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- MATERIALS AND METHODS
Summary: Purpose: Status epilepticus (SE) is more common in children than adults and has a high mortality and morbidity rate. SE in adult rats results in long-term disturbances in learning and memory, as well as an enhanced seizure susceptibility to further seizures. In contrast, a number of studies suggest that the immature brain is less vulnerable to the morphologic and physiologic alterations after SE. The goal of this study was to determine whether the long-term consequences of SE during development on hippocampal plasticity and cognitive function are age and model specific.
Methods: We used lithium-pilocarpine (Li-PC) to induce SE at different age points during development (P12, P16, P20) and evaluated the effects of this abnormal neural activity on spatial memory performance and seizure susceptibility in the animals beginning at P55, corresponding to young adulthood.
Results: We demonstrated that SE at P12 did not result in any structural or functional changes detectable in adulthood, whereas SE at both P16 and P20 induced cell loss and mossy fiber sprouting within the hippocampus and cognitive impairment when the animals were tested as adults.
Conclusions: Whereas the seizure threshold to generalized seizures was not altered, animals with SE at P20 showed an increased susceptibility to kindling in adulthood.
Status epilepticus (SE), an acutely life-threatening event characterized by repetitive or prolonged seizures, is a common pediatric emergency. Approximately 10% of children and adults who have a first unprovoked seizure or newly diagnosed epilepsy have SE (1,2). SE is more common in children than in adults, with half of the total cases occurring in those younger than 2 years (3). In the clinical literature, some reports indicate that SE in the immature brain is less harmful than SE in adults (4,5). However, recent studies suggest that SE can result in brain damage, resulting in long-term cognitive impairment and permanent susceptibility to future seizures (6–8).
Likewise, in rodent studies, the pathophysiologic effects of SE appear to vary as a function of age (9–12). Severe seizures in pubescent rats can produce persistent neuronal dysfunction, resulting in deficits in learning and memory (13–15) and an enhanced susceptibility to further seizures (16,17). Damage in the developing hippocampus, a structure implicated in both memory acquisition and seizure expression, may be responsible for these effects. Abnormal neural activity such as seizures during its development might have effects on the developing circuitry, particularly in the dentate gyrus (DG), modifying the development of synaptic connections and therefore influencing hippocampal function in adulthood. Although the immature hippocampus is highly susceptible to seizure, and experimental seizures induced in immature rats are typically more severe than those in adults (18), a number of studies suggest that the immature brain is less vulnerable to the morphologic and physiologic alterations after SE (15,19,20). Before therapeutic interventions can be studied in developmental models of SE, a greater understanding of the relation between age and SE damage is necessary.
A variety of animal models developed in the past 20–25 years share important characteristics with human SE. Among many techniques using chemoconvulsants, the lithium-pilocarpine (Li-PC) model has been especially popular because it replicates temporal lobe epilepsy (TLE) pathology (21–23). The cholinergic agent pilocarpine (PC) produces limbic and generalized SE in rodents. Pretreatment with lithium (Li) has been shown to potentiate the epileptogenic action of PC, reduce mortality, and avoid many of the peripheral cholinomimetic side effects of PC (24). This is a good experimental model with which to test the long-term sequelae on hippocampal circuitry, and particularly visuospatial learning and memory. These animals exhibit many of the characteristic sequelae observed in humans after SE, such as interictal epileptiform discharge on EEG (25,26), the development of spontaneous recurrent seizures (22,25), decreased γ-aminobutyric acid (GABAA)-receptor function and expression (27), and hippocampal sclerosis. Many previous studies provided evidence that the long-term effects of seizures during development are age specific (17,25,28,29).
Whereas the long-term effects of Li-PC–induced SE on hippocampal neuronal loss and synaptic reorganization have been extensively studied in the adult and in the immature animal (25,30), no studies have evaluated the long-term consequences of Li-PC seizures during development on spatial memory performance and seizure susceptibility. Because of the potential clinical importance of the long-term effects of seizures on hippocampal plasticity and cognitive function, we addressed the question whether age at the time of SE in immature rats is a determining factor for whether cognitive impairment and increased seizure susceptibility occur in adulthood.
