarpine-treated rats (Figs. 4A,B). Small numbers of dark neurons were inconsistently found in the corpus striatum and CA3 hippocampal field. Exact quantification of the changes was precluded by the thickness of the slices; however, the number of affected neurons appeared to be smaller in comparison with the numbers 1 and 2 weeks after NCSE [18,19]. No obvious changes were observed in any other brain structure.
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Evaluation of Timm-stained sections showed no sprouting of mossy fibers in the hippocampus of pilocarpine-treated animals, i.e., level 0 according to Cavazos et al. [23]. 3.4.2. Immunohistochemistry In control animals, PV-IR cells were found in all cortical layers except layer I, and exhibited a variety of somatic and dendritic morphologies (Fig. 4C). Multipolar cell bodies predominated in the upper layers while small pyramidal and bipolar neurons were less frequent. In layers V and VI, some bipolar and triangular (pyramidal-like) neurons were distinguished in addition to the prevailing multipolar neurons. Most PV-IR cells were moderately to heavily labeled. The highest density of PV-IR neurons was located in the supragranular layers of the motor area. Two populations of CB-IR cells could be distinguished. First, weakly to moderately positive neurons predominated in layers II and III and could also be found in layer V. Many weakly and moderately CB-IR cells were obscured by dense diffuse staining of the neuropil in layers I–III. Second, heavily labeled CB-IR neurons were found scattered in layers II–VI but most of them were localized in two belts corresponding to layers II–III and V–VI. Both bipolar and multipolar perikarya were present in the supragranular layers, while the majority of heavily labeled cells in layer V–VI were multipolar (Fig. 4E). In pilocarpine-treated animals, a significant reduction in both PV-IR and CB-IR neurons in the motor neocortical area was evident in comparison with controls (Figs. 4D,F). The number of PV-IR neurons throughout layers II–VI was 47.7 2.88 (mean SEM)
Fig. 3. Motor performance in the rotorod test (left, expressed as the time spent on the rod without falling down) and the bar-holding test (right, expressed as the time of grasping). Data are expressed as means + SEM. Open bars: control group (n ¼ 22 and 14, respectively); crosshatched bars: pilocarpine group (n ¼ 25 and 14, respectively). Statistical significance, P < 0:05 (two-tailed): Pilocarpine versus control group.
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Fig. 4. Photomicrographs demonstrating parvalbumin-containing cells in the sensorimotor cortex (A,B), calbindin-containing cells in the motor cortex (C,D), and parvalbumin-containing cells in the dentate gyrus (E,F). Control rats: A, C, E; animals after nonconvulsive status: B, D, F. Bars ¼ 500 lm. Roman numerals denote cortical layers. WM, white matter. CA3, hippocampal field CA3; sm, stratum moleculare; sg, stratum granulare. Arrowheads in (E) and (F) denote parvalbumin-positive cells on the borderline between the granular layer and hilus of the dentate gyrus.
in pilocarpine-treated and 94.3 4.72 in control animals. A decline in PV-IR cells was particularly prominent in layers II and III. Some of the persisting neurons exhibited weaker immunopositivity of perikarya and dendrites when compared with controls. The average number of CB-IR neurons in a 0.3 0.3-mm area localized randomly in layers V and VI was 4.57 0.13 in pilocarpine-treated rats and 6.33 0.26 in the control group (Fig. 5). In control animals, PV-IR neurons predominated in the stratum pyramidale and oriens of the hippocampus and in the stratum granulosum and hilus of the dentate gyrus (Fig. 4G). A decline in the number of PV-IR neurons was demonstrated in both blades of the dentate gyrus in pilocarpine-treated rats (Fig. 4H). The average
number of PV-IR cells in the dentate gyrus was 6.75 0.49 in pilocarpine-treated and 10.27 0.82 in control animals. On the other hand, no significant differences in the numbers of PV-IR neurons were found in the CA1 (17.6 1.78 pilocarpine-treated vs 20.7 1.0 controls) and CA3 (12.2 0.72 pilocarpine-treated vs 12.4 0.89 controls) hippocampal fields (Fig. 6).
