Prophylactic treatment with levetiracetam after status epilepticus: Lack of effect on epileptogenesis, neuronal damage, and behavioral alterations in rats

Neuropharmacology 53 (2007) 207e221 www.elsevier.com/locate/neuropharm

Prophylactic treatment with levetiracetam after status epilepticus: Lack of effect on epileptogenesis, neuronal damage, and behavioral alterations in rats Claudia Brandt, Maike Glien1, Alexandra M. Gastens, Maren Fedrowitz, Kerstin Bethmann, Holger A. Volk2, Heidrun Potschka3, Wolfgang Lo¨scher* Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Bu¨nteweg 17, D-30559 Hannover, Germany Received 16 October 2006; received in revised form 28 February 2007; accepted 3 May 2007

Abstract Levetiracetam (LEV) is a structurally novel antiepileptic drug (AED) which has demonstrated a broad spectrum of anticonvulsant activities both in experimental and clinical studies. Previous experiments in the kindling model suggested that LEV, in addition to its seizure-suppressing activity, may possess antiepileptogenic or disease-modifying activity. In the present study, we evaluated this possibility by using a rat model in which epilepsy with spontaneous recurrent seizures (SRS), behavioral alterations, and hippocampal damages develop after a status epilepticus (SE) induced by sustained electrical stimulation of the basal amygdala. Two experimental protocols were used. In the first protocol, LEV treatment was started 24 h after onset of electrical amygdala stimulation without prior termination of the SE. In the second protocol, the SE was interrupted after 4 h by diazepam, immediately followed by onset of treatment with LEV. Treatment with LEV was continued for 8 weeks (experiment #1) or 5 weeks (experiment #2) after SE, using continuous drug administration via osmotic minipumps. The occurrence of SRS was recorded during and after treatment. In addition, the rats were tested in a battery of behavioral tests, including the elevated-plus maze and the Morris water maze. Finally, the brains of the animals were analyzed for histological lesions in the hippocampal formation. With the experimental protocols chosen for these experiments, LEV did not exert antiepileptogenic or neuroprotective activity. Furthermore, the behavioral alterations, e.g., behavioral hyperexcitability and learning deficits, in epileptic rats were not affected by treatment with LEV after SE. These data do not support the idea that administration of LEV after SE prevents or reduces the long-term alterations developing after such brain insult in rats. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Epilepsy; Antiepileptic drugs; Learning; Hippocampus; Elevated-plus maze

1. Introduction Epileptogenesis, i.e., the process leading to epilepsy, is a common sequel of brain insults such as head trauma, cerebrovascular disease, brain tumors, neurosurgical procedures, neurodegenerative conditions, status epilepticus, and febrile seizures (Herman, 2002; Pitka¨nen, 2004). Following such brain * Corresponding author. Tel.: þ49 511 856 8721; fax: þ49 511 953 8581. E-mail address: [email protected] (W. Lo¨scher). 1 Present address: Sanofi-Aventis Pharma Deutschland, Frankfurt am Main, Germany. 2 Present address: Department of Veterinary Clinical Sciences, Neurology, The Royal Veterinary College, University of London, London, UK. 3 Present address: Institute of Pharmacology, Toxicology and Pharmacy, University of Munich, Munich, Germany. 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.05.001

insults, there is a cascade of morphologic and functional changes in the injured area over months to years before the occurrence of spontaneous recurrent seizures (SRS), i.e., the hallmark of epilepsy. This latent period may offer a therapeutic window for the prevention of epileptogenesis and the development of unprovoked seizures and epilepsy (Pitka¨nen, 2004). However, in clinical trials, administration of conventional antiepileptic drugs (AEDs) such as phenytoin, carbamazepine or valproate following acute brain insults has thus far failed to prevent epileptogenesis (Temkin, 2001, 2004). Based on data from the kindling model of temporal lobe epilepsy (TLE), the novel AED levetiracetam (LEV; KeppraÒ) has been suggested to exert antiepileptogenic properties (Klitgaard and Pitka¨nen, 2003). LEV seems to act by a unique mechanism, i.e. modulation of synaptic release of neurotransmitters by

