Address correspondence and reprint requests to Dr. L-T. Huang at Department of Pediatrics, Chang Gung Memorial Hospital, No. 123, Ta Tei Road, Niao Sung Hsiang, Kaohsiung Hsien, 833, Taiwan. E-mail: Huang_li@taiwan.com
Summary: Purpose: Recurrent seizures in infants are associated with a high incidence of neurocognitive deficits. Animal models have suggested that the immature brain is less vulnerable to seizure-induced injury than is that in adult animals. We studied the effects of recurrent neonatal seizures on cognitive tasks performed when the animals were in adolescence and adulthood.
Methods: Seizures were induced by intraperitoneal injection of pentylenetetrazol (PTZ) for 5 consecutive days, starting from postnatal day 10 (P10). At P35 and P60, rats were tested for spatial memory by using the Morris water maze task. In adulthood, motor performance was examined by the Rotarod test, and activity level was assessed by the open field test. Seizure threshold was examined by inhalant flurothyl. To assess presence or absence of spontaneous seizures, rats were video recorded for 4 h/day for 10 consecutive days for the detection of spontaneous seizures. Finally, brains were examined for histologic evidence of injury with cresyl violet stain and Timm staining in the supragranular zone and CA3 pyramidal cell layers of the hippocampus.
Results: PTZ-treated rats showed significant spatial deficits in the Morris water maze at both P35 and P60. There were no differences in seizure threshold, motor balance, or activity level during the open field test. Spontaneous seizures were not recorded in any rat. The cresyl violet stain showed no cell loss in either the control or experimental rats. PTZ-treated rats exhibited more Timm staining in the CA3 subfield. However, the control and experimental rats showed similar Timm staining within the supragranular zone.
Conclusions: Our findings indicate that recurrent PTZ-induced seizures result in long-term cognitive deficits and morphologic changes in the developing brain. Furthermore, these cognitive deficits could be detected during pubescence.
Seizures occur more frequently in the neonatal period and early childhood than at any other time in life. The high incidence of seizures in the first decade of life and the propensity of children toward febrile seizures and status epilepticus (SE) are reflective of the increased susceptibility of the immature brain to seizures (1). In addition to the increased risk for seizures in children, such seizures during development may be more detrimental to the organism than when they occur during adulthood. The mean IQ score in children with seizures is lower than that in the normal population (2,3). However, even when they have normal IQs, children with seizures have learning and behavioral problems (2,3).
Studies on the long-term consequences of recurrent early-life seizures in young animals are limited. Available reports were based on restricted sampling of various seizure models and developmental ages (4–15), or they had examined specific aspects, such as behavior (4,13,14), metabolism (6), or histopathology (10,15). The results of these studies are still controversial. For example, Sarkisian et al. (5) reported that immature rats are spared from long-term cognitive and pathologic sequelae after repeated doses of kainate-induced SE. In contrast, previous studies demonstrated that repeated flurothyl-induced seizures in young rats resulted in significant subsequent cognitive deficits (4,7). Pentylenetetrazol (PTZ), a γ-aminobutyric acid (GABA) antagonist, is one of the most commonly used pharmacologic agents for inducing seizures. Here we used PTZ to test the hypothesis that recurrent seizures in early life can have adverse long-term effects. Because information regarding the time course of cognitive deficits after neonate recurrent seizures is unavailable, an additional aim of this study was to examine whether the cognitive impairment appears at adolescence.
MATERIALS AND METHODS
Sprague–Dawley male and female rats were used throughout the experiments. Attempts were made to minimize the numbers of animals used. The delivery day was designed as postnatal day 0 (P0). The rats were purchased from the National Science Counsel and housed in Chang Gung Memorial Hospital, Kaohsiung, Taiwan. Animals had access to food and water ad libitum and were housed with their littermates until weaning at P21, when they were group-housed in plastic cages on a standard 12 h/12 h light/dark cycle.
