The aim of this study was to characterize a novel animal model hyperthermia-induced febrile seizure and to investigate the impacts of repetitive febrile seizures on spatial learning and memory performances in immature rats.
Epilepsy is a chronic neurological disorder which affects about 1% of the world population and which is more common during childhood than at any other age [
Epilepsy in infants has the particularity of occurring in a developing brain. This could be the origin of possible cognitive-behavioral consequences [
These particular cognitive deficits are due to the location of a brain epileptogenic zone and to the mode of propagation of the electrical discharge. The neurons responsible for the electrical discharge are found in the temporal lobe, in the hippocampus more specifically. In some patients, the origins of this disorder are brain injuries, but in most cases there is no obvious organic cause. Temporal lobe epilepsy (TLE) is the most common and the most severe form of epilepsy in adults. It results in the loss of contact with reality and affects the memory. In a lithium-pilocarpine rat model of TLE, Ye et al. [
Epilepsy appears in infants with a more heterogeneous semiology [
Most patients that underwent surgery for temporal lobe refractory epilepsy have suffered a prolonged febrile seizure during childhood [
Fever is the most common symptom observed in the infant pathology [
Several animal models have been developed to investigate the pathogenesis of febrile seizures and their consequences [
Neonates from pregnant Wistar rats were obtained from an inbred stain (Faculty of Sciences, Bizerta). The day of birth was considered as day 0 of postnatal life. When weaned (on P21), rats were housed 6 per cage and were kept at 25°C, under a 12:12 light/dark cycle with free access to food and water. Animals were cared of under the Tunisian code of practice for the care and use of animals for scientific purposes. The experimental protocols were approved by the Faculty Ethics Committee.
On postnatal day 11 (P11) [
Following a trigger of myoclonic jerks, the rat was then transferred to the surface at room temperature. A few seconds later, the seizure turned into a tonic-clonic generalised seizure with vigorous shaking of the head, ears, and upper and lower limbs and an especially violent vibration of the tail. Most animals showed a reaction to the myoclonic febrile seizures and developed tonic-clonic generalised seizure.
A 30 min observation was then necessary to monitor the postictal state of the animals, which required less than 20–25 min to return to normal.
Each animal was rapidly cooled down with a few drops of water and dried out with paper towel in order to help it reach its natural body temperature. It then gradually resumed its activity and was returned to the cage.
Rats were exposed to one, two, or three hyperthermia episodes in 10-day intervals at postnatal days 11, 21, and 31 (P11, P21, and P31). Control rats were exposed to the same experimental conditions, excluding hyperthermia. These rats did not suffer any seizures, only moderate agitation due to the new environment.
Animals’ spatial memory was tested with the Morris water maze [
Treated and control rats underwent a spatial test after 2 days’ rest in order to evaluate the impacts of febrile seizures on the spatial memory in the Morris water maze [
The test was carried out using a round pool (90 cm in diameter and 50 cm high) filled to a depth of 25 cm with water (22°C) and placed in the center of a room surrounded by several cues. The experiment was divided into various phases carried out in the following order (Figure
Treatment and behavioural testing protocol for treated group 1 (Tr1). The same procedure was applied for treated 2 and 3 groups. P11: postnatal day 11; FS: febrile seizure; NW: northwest; SW: southwest.
During pretraining the platform (20 cm in diameter) was located in a fixed position of the pool (middle of the northwest quadrant) and was made invisible to the rat by placing it 2 cm below the water surface covered by small polystyrene pieces.
On day 4 of the pretraining session and 20 min after the last trial, a retention test was performed. Latency to find the submerged platform was measured. Because a similar decrease of latency was recorded from the four starting positions, only north and west points were subsequently carried. Twenty-four hours after the retention test, a probe test was conducted. Rat was placed in the middle of the pool and the time spent in the quadrant that the platform was previously located in was recorded.
Twenty-four hours after the probe test, a second trial acquisition session was given under the same experimental conditions as the first test but with a visible platform lifted 1 cm above water level and placed in the southwest quadrant. On day 4, escape latency was measured after north or west introduction for each rat.