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binding to the synaptic vesicle protein, SV2A (Lynch et al., 2004; Gillard et al., 2006). In the kindling model, LEV was reported to markedly suppress kindling development at doses devoid of visible adverse effects (Lo¨scher et al., 1998). After termination of treatment, all rats developed fully kindled seizures, but the duration of these seizures was significantly lower compared to controls, indicating a disease-modifying effect of the treatment (Lo¨scher et al., 1998). Our initial observation was subsequently confirmed by Stratton et al. (2003) who reported that, while both treatment with LEVor lamotrigine retarded kindling, only prior treatment with LEV prevented the increase in seizure duration upon further kindling following cessation of treatment. However, whether this effect of LEV really represents an antiepileptogenic or disease-modifying action is difficult to ascertain, because traditional kindling does not lead to epilepsy with SRS. More recently, Yan et al. (2005) reported that LEV retarded the development of SRS in a spontaneously epileptic rat mutant and suggested that LEV possesses not only antiseizure effects but also antiepileptogenic properties. For further evaluating whether LEV exerts antiepileptogenic activity, we used a rat model in which SRS develop after a status epilepticus (SE) induced by sustained electrical stimulation of the basolateral amygdala (BLA) (Brandt et al., 2003a). Our goal was to evaluate LEV by a clinically driven study design. Such a design should ideally satisfy the following criteria: (1) the chosen animal model should recapitulate most, if not all, features of a given type of epilepsy such as TLE, including its progressiveness and its pathological landmark, i.e. hippocampal sclerosis; (2) administration of the drug candidate should begin shortly after the epileptogenesis-inducing brain insult to mimic the clinical setting; and (3) the dosing protocol of the drug candidate should take the differences in pharmacokinetics between rodents and humans into account. Based on these considerations, LEV was tested by two experimental protocols. In the first protocol, which was based on a previous study by Bolanos et al. (1998) with valproate in the kainate model, LEV treatment was started 24 h after onset of electrical BLA stimulation without prior termination of the SE. In the second protocol, the SE was interrupted after 4 h by diazepam, immediately followed by onset of treatment with LEV. This duration of SE has been shown to be sufficient for inducing development of SRS and histological damage in the hippocampal formation, particularly in the dentate hilus (Brandt et al., 2003a). Treatment with LEV was continued for 8 weeks (experiment #1) or 5 weeks (experiment #2) after SE, using continuous drug administration via osmotic minipumps. The occurrence of SRS was recorded during and after treatment. In addition to recording SRS, the rats were tested in a battery of behavioral tests, including the elevated-plus-maze and the Morris water maze. Finally, the brains of the animals were analyzed for histological lesions in the hippocampal formation. 2. Materials and methods 2.1. Animals Fifty-nine female SpragueeDawley rats were purchased at a body weight of 200e220 g (Harlan-Winkelmann Versuchstierzucht, Borchen, Germany).

Following arrival, the rats were kept under controlled environmental conditions (24e25  C; 50e60% humidity; 12:12 h light/dark cycle; lights on at 06:00 h) with free access to standard laboratory chow (Altromin 1324 standard diet) and tap water. All experiments were done in compliance with the European Communities Council Directive of 24th November 1986 (86/609/EEC), and approval by an institutional review board was obtained for all procedures used in this study. All efforts were made to minimize animal suffering and to reduce the number of animals used.

2.2. Electrode implantation For electrode implantation, 51 rats were anesthetized with chloral hydrate (360 mg/kg i.p.). A Teflon-isolated bipolar stainless steel electrode was stereotaxically implanted into the right anterior basolateral amygdala (BLA) and served as stimulation- and recording-electrode. The stereotaxic coordinates in millimeter relative to bregma according to the atlas of Paxinos and Watson (1998) were: AP, 2.2; L, 4.7; V, 8.7. One screw, placed above the left parietal cortex, served as the indifferent reference electrode. Additional skull screws and dental acrylic cement anchored the entire headset. After surgery, the animals were allowed to recover for a period of 4e5 weeks. For the first week after surgery, the rats were treated with antibiotics to prevent infection. Two rats died during anesthesia, so that 49 rats with BLA electrodes could be used for SE induction.