Induction of seizures
Pentylenetetrazol (PTZ; Sigma, St. Louis, MO, U.S.A.), a GABAA-receptor antagonist, was used as the convulsant to evaluate the long-term morphologic and behavioral effects of recurrent seizures in the immature brain. Beginning at P10, the experimental group received repetitive subconvulsive doses of PTZ, modified from previous studies (16–18). First, a dose of 90 mg/kg was injected intraperitoneal (i.p.), followed 10 min later by an injection of 40 mg/kg. Thereafter, additional doses of 20 mg/kg were administered to the rat every 10 min until the onset of SE, which was characterized by clonic movements of the four limbs and loss of posture. PTZ was administered daily from P10 to P14. Control rats were given an equal volume of saline and were separated from their dams for the same duration of time. The animals were kept under a heating lamp to maintain their temperatures within the normal range.
Morris water maze
Two separate groups of rats, 14 rats in each (PTZ treated, n = 8; control, n = 6), were evaluated for spatial memory in the Morris water maze at two different time periods (P35–P40 and P60–P65). A circular tank (180 cm diameter × 50 cm height) 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, which prevented visualization of the platform. Room lights illuminated the pool, and multiple distant cues around the room (window, cabinets, furniture) were kept in the same location throughout the experiments. Four points on the perimeter of the pool were designed north (N), east (E), south (S), and west (W), thus dividing the pool into four quadrants (NW, NE, SE, SW).
A 10 × 10 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. A video camera was set above the center of the pool and connected to a videotraction system (EthoVision, Noldus, The Netherlands). On the first day, each rat was placed in the pool for 60 s without the platform present; this free swim enabled the rat to become habituated to the training environment. On days 2 to 5, rats were trained for 24 trials (six trials a day) to locate and escape onto the submerged platform. For each rat, the quadrant in which the platform was located remains 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. Thus the rat was not able to predict the platform location from the point at which it was placed in the pool.
On mounting the platform, the rats were given a 30-s rest period, after which the next trial was started. If the rat did not find the platform in 120 s, it was manually placed on the platform for a 30-s rest. At the start of each trial, the rat was held facing the perimeter and dropped into the pool to ensure immersion. Latencies to escape onto the platform were recorded. On day 6, the platform was removed. The rat was allowed 60 s of free swimming. The time spent in the quadrant where the platform was previously located was measured (probe trial). The testing procedure used during the 4 days of locating the hidden platform is considered a measurement of spatial reference memory, whereas the probe trial is considered to measure the strength of spatial learning (19).
Twelve rats (PTZ treated, n = 6; control, n = 6) were evaluated for motor ability (locomotion) by using the Rotarod at P80. The Rotarod was used to test the limb motor coordination and balance aspects of motor performance in rats (20). Animals were placed on the rotating rod (Ugo, Basile, Italy) with a diameter of 7 cm (speed, 12 rpm). Time spent on the rod was measured up to 180 s.
Open field test
The open field test was used to check the behavioral response to a novel environment, motor abilities, and habituation (21). The floor of a square plastic board (50 × 50 cm) with plastic sides (30 cm high) was divided into 16 squares. Activity level was expressed as the total number of squares crossed during the 5-min testing period. Exploratory activity was expressed as the total number of rears. The open field test was done at ∼P90.
Seizure threshold to flurothyl
Seizure susceptibility was studied in two subgroups of rats (PTZ treated, n = 6; control, n = 6) by exposing the rats to flurothyl inhalation as previously described (4). The rats exposed to flurothyl were killed immediately. Furthermore, experimental and control rats were paired in the chamber. The transparent plastic airtight box (40 × 20 × 20 cm) was divided into two equal parts with side doors. Flurothyl was administered at the rate of 0.04 ml/min (Medfusion 2001; Medex, U.S.A.) through a hole in the center of the chamber. The latencies to first myoclonic jerk and generalized tonic seizures were recorded. Animals were removed from the side door as soon as a tonic seizure was induced.