It has been proposed that the 7- to 10-day-old rat may be equivalent to a human newborn (31,32). Although the exact correspondence between developmental stages across species is not readily describable, P12 in rats can be reasonably considered as corresponding to the newborn period, and P16 and P20, to infancy and early childhood in humans, respectively. Therefore, we induced Li-PC SE on specific days during development (P12, P16, and P20) and tested the animals for water maze performance and seizure susceptibility to flurothyl and to kindling, beginning on P55, an age that corresponds to young adulthood.
MATERIALS AND METHODS
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- MATERIALS AND METHODS
Sprague–Dawley male rats (Charles River Laboratory, Boston, MA, U.S.A.) were used throughout the study and were treated in accordance with the guidelines set by the National Institutes of Health for the humane treatment of animals. Animals had access to food and water ad libitum and were group housed in plastic cages under diurnal lighting conditions, with lights on from 8:00 to 20:00.
Rat pups of 12, 16, and 20 postnatal (P) days were given 3 mEq/kg lithium chloride (Sigma, St. Louis, MO, U.S.A.) intraperitoneally (i.p.) on the day before the induction of SE. Seizures progressing to SE were induced by subcutaneous injection of 80 mg/kg in the P12 and P16 rat pups and of 60 mg/kg in the P20 rat pups of pilocarpine hydrochloride (Sigma). These dosages were determined from a pilot study (data not shown) in which we used various doses of PC to determine the dosage required to induce SE without resulting in excessive death. Control animals were handled and housed in the same manner as the experimental animals and received an equal volume of i.p. saline. Rats were put in individual plastic cages for behavioral observation after PC injection. Only rats displaying behavioral manifestation of seizures for ≥1 h were used for the study. Rats were allowed to recover from SE spontaneously. Some additional rat pups subjected to Li-PC–induced SE at P20 were used only for the implantation of electrodes for kindling at P60.
Water maze testing
To assess the long-term behavioral consequences of seizures induced early in life, we evaluated the performance on visuospatial learning and memory in P55 rats previously subjected to SE at P12, P16, or P20 by using a modified water maze (33,34). This performance requires integrity of the hippocampus.
Rats were tested from days P55–60. A circular steel tank (117 cm diameter × 50 cm high) was filled with water (26 ± 1°C) to a depth of 25 cm. The water was made opaque by addition of 100 ml evaporated milk to prevent visualization of the platform. The pool was illuminated by room lights, and its location was kept constant from day to day. Four points on the perimeter of the pool were designated north (N), east (E), south (S), and west (W), thus dividing the pool into four quadrants (NW, NE, SE, SW). An 8 × 8-cm plexiglass platform, onto which the rat could escape, was positioned in the center of one of the quadrants, 1 cm below the water surface. On day 1 of the testing (P55), each rat was placed in the pool for 60 s without the platform present. This free swim enabled the rats to become accustomed to the training environment. On days 2 to 5 (P56 to P59), rats were trained for 24 trials (six trials per day) to locate and escape onto the submerged platform. For each rat, the quadrant in which the platform was located remained constant, but the point of immersion into the pool varied between N, E, S, and W in a quasi-random order for the 24 trials, so that the rat would not be able to predict the platform from the location of the point at which it was placed into the pool. The latency from immersion into the pool to escape onto the platform was recorded for each trial by one observer, while another observer manually recorded the swim route taken by the rat to reach the platform. On mounting the platform, the rat was given a 30-s rest period, after which the next trial was started. If a rat did not find the platform in 120 s, the animal was placed on the platform for a 30-s rest. If a spontaneous seizure occurred during the testing, the rat was allowed a 60-min rest before testing was resumed. After 4 days of training, rats underwent the probe test on P60. The rats were placed in the pool into the quadrant opposite the prior location of the platform. Rats were allowed to swim in the tank for 60 s without the platform present. Time spent in each quadrant was recorded. During this procedure, the rats typically spend more time swimming in the quadrant where the platform had previously been placed than in the other three quadrants. The testing procedure used during the 4 days of locating the hidden platform provides a measure of spatial reference memory, whereas the probe trial is considered to measure the strength of spatial learning (35).