2.3. Electrical induction of a self-sustained status epilepticus (SSSE) About 4e5 weeks after electrode implantation, 49 rats were electrically stimulated over a time period of 25 min via the BLA electrode for induction of a SSSE as described previously (Brandt et al., 2003a). For the stimulation, an Accupulser A310C stimulator connected to a Stimulus Isolator A365 (World Precision Instruments, Berlin, Germany) were used. The stimulus consisted of 100 ms trains of 1 ms alternating positive and negative square wave pulses. The train frequency was 2 Hz and the intra-train pulse frequency was 50 Hz. The intensity of the stimulus was 700 mA. Following termination of BLA stimulation, the EEG was recorded via the BLA during and after SSSE for up to 20 h (see below). As reported previously (Brandt et al., 2003a), most (45/49) rats developed a self-sustained SE (SSSE) upon BLA stimulation. Based on seizure type and severity, which was rated by a modified Racine scale (Racine, 1972), three types of SSSE could be distinguished as described elsewhere (Brandt et al., 2003a): type 1, partial SSSE (nonconvulsive limbic seizure activity); type 2, partial SSSE frequently interrupted by generalized convulsive seizures; and type 3, generalized convulsive SSSE. The typical paroxysmal EEG alterations occurring during these types of SSSE have been described in detail by us previously (Brandt et al., 2003a). Type 2 and 3 SSSE have previously been shown by us to induce the development of spontaneous recurrent seizures (SRS) in >90% of rats after a latency period of about 4 weeks (Brandt et al., 2003a), so that only rats with type 2 or 3 SSSE were used for studying the effects of LEV on long-term consequences of SE. The effect of LEV was studied in two separate experiments.

2.4. Experiment #1 The experimental protocol of this experiment is illustrated in Fig. 1. Of the 20 rats used for this experiment, two rats developed a type 2 and 18 rats a type 3 SSSE upon prolonged BLA stimulation. The duration of SSSE was not terminated by anticonvulsant treatment, but rats were allowed to spontaneously recover from SSSE. The SSSE in these rats lasted at least 4.5 h, but in no case longer than 22 h. On the morning of the next day after induction of SSSE, none of the animals exhibited ongoing seizure activity. The rats were randomly distributed to the LEV-treated and the control group. Twenty-four hours after start of the electrical BLA stimulation, rats were anesthetized with methohexital (30 mg/kg i.p.) and fentanyl (0.05 mg/kg i.p.) and an osmotic minipump (Alzet, model 2ML2; Alza, Palo Alto, CA, USA) was implanted subcutaneously (s.c.) into the neck of the animals. The type of pump has a reservoir volume of 2 ml and delivers solutions

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Prophylactic treatment with LEV after SE -Experimental protocol #1Induction of status epilepticus Eight weeks

Four weeks

Prophylactic treatment with LEV* or vehicle after SE

No treatment

Blood sampling for drug analysis in plasma

Continuous drug administration via osmotic minipumps Recording of all observed seizures

Video monitoring of seizures (12 h per day, 5 days/week, every second week over 8 weeks

*Treatment started 24 h after onset of BLA stimulation; LEV loading dose 200 mg/kg, followed by infusion with 231-315 mg/kg/day for 8 weeks Fig. 1. Schematic illustration of the protocol of experiment #1.