Spontaneous recurrent seizures (SRSs)
To quantify SRSs after recurrent early-life PTZ-induced seizures, we used a closed-circuit video-monitoring system starting at the age of 9 months. A wide-angle lens was used to videotape all the rats (PTZ treated, n = 6; control, n = 6) simultaneously. Each rat was videotaped 4 h/day over 10 consecutive days. Monitoring was performed during the daytime (10 a.m. to 2 p.m.), and all the tapes were reviewed off-line.
On completion of the experiments, the animals were killed, and their brains were analyzed for (a) neuronal loss with cresyl violet stain, and (b) mossy fiber sprouting by using the Timm technique. After deep anesthesia with sodium pentobarbital (80 mg/kg), rats were perfused transcardially with 250 ml of sodium sulfide perfusion medium (2.925 g Na2S, 2.975 g NaH2PO4:H2O in 500 ml distilled H2O) followed by 250 ml 4% paraformaldehyde.
The brains were postfixed in paraformaldehyde 4% for 24 h followed by a 30% sucrose solution for 24 h or until the brains sank to the bottom of the container. Coronal sections through the entire extent of the hippocampus were cut at 30 μm on a freezing microtome, and sections were then stored in phosphate-buffered saline. Every fourth section was stained for mossy fibers with the Timm stain, with alternate sections stained with cresyl violet for cell counting.
Cresyl violet staining
The staining procedure consisted of mounting cryocut sections, dehydration of the tissue, followed by staining (6–10 min) and rehydration in a series of ethanol (70–l00%), xylene (5 min each), and coverslipping with Permount.
The sections were developed in the dark for 40–45 min in a solution of 50% arabic gum (120 ml), 10 ml of citric acid (51 g/100 ml H2O), 10 ml sodium citrate (47 g/100 ml H2O), 3.47 g hydroquinone in 60 ml distilled H2O, and 212.25 mg AgNO3. After washing, the slides were dehydrated in alcohol, cleared in xylene, and mounted on slides with Permount. Timm scores in CA3 were counted as previously reported (7,10,11). In brief, 0 indicated no granules in the stratum pyramidal (SP) or stratum oriens (SO), 1 indicated occasional granules in the SP or SO in discrete bundles, 2 indicated occasional to moderate granules in the SP or SO, 3 indicated prominent granules in the SP or SO, 4 indicated prominent granules in the SP or SO in near-continuous distribution along the entire CA3 region, and 5 indicated continuous or near-continuous dense laminar band of granules in the SP or SO along the entire CA3 region. Timm staining for supragranular sprouting also was evaluated, as previously reported (7,10,11).
Analysis of results
Comparisons of the escape latencies during the 24 trials of place learning of the Morris water maze were analyzed by one-way analysis of variance (ANOVA) with repeated measurements. The swimming speed, the probe trial, the Rotarod test, open field test, seizure threshold, and Timm scores were analyzed with the nonparametric Mann–Whitney U test. Values are expressed as mean ± SEM, and significance is defined as p < 0.05 for all tests.
Behavioral features of PTZ-induced seizures
All rats in the experimental group had seizures induced by repetitive PTZ. The seizures were characterized by head shaking, squealing, crawling, wild running, loss of righting reflex, and generalized tonic–clonic convulsions. In most animals, the duration of convulsions ranged between 10 and 40 min. The rats returned to their baseline activities by 180 min after the seizures. The total mortality rate in PTZ-treated rats was 30%. There was no difference in mortality by postnatal ages.
Morris water maze
Spatial learning was assessed using the Morris water maze task. Figure 1A shows the mean escape latencies in the Morris water maze as a function of day for P35 and P60 rats. Both groups had significant decreases in latencies in time-to-platform over 4 testing days. It is noteworthy that, compared with the control group, the differences of mean escape latencies in PTZ-treated rats at P35 were significant over the 4 testing days, with the greatest difference occurring on the first day of testing (F1,12 = 6.79; p = 0.023). The P60 rats had significant impairment only in the first day (F1,12 = 19.164; p = 0.01). At both ages, there was no significant difference in swimming speed between groups on any test day (p > 0.05).