Flurothyl seizure threshold
We used flurothyl to first test the seizure susceptibility on generalized, nonlimbic seizures after an episode of SE during brain development. On P61, the rats were tested for seizure threshold by using flurothyl. Experimental and control rats were paired according to their weight into a plastic chamber with two separate compartments of equal size. Liquid flurothyl was delivered through a plastic syringe at a rate of 0.1 ml/min and dripped onto filter paper in the center of the container, where it evaporated. Latencies to the first myoclonic jerk and generalized tonic seizure were recorded. Animals were removed from the chamber as soon as both animals developed tonic seizures.
Hippocampal plasticity also was evaluated in young adults rats (P61) by assessing susceptibility to amygdala kindling in a subset of rats that had SE at P20 and normal age-matched controls (36,37). Kindling is a form of pathological neural plasticity in which repeated activation of brain pathways by electrical or chemical methods induces progressive susceptibility to evokes seizures (38). Kindling is a model of partial seizures with secondary generalization (39), and susceptibility to kindling can be regarded as a measure of vulnerability to epileptogenesis. Rats were first anesthetized with pentobarbital (PTB; 45 mg/kg) and chloral hydrate (200 mg/kg) and then implanted with an insulated stainless steel twisted bipolar electrode for stimulation and recording. The electrode was placed in the amygdala by using the following coordinates: 4.6 mm lateral to midline; 1.88 mm posterior to bregma; 8.5 mm depth below skull. Electrodes were held in position with skull screws and dental acrylic. Kindling stimuli were initiated after a week of postoperative recovery. All animals were stimulated with a Grass stimulator connected serially to two constant-current stimulus isolation units. To determine whether a single bout of SE early in life alters seizure susceptibility, we first measured the afterdischarge threshold (ADT) in the amygdala. The stimulus consisted of a 1-s train of 60-Hz, 1-ms, biphasic current pulse beginning at 25 μA (peak to peak). The voltage was then increased every 2 min in increments of 25 μA until an afterdischarge (AD) was elicited on the EEG. The ADT was defined as the lowest current intensity that evoked an AD of ≥3 s. Once the ADT for each animal was determined, the current was increased by 50 μA (suprathreshold) and used for kindling. Kindling seizure behavior was scored by using the five-stage classification scale of Racine (40): 1, immobility, eye closure, twitching of vibrissae, facial clonus; 2, head nodding, associated with more severe facial clonus; 3, clonus of forelimb; 4, rearing, mostly accompanied by bilateral forelimb clonus; and 5, rearing with loss of balance and falling accompanied by generalized clonic seizures. Beginning on P61, all rats were kindled over 5 consecutive days by using twice-daily (6 h apart) stimulations until three consecutive stage five seizures occurred. Latency to all kindling stages was recorded and means compared between rats with SE at P20 and their age-matched controls.
Rats were killed at P73 after completion of the experiment and prepared for histologic examination and electrode-positions verification.
To assess whether SE experienced in early life induced overt hippocampal reorganization and neuronal damage that could be detected in young adulthood, the hippocampus of SE rats and controls was examined with Timm and thionine staining methods. The aberrant growth of the granule cell axons, so-called “mossy fibers,” was visualized through the Timm staining method, and neuronal loss, with thionine staining.