continuously for 14 days with a pumping rate of 5.0 ml/h. In order to assure immediate start-up of the pumps after implantation, all pumps were preincubated for at least 4 h in sterile saline at 37  C. Ten rats received pumps filled with 1000 mg LEV dissolved in 2 ml distilled water, and an i.p. bolus injection of 200 mg/kg LEV dissolved in saline (injection volume 3 ml/kg). Another group of 10 rats, which served as control group, received minipumps filled with saline as well as a single injection of 3 ml/kg saline. As described in detail previously (Glien et al., 2002), bolus injection and continuous infusion rate of LEV were calculated on basis of the rapid elimination of LEV in rats (halflife 2e3 h; Lo¨scher et al., 1998), to assure that plasma drug concentrations around the maximal plasma levels of LEV (w40 mg/ml) determined during twice-daily treatment of epilepsy patients with 1000 mg of this drug (Patsalos, 2002) were achieved in rats. The pumps were replaced three times every 14 days under anesthesia, so that rats were continuously treated with LEV over 8 consecutive weeks. For calculating the average pumping rate, the residual amount of solution in each removed pump was determined. Pumping rates proved to correspond well to the pumping rates given by the manufacturer, indicating that all minipumps had worked with sufficient accuracy. During the first replacement of the pumps under anesthesia with methohexital and fentanyl, two rats of the control group died of asphyxia, so that the anesthetic was changed to chloral hydrate (360 mg/kg i.p.) for the subsequent pump replacements. After 2, 4, 6, and 8 weeks of drug treatment, blood (0.5 ml) was withdrawn in each rat by retro-orbital puncture (under anesthesia during replacement or removal of the minipumps). LEV concentration in plasma samples was determined by high pressure liquid chromatography (HPLC) as described previously (Doheny et al., 1999). For monitoring of spontaneous recurrent seizures during and after treatment with LEV or saline, the rats were randomly distributed in two groups (8 and 10 animals). The observer, who counted and scored seizures in these animals, was not aware which animals belonged to the control group or the treated group. The osmotic minipumps were prepared by a different person. Seizure recordings were performed as follows (Fig. 1). First, all seizures observed during handling or other manipulations of the animals were noted. Second, starting 34 days after the electrical BLA stimulation, animals were video

monitored. Each group was monitored every second week over 2 months from Monday to Friday 12 h per day from 06:00 to 18:00 h (diurnal period), i.e. 2 weeks during treatment (drug period) and 2 weeks during the postdrug period. The video recordings (using 4 h videotapes) were analyzed for seizures by using the fast-forward speed (7 times normal speed) of the video recorder. Once a seizure-like activity was seen, the videotape was rewound to the beginning of the behavior and examined at real-time speed. Furthermore, EEGrecordings were made from each rat on at least 2 days per monitored week. For data analysis, all seizures were included which were recorded or observed till the last day of video monitoring. Seizure severity was scored according to the Racine scale (Racine, 1972). Seizure duration was the time period of partial (stage 1e2; nonconvulsive) and/or secondarily generalized convulsive (stage 3e5) seizures. Cumulative seizure duration was the sum of all seizure durations of a period, which was separately calculated for each rat.

2.5. Experiment #2 The experimental protocol of this experiment is illustrated in Fig. 2. Twenty-nine rats were electrically stimulated. Four rats did not develop a SSSE. Two rats developed a type 1, eight rats developed a type 2 and 15 rats a type 3 SSSE upon prolonged BLA stimulation in this experiment. Only rats with type 2 or 3 SSSE were used for the pharmacological experiment. In contrast to experiment #1, the duration of SSSE was terminated after 4 h by i.p. injection of 10 mg/kg diazepam. If necessary, the application of this dose of diazepam was repeated, which was needed in 6 (24%) of the 25 rats with SSSE. By this treatment protocol, diazepam completely stopped motor and EEG seizure activity (recorded up to 20 h after SSSE) in all rats, which we previously described in detail for this model (Brandt et al., 2003a). For treatment with LEV after SE, the rats were randomly distributed to two groups (vehicle group; LEV group), so that they did not differ in severity of SSSE between groups. To further ascertain that SE was similar in the treatment groups, spike counting was conducted in the animals during SSSE as reported previously (Brandt et al., 2003a; Volk et al., 2006). Two rats died 1 day after induction of SSSE (one from the vehicle group and one from the LEV group)