When the probe trial was tested at adolescence and adulthood, both groups of rats spent more time in the goal quadrant. However, there was no significant difference in time spent in the goal quadrant between groups (P40, Mann–Whitney = 11, p > 0.05; P65, Mann–Whitney = 15, p > 0.05; Fig. 1B).
There was no significant difference in Rotarod test between rats subjected to recurrent PTZ-induced seizures and controls (Mann–Whitney = 11, p > 0.05).
Open field test
We measured the total number of blocks crossed and number of rears in a 5-min period. There was no significant difference in either blocks crossed (Mann–Whitney = 8, p > 0.05) or rears (Mann–Whitney = 14.5, p > 0.05).
Seizure threshold to flurothyl
There was no significant difference between groups in the latency to myoclonic jerks (Mann–Whitney = 7, p > 0.05) or in tonic seizures (Mann–Whitney = 10, p > 0.05; Fig. 2).
Spontaneous recurrent seizures
There was no SRS detected in a rat from either group. Because rats were recorded for only 4 h during the day, it is of course possible that some spontaneous seizures were not observed.
Cresyl violet staining revealed no apparent cell loss in the histologic examination of specimens from the rats with or without recurrent seizures (Fig. 3A and B).
The Timm score in CA3 was significantly lower in the controls (Fig. 3C) than in the PTZ-treated rats (Fig. 3D; PTZ treated, 1.83 ± 0.15; controls, 0.52 ± 0.29; Mann–Whitney = 1, p = 0.016). The Timm staining in the supragranular region was barely visible in either the controls (Fig. 3E) or the PTZ-treated rats (Fig. 3F).
The purpose of the present study was to investigate the long-term behavioral, motor, and morphologic effects of recurrent PTZ-induced seizures on the immature brain. PTZ-treated rats had spatial memory deficits, which could be detected at adolescence. When examined as adults, rats with a history of recurrent seizures had significant deficits in spatial memory and showed more mossy-fiber sprouting in the CA3 subfield than did controls. However, there were no significant differences in the activity levels, motoricity and balance, or threshold to flurothyl-induced seizure between control and experimental rats. Neither neuronal loss nor SRSs were found after recurrent neonatal seizures.
Previous studies have shown that recurrent early-life seizures caused long-term alterations of cerebral metabolism (6), reduction in dendritic spine density (8), reduced neurogenesis in the dentate gyrus (15), reduced seizure susceptibility (4,7,11), alterations in the firing properties of hippocampal CA1 pyramidal neurons (22), and abnormalities in the CA3 terminal field of the mossy-fiber pathway (7,10,11,14). However, there is still controversy about whether recurrent seizures in the developing brain are harmful (23,24). A previous study reported that the immature rat was spared from the long-term cognitive and pathologic sequelae after repeated doses of kainate-induced SE (5). However, other studies have indicated that recurrent seizures in the immature brain resulted in long-term cognitive deficits (4,7,11–14). Our findings add to the limited evidence that recurrent seizures in the developing rat brain can cause long-term cognitive deficits.
It is generally accepted that rat pups of P10 to P12 roughly correspond to a term human infant. The P15 to P17 period is roughly equal to a 6-month to 1-year-old human infant, and at ∼35 days is equivalent to human adolescence (25,26). It still remains unclear whether recurrent PTZ-induced seizures at different ages result in reduced seizure threshold and mossy-fiber sprouting. Holmes et al. (10) found that injecting rats daily with PTZ from P0 to P15 could cause significant changes in the distribution of mossy-fiber terminals in the CA3 subfield. In addition, rats receiving twice-daily injections of PTZ between P10 and P25 showed reduced seizure threshold and Timm staining in the supragranular region. In the present study, recurrent PTZ-induced seizures were performed from P10 to P14 and showed no supragranular Timm staining or reduced seizure threshold. Because the dentate gyrus plays a role in learning (27) and undergoes the majority of its development during P1 through P14 (28), we limited the treatment of our animals to the age range P10–P14. Our data extended the previous study (10) and showed that brief recurrent seizures in rat pups resulted in spatial deficits, as measured by the Morris water maze task. Taken together, these data suggest that the seizure number and/or duration are correlated with supragranular staining and may eventually lead to reduced seizure threshold.