The ANOVA parametric test was used, complemented by Tukey’s test as a post hoc test to study the significance of differences between groups. Data are given as mean ± SEM values.
As explained in the methods section, neonates (P11) were subjected to hyperthermia until they suffered a generalised tonic-clonic seizure (GTCS). In order to reach this stage, the rats had previously undergone myoclonic jerks. The time for myoclonic jerks to first appear (MJAT), the time for a GTCS to appear (GTCSAT), and the back to normal recovery time (RT) were recorded.
Figure
Myoclonic jerks appearance time (MJAT), generalised tonic-clonic seizure appearance time (GTCSAT), and recovery time (RT) after a first exposure (
To examine the effect of repetitive hyperthermia exposure on time appearance of seizures, animals treated by a first exposure (Tr1) were exposed in an interval of 10 days a second (Tr2) and a third time (Tr3) to hyperthermia. In Tr3 group, the GTCS took less time to appear than in those exposed only once or twice to hyperthermia. The average GTCSAT for this group was
Generalised tonic-clonic seizure appearance time (GTCSAT) in treated group 1 (Tr1,
Group | Tr1 | Tr2 | Tr3 |
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Time (min) |
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The decrease of rat body weight before and immediately after seizures averaged 2.37, 2.20, and 1.94%, respectively, in Tr1, Tr2, and Tr3 groups. This decrease which was not significantly different compared to that of control rats (2.32, 1.96, and 1.14%, resp., in C1, C2, and C3) may be explained by fecal and urinary losses during confinement conditions.
To examine the effect of repetitive febrile seizures on spatial learning and memory, we evaluated control and hyperthermia-treated rats for their performance in the Morris water maze which is employed in experiments as a basic assay of spatial memory and associated learning ability [
Our results indicated that, by the fourth day of the acquisition phase of the task, Tr1 and Tr2 groups displayed more time than the corresponding controls to reach the submerged platform after a north or west introduction, suggesting that, after one or two hyperthermia exposures, rats did not improve their performance during the 4 days of pretraining (Table
Average latency to reach the hidden platform from the north and west starting points for control (C1,
Group | North | West | |
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Time (sec) | C1 | 19.57 ± 3.22a | 17.85 ± 3.38a |
Tr1 | 38.75 ± 5.04b | 30.23 ± 3.88b | |
C2 | 20.50 ± 5.33a | 14.50 ± 1.38a | |
Tr2 | 44.12 ± 5.75b | 36.80 ± 7.35b | |
C3 | 7.50 ± 1.70c | 4.30 ± 0.65c | |
Tr3 | 5.54 ± 1.03c | 7.72 ± 2.37c |
During the probe test, both control and treated groups spent more time in the quadrant where the platform was located than would be predicted by chance (>15 min). However, treated rats Tr1 and Tr2 spent less significant time than corresponding controls in the target quadrant, while Tr3 group spent an equivalent time in this quadrant as C3 group. Nevertheless, in both hyperthermia-exposed and nonexposed groups, the quadrant dwelling time increased gradually with age (Table
Average time spent by control (C1, C2, and C3) and treated (Tr1, Tr2, and Tr3) groups in target quadrant. Data are shown as mean values ± SEM. Values not sharing the same letter are significantly different;
Group | C1 | Tr1 | C2 | Tr2 | C3 | Tr3 |
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Time (sec) | 22.31 ± 1.90a | 17.13 ± 2.20b | 28.18 ± 4.93a | 19.07 ± 3.10b | 51.44 ± 5.94c | 47.53 ± 5.60c |
To check for the ability to learn a new location of the platform, we conducted a memory transfer test in which the platform was moved to the southwest quadrant and lifted 1 cm above water surface. Our results indicated that treated groups required generally more time to change the spatial reference of the previous environment to attain the emerged platform than corresponding controls, but the difference was significant only for Tr3 group after both north and west introductions as shown by the important increase of the latency to escape (Table
Average latency to reach the visible platform (southwest quadrant) for control (C1, C2, and C3) and treated (Tr1, Tr2, and Tr3) groups after north or west introduction. Data are shown as mean values ± SEM.