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Prophylactic treatment with LEV after SE -Experimental protocol #2-

Induction of status epilepticus Five weeks

Five weeks

One week

Prophylactic treatment with LEV* or vehicle after SE

No treatment

No treatment

Continuous EEG and video monitoring of seizures

Daily drug administration Blood sampling for drug analysis in plasma

Nine months

Four weeks Evaluation of alterations in behavior and memory**

Recording of all observed seizures

Histological analyses of neuronal damage**

Recording of all observed seizures

**Age-matched control rats used as non-epileptic controls

*Treatment started after 4 h of SSSE by 3 times daily 200 mg/kg for 5 days, followed by continuous infusion with 231-315 mg/kg/day for 4 weeks Fig. 2. Schematic illustration of the protocol of experiment #2.

and one rat died about 3 weeks after SSSE (LEV group), so that group size was reduced to 10 rats in each group. Over the first 5 days after SSSE, LEV was administered i.p. at a dose of 200 mg/kg in 3 ml/kg saline every 8 h. LEV application started immediately after the termination of SSSE by diazepam. Vehicle controls received saline (3 ml/kg) instead of LEV. At the 5 th day, osmotic minipumps (Alzet type 2ML2) filled with 1000 mg LEV in 2 ml saline were implanted after preincubation of the pumps as described for experiment #1. In the vehicle group, the minipumps were filled with 2 ml saline only. Blood samples for drug analysis were taken 1e2 h after the last LEV i.p. injection while the rats were anesthetized (360 mg/kg chloral hydrate) for minipump implantation. Drug delivery with minipumps was performed over a period of 4 weeks. Since type 2ML2 minipumps continuously release the drug for 2 weeks, the minipumps had to be replaced after 2 weeks under anesthesia. At this time point, a second blood sample was taken. At the end of the 4-week period, the minipumps were removed and a third blood sample was taken under anesthesia. During anesthesia for the minipump implantation, two rats from the LEV-treated group and one rat from the vehicle-treated group died. The pumping rate of the minipumps was determined as described for experiment #1. During treatment and in the five weeks following treatment, all spontaneous seizures observed during handling or other manipulations of the animals were noted. Ten weeks after the induction of SSSE (5 weeks after termination of the drug treatment), rats were continuously EEG- and video-monitored for 7 days (24 h/day) by a combined video- and EEG-detection system. For the EEG-monitoring, an 8-channel amplifier (CyberAmp 380, Axon Instruments Inc., Foster City, CA) and eight 1-channel amplifiers (BioAmp, Axon Instruments) and an analogue-to-digital converter (PowerLab/800 s, ADInstruments Ltd, Hastings, East Sussex, UK) were used. The data were recorded and analyzed with the Chart4 for windows software (ADInstruments). The sampling rate for the EEG-recording was 200 Hz. A high pass filter for 0.1 Hz and a low pass filter for 60 Hz was used. The typical ictal discharges occurring during spontaneous seizures in this model have been described in detail previously (Brandt et al., 2003a). Simultaneously to the EEG-recording the rats were video-monitored with light-sensitive black-and-white cameras (CCD-