Animals used for the behavioral tests were killed on P61. After deep anesthesia with PTB (80 mg/kg, i.p.) rats were perfused transcardially with 200 ml of sodium sulfide perfusion medium (2.925 g Na2S, 2.975 g NaH2PO4; H2O in 500 ml distilled H2O) followed by 200 ml 4% paraformaldehyde (PFA). The brains were postfixed in PFA for 24 h and then placed in a 30% sucrose solution until the brains sank to the bottom of the chamber. Coronal sections through the entire extent of the hippocampus were cut at 30 μm on a freezing microtome, and sections were stored in phosphate-buffered saline (pH 7.3). Every fourth section was stained for mossy fibers with Timm stain, and alternate sections were stained with thionine for cell loss. In addition, another series of sections was Timm stained and then counterstained with thionine. These slices were not used for Timm score or cell count. The sections for Timm stain were developed in the dark for 45 min in a solution of 50% arabic gum (120 ml), 10 ml citric acid (51 g/100 ml H2O), 10 ml sodium citrate (47 g/100 ml H2O), 3.47 g hydroquinone in 60 ml, and 212.25 mg AgNO3. Slices from control animals were always stained at the same time as those from the experimental animals. After washing, the slices were dehydrated in alcohol, cleared in xylene, and mounted on slides with Permount. A minimum of 10 sections (20 hippocampi) stained with Timm and 10 cresyl violet–stained sections were analyzed for each rat by an investigator blind to experimental group (G.L.H.). Assessment of the Timm score in the supragranular layer was done in the inferior blade of the dentate, avoiding the edge and the crest of the gyrus. Timm staining in the pyramidal and infrapyramidal CA3 regions and supragranular region was assessed on each section from the septal area, where the two blades of the dentate were equal and formed a V shape (2.8 mm posterior from the bregma) to a point ∼3.8 mm posterior to the bregma (41). Timm staining was analyzed by using a semiquantitative scale for terminal sprouting on CA3 pyramidal cell region and supragranular layer of the DG. The scales used for visual analysis are given in Tables 1 and 2. Table 2 is a modification of a scale proposed by Sutula et al. (42,43). Both the CA3 and supragranular scales have been used previously in assessing sprouting (44,45). Both hippocampi of the specimens were analyzed, and the score given for each animal to the CA3 and supragranular regions reflected the mean for the two sides and was used for the analysis.
Table 1. Scoring system for CA3 mossy fiber sprouting
|0||No granules in the stratum pyramidale or stratum oriens along any portion of the CA3 subregion|
|1||Occasional granules in the stratum pyramidale or stratum oriens occurring in discrete bundles|
|2||Occasional to moderate granules in the stratum pyramidale or stratum oriens|
|3||Prominent granules in the stratum pyramidale or stratum oriens|
|4||Prominent granules in the stratum pyramidale or stratum oriens occurring in near-continuous distribution along the entire CA3 region|
|5||Continuous or near-continuous dense laminar band of granules in the stratum pyramidale or stratum oriens along the entire CA3 region|
Table 2. Scoring system for supragranular mossy fiber staining
|0||No granules noted between crest and tips in the supragranular region|
|1||Occasional granules in the supragranular region occurring in patchy distribution|
|2||Numerous granules in the supragranular region occurring in patchy distribution|
|3||Granules in the supragranular region occurring in near-continuous distribution|
|4||Highly concentrated band of granules appearing either in continuous or near-continuous distribution|
|5||Continuous dense laminar band of granules from crest to tip of dentate|
Slides stained with thionine were analyzed for cell loss in the hilus and CA1 and CA3 subfields of the hippocampus. The severity of the observed lesion was established on a scale of 0 to 3 by using a method previously described in our laboratory (46,47), and a mean score was calculated for each animal. A score of 0 indicated no lesion. A score of 1 indicated a minimal lesion localized in the hilus, CA1, or CA3 region of the hippocampus. A score of 2 indicated cell loss in the hilus, CA1, or CA3, with preservation of the general cellular architecture and some normal cellular elements. A score of 3 indicated complete disruption of the normal cellular architecture. The mean of the scores for the hilus, CA1, and CA3 subfields was calculated for each animal and used for the analysis. Scoring was conducted blindly with respect to the treatment status of the animals by G.L.H.