Group | North | West | |
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Time (sec) | C1 | 24.16 ± 7.08 | 13.21 ± 6.92 |
Tr1 | 25.61 ± 7.49 | 18.13 ± 7.90 | |
C2 | 25.60 ± 3.98 | 12.60 ± 3.84 | |
Tr2 | 29.01 ± 5.99 | 18.30 ± 5.49 | |
C3 | 23.45 ± 2.44 | 13.18 ± 2.95 | |
Tr3 |
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Febrile seizures occurred commonly in 2–5% of children under the age of 5 years and can be simple or complex [
In rats, as well as in humans, susceptibility to febrile convulsions is age-related. The highest sensitivity is found in young animals and it sharply decreases with age [
Using this novel model, we showed that 30 min of hyperthermia hot water was sufficient to induce febrile seizures in P11, P21, and P31 rats. All hyperthermia-treated rats developed rapidly generalised seizures. This result is consistent with previous data suggesting that, in the immature healthy brain, a high and prolonged temperature is required for convulsive seizures to appear [
In accord with a previous study [
In rats subjected to hyperthermia for two or three episodes in 10-day intervals, the generalised seizure appearance time was reduced by 18 and 23%, respectively, indicating an enhancement of seizure susceptibility with repetitive hyperthermia exposure, probably due to thermoregulatory centres maturation.
Retrospective studies have shown that febrile seizures induced damage in the medial temporal structures and thus led to hippocampal sclerosis and to TLE [
Our research was directed to check the impact of single or repetitive prolonged febrile seizures on memory function in immature rats. To this end, we have used the Morris water maze which is mainly a hippocampus-dependent spatial learning and memory task [
By the fourth learning day, only rats of control group C3 improved their performance to attain the aim after north or west introduction. In contrast, Tr1 and Tr2 groups displayed significantly more time to reach the submerged platform suggesting that after one or two hyperthermia exposures, rats did not improve their performance during the 4 days of pretraining and solely Tr3 group displayed the same ability as the corresponding control after north or west introduction. It may be concluded that, by the third episode of hyperthermia, rats acquired the task similarly as untreated groups. Indeed, hyperthermia-induced seizures at P11 and P21 had a significant but transient effect on the Morris water maze spatial learning, since additional hyperthermia episode failed to induce cognitive deficit and Tr3 rats became indistinguishable from corresponding controls. These findings correlated with those of Notenboom et al. [
In the probe test, Tr1 and Tr2 rats spent also less significant time in the target quadrant than corresponding controls, whereas Tr3 and C3 groups spent the same time in this quadrant. Moreover, with age, control and treated groups tended to spend more time in the target quadrant, but the effect was significant only for C3 and Tr3. Indeed, hyperthermia-induced seizures early in life may impair spatial memory consolidation. These observations could be correlated with studies showing that rats with hippocampus, dentate gyrus, subiculum, or combined damage did not perform well in tests without a platform [
Memory transfer test showed that control rats were generally more efficient in the transfer of learning and memorizing the new position of the visible platform (southwest quadrant) than corresponding treated groups, but the differences were only significant for C3 group. These observations indicated that repetitive febrile seizures might affect the ability to memorize and use spatial information to learn the novel location of the platform.
No sex differences were observed in learning and visuospatial memory in the Morris water maze [
Taken together, our results showed that febrile seizures in low age affected mainly spatial acquisition of cognitive abilities and the alterations seemed to disappear later, confirming in part previous behavioural animal studies demonstrating that early-life prolonged febrile seizures resulted in moderate or absence of alteration of cognitive performance at adulthood [
It has been shown that duration of febrile seizures influenced the probability of developing subsequent epilepsy and severity of the spontaneous seizures. Thus, febrile status averaging 64 min was more efficient compared to 24 min to increase the severity and the duration of febrile seizures [
With a novel methodology using hyperthermia hot water, we showed that the possible hippocampus damage being caused by repetitive hyperthermia-induced seizures in immature rats might be moderate and transient. Thus, further investigations using especially biochemical and histopathological markers of hippocampal alteration are required to better characterize this model.
The authors are solely responsible for the content of this paper.
The authors report no conflict of interests.
This work was supported by the Tunisian Ministry of Higher Education and Scientific Research.