Kamera-Modul; Conrad Electronic, Hannover, Germany). For detection of spontaneous seizures, the EEG-recording was visually analyzed for characteristic seizure-like activity. To evaluate the severity of motor seizure activity during a detected seizure in the EEG, the corresponding video-recording was viewed. Sixteen rats (i.e. 8 rats of each group) could be EEG/video-monitored at the same time. As shown in Fig. 2, about 9 months following the EEG-video recording session, the rats were used for behavioral testing. Two of the rats of the LEV group had died about 6 months after the SE, so that group size was reduced to 6 for the behavioral experiments. One of these 6 rats was so difficult to handle that it had to be omitted from the behavioral experiments. Before starting the behavioral tests, the rats were continuously observed for SRS for 5 h per day over 6 consecutive days. Furthermore, all seizures which were observed during handling or other manipulations of the animals over the four weeks of behavioral testing were noted. The aim of this SRS observation was twofold. First, we were interested in characterizing the progression of epilepsy in vehicle-controls in the post-SE model used in this study. Second, we were interested in determining the long-term effects of LEV on the progression of epilepsy. For rating of seizure severity of spontaneous seizures, Racine’s scale (Racine, 1972) was used. Based on this scale, seizures were subdivided into nonconvulsive (stages 1 and 2) and convulsive (stages 3e5). In addition to determining the effects of LEV on development and progression of SRS, we evaluated whether LEV altered the behavioral changes induced by SE. All behavioral tests were performed within a time period of 4 weeks. In addition to the LEV and saline treated epileptic groups, we included an age-matched group of 8 nonepileptic control rats (without electrodes) in the behavioral testing. The tests were performed in the following sequence: elevated-plus maze, open field, Morris water maze, hyperexcitability tests, and Porsolt swim test. Before each test, the rats were transferred to a room next to the room in which the experiments were performed. The two rooms were connected through a door, which was closed during the behavioral trials, so that the respective rat that was subjected to a behavioral test was not affected by the presence of an experimenter (except that handling of the rats

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221 was needed in the test) or the other rats. The experiments were performed between 09:00 and 13:00 h. For the elevated-plus maze, open field, and water maze, a video tracking system with the EthoVision software from Noldus (Wageningen, Netherlands) was used. Behavioral testing was performed only if no motor seizure was observed for at least 1 h before the test. If a spontaneous seizure occurred during a trial, the trial was disrupted, the rat was placed back into its home cage, and the trial was repeated after a time interval of 1 h.

2.6. Elevated-plus maze The elevated-plus maze is a validated model to assess the level of anxiety in rodents (File, 1993). The apparatus was constructed with black plastic. It comprises two open arms (50  10 cm), two enclosed arms (50  10  30 cm), and a central platform (10  10 cm). The configuration has the shape of a plus sign, and the apparatus is elevated 86 cm above the floor level. Grip on the open arms is facilitated by inclusion of a small edge (0.5 cm high) around their perimeter, made of transparent Plexiglas. Before each trial, the maze was cleaned thoroughly with 0.1% acetic acid solution. At the beginning of the test, rats were placed on the central platform always facing the same closed arm. The test lasted 5 min. The behavior of rats in the test was analyzed using the EthoVision Software. Activity and anxiety-related behaviors were assessed. Standard measures comprised: the total distance moved (cm), the time spent in different sections of the maze (open and closed arms) and rearing frequency.

2.7. Open field The open field is a very popular animal model of anxiety-like behavior (Prut and Belzung, 2003). The procedure consists of subjecting an animal to an unknown environment from which escape is prevented by surrounding walls. In such a situation, rodents spontaneously prefer the periphery of the apparatus to activity in the central parts of the open field. In the present study, the test was performed in a round open field with gray-painted wall and floor (diameter 83 cm). The animals were placed individually in the center of the open field. Behavior was observed for 10 min. Before each trial, the field was cleaned thoroughly with 0.1% acetic acid solution. The open field was divided into three zones: center, internal ring, and outer ring. The center and internal ring were considered aversive places for the rats. For each rat, the total distance moved, and the time spent in each zone were measured by a computerized tracking system (EthoVision). Rearings and grooming were recorded manually by the experimenter.