4. Discussion Classic pilocarpine-induced SE represents a model of secondary generalized convulsive status epilepticus [25,26]. Studies dealing with subconvulsive doses of pilocarpine are rare. Two original studies described
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Fig. 5. Average number of calbindin-positive neurons counted in the square of 0.3 0.3 mm localized randomly in layers V and VI of the motor neocortex (left); average number of parvalbumin-positive neurons counted in the whole range of layers V and VI of the motor neocortical fields Fr 1– 3 of both hemispheres (right). Open bars: control group (n ¼ 5); crosshatched bars: pilocarpine group (n ¼ 11). Data are expressed as means + SEM. Statistical significance, P < 0:05 (two-tailed): pilocarpine versus control group.
Fig. 6. Average number of parvalbumin-positive neurons in the dentate gyrus (counted in the whole extent of both blades) and in the CA1 and CA3 hippocampal fields (counted in a square of 0.3 0.3 mm). Open bars: control group (n ¼ 5); crosshatched bars: pilocarpine group (n ¼ 11). Data are expressed as mean + SEM. Statistical significance, P < 0:05 (two-tailed): Pilocarpine group versus control group.
effects of nonconvulsant pilocarpine doses in rats not pretreated with lithium chloride [27,28]. Sixty to one hundred twenty minutes of abnormal behavior concurrently with EEG changes (theta rhythm and spiking in
the hippocampus and low-voltage fast activity in the cortex) were observed. However, we found only one subsequent study that focused on these phenomena and reported expression of the c-fos protein in certain limbic areas induced by a subconvulsant dose of pilocarpine [29]. Our previous results demonstrated that the model of NCSE induced by a subconvulsant dose of pilocarpine in lithium chloride-pretreated rats, characterized by behavioral automatisms and spikes (isolated as well as in series) in hippocampal and cortical EEGs, is equivalent to human CPSE [18,19]. The striking result of the analysis of this model was the finding of morphological damage in the motor neocortex 1 and 2 weeks after NCSE. As one of the most critical questions regarding NCSE in humans concerns the risk of long-term harmful sequelae to the brain, we turned our attention to analyzing both behavioral and morphological consequences of nonconvulsive seizures in our model. The possibility of behavioral sequelae was suggested by the study of Cook and Persinger [20], a pioneering morphological study. Video EEG monitoring did not reveal any epileptic activity in our animals. This contrasts sharply with data for convulsive status epilepticus (lithium–pilocarpine model with pilocarpine doses of about 40 mg/kg and/or high-dose pilocarpine model) where life-long spontaneous seizures were recorded under conditions similar to those for our monitoring [30]. Behavioral tests revealed some deficits 2 months after NCSE. Conversely to the prominent disturbances observed in rats during pilocarpine-induced NCSE [18,19], discernible long-term behavioral consequences were only subtle. No long-term structural disintegration
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of spontaneous behavior was observed. However, the animals experiencing NCSE displayed more face washing. In intact animals, this behavioral component occupies the second position in the sequential organization of grooming behavior [31,32]. An increased display of face washing in animals experiencing NCSE probably reflects different levels of responsiveness following exposure to a stressor when placed into a new environment. It has been proved that the grooming response is related to age [33,34] and, among others, also to neurohormonal modulation [35,36]. The social interaction test revealed decreased investigation by both pilocarpine-treated and control animals reexposed to the same juvenile 30 minutes after the initial interaction, suggesting that the animals are able to recognize juvenile conspecifics [37–40]. However, duration of investigation during the initial exposure was markedly decreased in animals after NCSE. One possible explanation is that the decreased interest in juveniles by pilocarpine-treated animals might reflect deficits in sensitivity as well as in responsiveness of animals exposed for the first time to social novelty. On the other hand, pilocarpine-treated rats remembered their juvenile conspecifics; there was a significant reduction in duration of investigation during the second exposure. The reduction in the transfer latency in the elevated plus-maze test during the second session showed that spatial orientation and/or short-term memory of pilocarpine-treated animals were not impaired. This, to a certain extent, contrasts with the finding of Cook and Persinger [20], who reported long-term memory disturbance in animals 5 months after treatment with a subconvulsive dose of pilocarpine. However, it is necessary to note that animals tested on the elevated plus-maze were apparently submitted to a less difficult memory task in comparison with the eight-arm radial maze used by Cook and Persinger. Long-term motor deficits were observed in animals that experienced NCSE. These may be caused by either a muscular tone deficiency or impaired skillfulness of the pilocarpine-treated animals. The static component of the equilibrium, in particular, seemed to be impaired. Moreover, pilocarpine-treated animals attempted to turn the body in the direction of the rotation on the rotorod and therefore fell. Equilibrium behavior depends on a number of sensory cues and on training [41,42]. The different behavioral strategy of the pilocarpine-treated animals could be related to deficits in perception, for example, of visual, proprioceptive, vestibular, and tactile stimuli. It has been demonstrated that the olivocerebellar pathway is involved in the temporal organization of motor learning and skills [43,44]. However, no discernible damage was observed in the brainstem (including oliva inferior) in our experimental rats. Therefore, a more plausible explanation is that the motor deficits reflect damage to the motor neocortical areas.