Meilleur et al. (29) studied PTZ-induced seizures in P20 rats. Field potentials were obtained from CA3 of hippocampal slices after a short (10 days) or a long (>40 days) interval. They observed abnormal cholinergic transmission in six of 15 rats after 10 days and in eight of eight rats after 40 days (29). Although their findings suggest that PTZ-induced seizures in immature rats might have a progressive and permanent effect, the time course of cognitive changes after recurrent early life seizures has not been well studied. We have shown here that rat pups exposed to recurrent PTZ-induced seizures had significant spatial memory deficits in the Morris water maze task, which was detected in adolescence (P35) and persisted into adulthood (P60). The age at which cognitive deficits first appear has clinical implications and requires further study.
Motor performance is one of the most important indices of brain development, but there are few data regarding motor consequences of SE or recurrent seizure in developing animals. Kubová et al. (30) induced SE in P12 and P25 rats by using lithium-pilocarpine, and both age groups exhibited permanent changes in motor performance after a silent period. The authors suggested that the subsequent motor-function impairment might be related to spontaneous epileptic activity. In a subsequent study (31), they found thalamic neuronal damage in P12 rats. In contrast, our results demonstrated that recurrent neonatal seizures did not cause motor or balance disturbances. Differences in seizure models, treatment periods, and morphology could account for the contrasting results. In addition to motor performance, the open field test measures the animal's reaction to a novel environment (21). A previous study demonstrated that rats with histories of recurrent flurothyl seizures between P0 and P4 were less responsive in the open field test (7). In our study, recurrent neonatal seizures did not appear to alter emotionality, exploratory behavior, or fear.
The relation between SRSs and cognitive deficit remains unclear. Mikati et al. (32) used phenobarbital (PB) to treat kainate-induced SRSs and found no protective effects on spatial deficits. We did not observe any SRSs in PTZ-treated rats. Although the cognitive test and recording seizures were not performed during the same time period, our results seem to support the suggestion that there is no significant correlation between cognitive deficits and SRSs in the rats with recurrent neonatal seizures seen in the present study.
Hussenet et al. (16) demonstrated that young rats subjected to neonatal seizures had long-term detrimental effects on cerebral glucose utilization in the absence of visible neuropathologic changes. This might explain why cognitive deficits and mossy-fiber sprouting occurred in our study in the absence of cell death. It has been assumed that the degree of mossy-fiber projections within the stratum pyramidale correlates to learning. Crusio et al. (33) found that the size of the hippocampal terminal field correlated inversely with error number in a radial maze. Lipp et al. (34) observed that the magnitude of the stratum pyramidale and infrapyramidal projections of mossy fibers correlated with the number of trials to criteria in two-way avoidance learning; the animals with fewer mossy-fiber terminals performed better than animals with more terminals. Previous studies also have shown that recurrent flurothyl-induced seizures in immature rats can cause mossy-fiber sprouting (7,11,14) and cognitive deficits (4,7,11,14). One recent study used flurothyl to induce recurrent neonatal seizures and found that there was an inverse correlation between the degree of mossy-fiber sprouting in the CA3 regions and performance in the Morris water maze (14). The current findings in the PTZ model are similar to the flurothyl model results, and suggest that recurrent seizures in the immature brain cause neuronal circuit abnormality in the hippocampus, as well as long-term cognitive deficits.
Acknowledgment: This research was supported by the Chang Gung Memorial Hospital (CMRP 1121) and New Century Health Care Promotion Foundation to Dr. Li-Tung Huang. We thank Prof. Gregory L. Holmes for reading this manuscript.