2.8. Morris water maze The Morris water maze (Morris, 1984), in which rats learn to escape from water onto a hidden platform, is a widely used test of visuospatial memory and hippocampal integrity. In the present study, the water maze apparatus was made of a black circular tank (diameter 150 cm, 60 cm deep) filled with water to a height of 27 cm. The water temperature was 19  1  C. The maze was surrounded by several visual cues. After a habituation trial (day 0, 2  60 s, no platform), a submerged black escape platform (1.5 cm below the water level, 10  10 cm) was placed in the middle of the northeast quadrant on 7 consecutive days (acquisition). This quadrant was not preferred or avoided by the rats during the habituation trial. Each day, the animals were placed into the pool at four different starting points. On day one beginning from the south turning clockwise until six trials were accomplished, on day 2 beginning from the west, day 3 from the north, and day 4 from the east, respectively. Animals, which did not find the escape platform within 60 s, were placed there by the experimenter. All rats remained on the platform for 10 s. Between the four trials per day, the animals were allowed to rest for 60 s in a transport cage. After the four trials, they were placed in a standard Plexiglas box with the floor covered with paper towels under a red light to dry. For each trial, the escape latency (seconds) was measured by a computerized tracking system (EthoVision). For each rat, the data of the four trials per day were averaged. For the spatial probe (day 8), the platform was removed and the crossings of the former platform position during a single trial over 60 s were recorded.

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2.9. Tests for behavioral hyperexcitability Rice et al. (1998) described four behavioral tests that potentially discriminate differences in behavioral excitability and sensory responsiveness between epileptic and control rats. These tests were taken from the functional observational battery described by Moser et al. (1988). The four tests, which are quick and easy to perform without prior training of the rats or special equipment, are described in the following. 1. Approach-response test: A pen held vertically is moved slowly toward the face of the animal. Responses were scored as 1, no reaction; 2, the rat sniffs at the object; 3, the rat moves away from the object; 4, the rat freezes; 5, the rat jumps away from the object; and 6, the rat jumps at or attacks the object. 2. Touch-response test: The animal is gently prodded in the rump with the blunt end of a pen. Responses were scored as 1, no reaction; 2, the rat turns toward the object; 3, the rat moves away from the object; 4, the rat freezes; 5, the rat jerks around toward the touch; 6, the rat turns away from the touch; and 7, the rat jumps with or without vocalizations. 3. Finger-snap test: A click noise with a clicker several centimeters above the head of the animal is performed. Responses were scored as 1, no reaction; 2, the rat jumps slightly or flinches or flicks the ear (normal reaction); and 3, the rat jumps dramatically. 4. Pick-up test: The animal is picked up by grasping around the body. Responses were scored as 1, very easy; 2, easy with vocalizations; 3, some difficulty, the rat rears and faces the hand; 4, the rat freezes (with or without vocalization); 5, difficult, the rat avoids the hand by moving away; and 6, very difficult, the rat behaves defensively, and may attack the hand. All tests were performed twice on two separate days with 2 days in between. There were four independent observers for each rat, and the means of their scores were calculated for each animal for each test. The tests were accomplished in the home cage.

2.10. Porsolt swim test A depressed state can be induced in rodents by forcing them to swim in a narrow cylinder from which they cannot escape (Porsolt et al., 1979). After a brief period of vigorous activity the animals adopt a characteristic immobile posture which is readily identifiable. In the present study, the animals were tested in a transparent Plexiglas tank (diameter 39 cm, height 60 cm) filled to a depth of 30 cm with water at 31  1  C (the animals could not touch the bottom). The glass tank was surrounded by dark walls. On the first experimental day, rats were gently placed in the water for a 15 min period of habituation. On removal from the water, they were placed in a standard Plexiglas box with the floor covered with paper towels under red light to dry. The next day, they were once more placed gently in the glass tank and observed for 5 min. The behavior of the animals was videotaped. At the end of the 5-min period, the rats were transferred to the red light warmed box and allowed to dry. Following the experiment, the videotapes were analyzed and the duration of the following behaviors was recorded: immobility (floating and making only those movements necessary to keep the nose above the water); swimming (when an animal exhibits active horizontal motions, i.e., moving around the tank including diving); climbing (when rats strongly move their forepaws in and out of the water, usually against the walls, laying perpendicularly in the water).