Data were analyzed by using GraphPad Prism 3.0 statistical software (GraphPad Software, San Diego, CA, U.S.A.). Time to reach the water maze platform was compared in the control and experimental groups by using analysis of variance (ANOVA) with repeated measures. In addition, the groups were compared on each testing day with the t test. Number of stimulations to reach each kindling stage was compared between SE and control rats with the t test. Histologic lesion scores were compared by using the nonparametric Kruskal–Wallis test. Latency to onset of tonic seizures during flurothyl inhalation in the experimental and controls rats was compared with the paired t test. Correlations were assessed by using the Spearman rank correlation test. A p value of <0.05 was considered statistically significant.
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- MATERIALS AND METHODS
This study was designed to determine whether the long-term consequences of SE during development on hippocampal plasticity and cognitive function are age and model specific. Because of the clinical importance of the long-term consequences of SE during development on hippocampal plasticity and cognitive function in adulthood, we used Li-PC to induce SE at different age points during development (P12, P16, P20), and evaluated the effects of this abnormal neural activity on spatial memory performance and seizure susceptibility in the animals beginning on P55, corresponding to young adulthood. We demonstrated that SE at P12 did not result in any structural or functional changes detectable in adulthood, whereas SE at both P16 and P20 induced cell loss and mossy fiber sprouting within the hippocampus and cognitive impairment when the animals were tested as adults. Although seizure threshold to generalized seizures did not seem to be altered, the animals with SE at P20 showed a markedly reduced susceptibility to kindling in adulthood.
Although previous studies suggested that the immature brain may be relatively resistant to seizure-induced morphologic and physiologic alterations compared with the adult nervous system (15,19,20), recent studies revealed that seizures in rat pups may induce abnormalities in the terminal field of the mossy fiber pathway and long-term impairment in hippocampus-dependent spatial memory task (29,44,45,48,49). Lynch et al. (29) showed that kainic acid–induced SE during early postnatal development (P1–P14) in the rat induced a long-term impairment in the radial arm maze performance, a hippocampus-dependent spatial memory task, and a reduced susceptibility to kindling in adulthood. Spontaneous seizures have been observed in rats subjected to Li-PC SE at ages 2 and 3 weeks only after a latent period of ≥2 months (25,50). Thus it is not surprising that we did not observe any spontaneous seizures during testing days from P55 to P61, <2 months after the induction of SE.
The present study shows that seizure-induced plasticity during development influences cognitive function in adulthood. We report that in rats subjected to SE at P16 and P20, impairment is shown in the water maze, a test of spatial memory, when the animals are tested as adults. After SE, rats were impaired both during the acquisition trials as well as during the probe test, demonstrating substantial impairment in visuospatial learning and memory. Moreover, in those rats with SE at P20, a negative correlation between performance in the water maze and the degree of supragranular sprouting suggests a role of synaptic reorganization in determining the cognitive impairment.
Several studies investigated the relation between hippocampal mossy fiber sprouting and learning. Some evidence indicates that the degree of CA3 stratum pyramidale mossy fiber projection correlates with learning (44,48,51). In a two-way avoidance learning task, animals having more CA3 mossy fiber terminals performed less well than animals with fewer terminals (51). Rats subjected to recurrent neonatal seizures developed mossy fiber sprouting in the CA3 region and cognitive impairment in the adulthood (44,48). The presence of increased sprouting in the animals showing cognitive impairment raises the question whether there is a causal relation between sprouting and learning. Whereas a positive correlation occurred between sprouting in the supragranular region of the DG and water maze performance in rats subjected to SE at P20, such a relation was not present in rats subjected to SE at P16. In neither age group was there a close relation between CA3 sprouting and cognitive impairment.