Both histological and immunohistochemical techniques demonstrated damage in different brain structures 2 months after NCSE, strongly supporting the view that short-term NCSE is able to damage the brain permanently. Damage to motor neocortical fields corresponding to motor deficits was the most conspicuous morphological finding. This is in accordance with our former observations [18]. It was suggested that the motor neocortex might be damaged by projection of the epileptic activity from the limbic structures via the amygdala [19]. Calcium-binding proteins like parvalbumin and calbindin play an important role as calcium transporters, a buffering system for intracellular calcium ions, and represent one of the most important calcium compartments in the brain [45]. The decrease in immunoreactivity (and probably in the concentrations of PV and CB) may thus increase neuronal susceptibility to calcium fluctuations, leading to structural and functional impairment. Analysis of tissue obtained from patients undergoing epilepsy surgery indicated reduced numbers of PV-IR and CB-IR neurons in human neocortical epileptic foci [46]. Loss of PV-IR neurons was also detected in the neocortex of the temporal pole in patients with intractable temporal epilepsy [47]. A decrease in glutamate decarboxylase and PV immunostaining was observed in the high-dose pilocarpine model in rats surviving 60 days after the onset of chronic seizures [48]. These findings support an involvement of neocortex in temporal lobe epilepsy with secondary generalization. The distribution of PV-IR and CB-IR neurons in the motor cortical areas (fields Fr1, Fr2, and Fr3) of control animals was similar to that reported in the studies of Celio [49] and van Brederode et al. [50]. In our pilocarpine-treated rats, a decline in both PV-IR and CB-IR neurons was demonstrated in layers II–VI and V–VI of the motor neocortical fields, respectively. This finding indicates that NCSE influenced neuronal expression of the calcium-binding proteins in the same direction as convulsive SE. PV-IR and CB-IR cells represent a subpopulation of cortical GABAergic inhibitory neurons [49]. PV is localized in fast-spiking, metabolically active large basket cells and chandelier cells in the neocortex. On the contrary, CB-IR cells are typically regular-spiking double-bouquet cells (some are fast-spiking ones) [51–53]. A reduction in neocortical PV and CB immunoreactivity thus suggests an impairment of GABAergic inhibition as a consequence of nonconvulsive SE. Our prior work failed to demonstrate an apparent effect on limbic structures, where the most prominent epileptic brain damage has repeatedly been described in humans [1,54], as well as in the majority of animal models of SE [2,16]. For that reason, discovery of the dentate gyrus lesion demonstrable by PV immunohistochemistry could be regarded as a crucial result of the
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present work. Although dark neurons were found in some pilocarpine-treated animals, examination of Nisslstained brain sections failed to reveal discernible damage to limbic structures 2 months after NCSE. However, it was suggested that conventional cresyl violet staining of the hippocampus might not be able to demonstrate cell loss because of a lack of sensitivity [55]. The pattern of PV-IR cells in the hippocampus in control rats was similar to that known from previous studies [56,57]. PV is present exclusively in interneurons of all hippocampal subregions [54]. These neurons can be classified as basket or chandelier cells [57]. Similarly to the neocortex, these cells are regarded as GABAergic interneurons [58] and constitute 25–31% of the GABAergic cell population in the normal hippocampus [56]. These cells innervate the somata, proximal dendrites, and axon initial segments of principal neurons. They may thus control the pattern and timing of output of principal cells and synchronize their action potential discharges [57,59]. Concerning the fascia dentata, the existence of two almost separate populations of GABAergic nongranular cells—somatostatin- and PV-containing neurons—has been reported. It was demonstrated that PV-IR neurons are less sensitive to seizure-induced calcium overload than somatostatin-containing cells [60]. Granular cell pathology was observed only in animals that exhibited a loss of adjacent hilar neurons [61]. A severe decrease in PV-IR cells was reported in the hippocampal formation of rats after convulsive pilocarpine-induced SE [62]. Partial loss of PV-IR neurons and markedly reduced