The possibility of seizure-induced brain damage and the concept of seizure-induced epileptogenesis in the developing brain remains a controversial issue in both clinical and experimental epilepsy. Previous research (52,53) showed that rat pups that survived SE induced by kainate or PC did not develop altered susceptibility to seizures as adults or show an induced resistance to kindling development when kainic acid was administered in the newborn rat (P1) (29). Conversely, Holmes et al. (45) found that rats subjected to recurrent seizures during the neonatal period had a decreased seizure threshold when tested as adults. However, in this study, the authors used flurothyl inhalation to induce neonatal seizures and pentylenetetrazol to evaluate the subsequent changes in seizure threshold: both are considered as models for primarily generalized seizures.
We chose to use two different models to assess seizure susceptibility, flurothyl inhalation, a model of generalized seizures, and kindling, a model of partial seizures with secondary generalization, because we wished to determine whether seizures in the immature brain could “prime” the brain for later seizure susceptibility.
Although the seizure threshold in the SE animals was not altered when it was evaluated by using flurothyl inhalation, a model of primarily generalized seizure, when seizure susceptibility was assessed with amygdala kindling, a validated model for focal and secondarily generalized seizures (36,54), the P20 SE animals demonstrated a lower seizure threshold, as they needed fewer stimulations to reach each kindling stage and had a significantly lower ADT compared with controls. Sankar et al. (50) made the important observation that activation areas after SE reflect age- and model-dependent plasticity. By using two models of seizure induction, Li-PC SE and perforant path stimulation (PPS), they demonstrated that at P21, PPS resulted in substantial induction of c-Jun immunoreactivity in the hippocampus and only mild induction in the amygdala and barely discernible Jun immunoreativity in the temporal neocortex or the thalamus, whereas Li-PC resulted in robust appearance of Jun immunoreactivity in these structures. The authors suggested that network recruitment as seen by the seizure-induced c-Jun immunoreactivity in extrahippocampal structures may be a function of synaptic maturation during development but also be related to the ability of the immature hippocampus to maintain GABA synthesis in the face of ongoing SE (55). Studies of seizure-induced expression of the immediate early gene products, c-Fos or c-Jun, showed a distinct pattern of neuronal activation after flurothyl (56) or Li-PC seizures (50). Whereas the expression of c-Fos indicates that the hippocampus is minimally activated by a single brief flurothyl exposure in adult mice, the pattern of regional c-Jun and c-Fos immunoreactivity seen after 2 h of Li-PC SE demonstrated hippocampal immunoreactivity with a high signal in the DG cells (50,57). Together these studies show that different structures of the brain are activated during a first episode of partial or generalized seizures.
Our experiment goes further and demonstrates that partial seizures at early age, primarily involving the limbic system, make the brain subsequently prone to seizures arising from the same modified circuitry. The specificity for a model of limbic seizure, such as kindling, suggests that the altered susceptibility to seizures as adult is not a result of a general increase in neuronal excitability throughout the brain but rather reflects seizure-promoting changes that we observed within the limbic system on circuitry previously activated by PC. These observations may have clinical implications for cognitive and memory dysfunction associated with epilepsy. Future studies will be necessary to determine whether discernible histologic damage is a prerequisite for reduction in seizure threshold. We elected to kindle only P20 rats because these animals had clear behavioral and histologic deficits. Whether younger rats with no observable damage (P12) or moderate damage (P16) would have altered kindling rates is not yet known.
In conclusion, we showed that disruption of normal neural activity during development produces structural and functional effects in the hippocampus. These changes are age dependent and can be seen after P12. We also showed that the mode of induction of the initial event during development plays an important role on its epileptogenicity because the affinity of the insult to specific neuronal circuits will influence the susceptibility to a certain type of future seizures. It remains unclear which mechanisms are causal, correlative, or consequential. Understanding these mechanisms is the key to preventing the onset of epilepsy.