pericellular innervation of dentate granular cells by PV-positive axons was recently described in the lithium–pilocarpine model as well as after intrahippocampal injection of kainic acid [63,64]. Similarly, there was a significant reduction in PV-IR and somatostatin-IR neurons in the hilar region accompanied by extensive mossy fiber sprouting after tetanic stimulation of the angular bundle [65]. A decrease in PV-IR neurons was detected in the dentate gyrus not only after stimulation of the angular bundle but also in systemic kainic acid-induced SE [66]. In contrast to the above-mentioned findings, a loss of PV-IR neurons occurred in the kainic acid model in areas CA1–CA3 of the ipsilateral hippocampus following a unilateral lesion, but not in the dentate gyrus [67,68]. A decrease in PV immunoreactivity does not necessarily mean cell death; it might be due to decreased PV expression. Anyway, it was suggested that a decrease in PV CB immunorectivity is associated with decreased phosphorylation and subsequent degradation of neurofilaments, leading eventually to neuronal degeneration [69]. On the other hand, a decrease in the number of PV-IR cells in the stratum oriens of region CA1 in pilocarpine-induced chronic seizures was ascribed to the death of these neurons [70]. It was, however, proven that PV- and CB-IR neurons are not spared in pilocarpine-induced brain damage.
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In contrast to a severe decrease in PV-IR cells reported in the hippocampal formation of rats experiencing convulsive pilocarpine-induced SE [62], the changes caused by NCSE were understandably less pronounced. Nevertheless, the loss of the specific and important population of nonpyramidal interneurons in the dentate gyrus might be responsible for a chronic alteration of inhibition. Our results support the view that no direct evidence had been obtained linking these calcium-binding proteins to specific protection against calcium-mediated cell injury [49]. It should be emphasized again that PV and CB immunonegativity can mean either that the cells have died or that neurons may be spared but do not express PV and CB [67]. No matter which of these is true, because calcium-binding proteins are present mainly in GABAergic interneurons, their disturbance could thus refer to an alteration of inhibitory mechanisms as the consequence of NCSE. Such an alteration was demonstrated in the dentate gyrus of rats exposed previously to convulsive pilocarpine-induced SE. Dentate granule cells exhibited prolonged EPSPs and discharged more action potentials in comparison with controls. In addition, IPSP conductances as well as frequency of GABA-A spontaneous and miniature IPSCs were decreased, thus confirming a loss of inhibition of granule cells [71]. This alteration might be responsible for the long-term deficits in equilibrium behavior and grasping observed in the rotorod and barholding tests. It is not easy to answer the question whether this effect on the dentate gyrus is directly responsible for the behavioral changes revealed by the social interaction test. As part of the hippocampal formation, the dentate gyrus participates in the processes of learning, memory, motivation, integration of cognitive functions, alerting responses, and awareness as well as in cardiovascular, endocrine, and reproductive functions [72]. Via the entorhinal cortex, the dentate gyrus integrates inputs from a variety of cortical regions [73]. The loss of inhibition in the dentate gyrus may cause hyperexcitability of the CA3 and CA1 subregions, and this changed excitability state in the whole hippocampal formation might cause diverse behavioral abnormalities. In conclusion, pilocarpine-induced NCSE represents a suitable model for studying possible consequences of complex partial SE (without secondary generalization) in the brain. It is necessary to study other well-defined neuronal populations (e.g., somatostatin-positive neurons), changes in excitability of at least neocortex and hippocampal formation, and memory functions in our model. The present finding of seizure-related brain damage associated with long-term behavioral deficits following this relatively subtle epileptic condition warns against underestimating the harmful effects of complex partial SE in patients.
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Acknowledgments This study was supported by Grants 309/00/1643 and 309/03/0770 of the Grant Agency of the Czech Republic and by Research Project AVOZ 5011922.
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