2.11. Histological determinations Following the behavioral tests in experiment #2, the two groups of post-SE rats and the age-matched non-epileptic controls were deeply anesthetized with chloral hydrate and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Six series of 40-mm-thick horizontal sections of the brain were cut on a freezing microtome. One of the series was Nisslstained with thionin for visualization of neurodegeneration in the hippocampal formation. Typically, in SE models the severity of damage varies along the septotemporal axis of the hippocampus, being more severe temporally than septally

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(Nissinen et al., 2000; Pitka¨nen et al., 2002), so that different standardized locations along the septotemporal (dorsoventral) axis of the hippocampal formation were examined in the present experiments. In a first step, thionin-stained sections of the following subregions of the hippocampal formation were visually inspected for damage: CA1, CA2, CA3a, CA3c/CA4, and dentate gyrus. Severity of neuronal damage in a section was semiquantitatively assessed by a grading system similar to that previously described by Halonen et al. (2001), Cilio et al. (2001) and Brandt et al. (2003b) as follows: score 0, no obvious damage; score 1, abnormal appearance of the structure without clear evidence of visible neuronal loss; score 2, lesions involving 20e50% of neurons; score 3, lesions involving >50% of neurons. In this respect, it is important to note that neuronal loss must exceed 15e20% before it is reliably detected by visual inspection (Fujikawa et al., 2000). In addition to scoring regions for neuronal damage, the granule cell layer of the dentate gyrus was visually analyzed for granule cell dispersion, which is a characteristic alteration in the dentate gyrus in both rat models of TLE and patients with TLE (Ribak and Dashtipour, 2002). Visual assessment was conducted blindly with respect to the treatment status of the animal. Compared to the extent of cell loss occurring after a SE in CA3 and CA1, which can be easily scored by microscopic examination, loss of neurons in the dentate hilus is more difficult to assess without cell counting. Therefore, in a second step, polymorphic neurons (i.e., mossy cells and interneurons) were counted in the dentate hilus of the hippocampal formation. Neuronal loss in the hilus was quantified in six sections per hippocampus in both hemispheres, i.e., two sections of the septal hilus (3800e4000 mm from Bregma), two sections of the mid-septotemporal hilus (5200e5700 mm from Bregma) and two sections of the temporal hilus (7000e7600 mm from Bregma) according to the atlas of Paxinos and Watson (1998). In case of shrinkage of the hippocampus, correct localization of the hippocampal subregions was defined on the basis of adjacent brain regions. The hilus was defined as the inner border of the granule cell layer and two straight lines connecting the tips of the granule cell layer and the proximal end of the CA3c region. In a first approach, all profiles >8 mm within these borders were counted at a 200 magnification (Axioscope, Carl Zeiss, Germany). The area of each hilus counted was measured with the KS400 software (Carl Zeiss). Neuronal densities (neurons per unit area) were calculated on the basis of neuronal counts in the hilus and hilus area. For the septal, mid-septotemporal and temporal hilus, per subregion a mean value from the two analyzed sections was calculated. Double-counting of cells was unlikely because of the distance (240 mm) between the 40 mm sections used for cell counting. All neuron counts were performed by a person who was blinded with respect to the origin of the sections. Similar to comparable studies of other groups (e.g., Andre´ et al., 2001; Gorter et al., 2001; Francois et al., 2006), unbiased stereological methods were not applied in this first approach, since the (biased) technique of cell counting as used in this first approach is a simple and reliable method to estimate group differences in hilar neuron number (Xu et al., 2004; Francois et al., 2006). However, to allow direct comparison of group differences obtained with this first (biased) approach of cell counting with data obtained by ‘‘unbiased’’ stereological methods for estimation of hilar cells, we repeated cell counting in the same six sections per rat by using the optical dissector method (Guillery and Herrup, 1997; West, 1999). In short, a counting grid with 40 squares was placed over the hilus, and 20 squares were randomly chosen for cell counting at 400 magnification. Only neurons that came into focus by passing from one optical plane to the next were counted. Neuronal densities were calculated in neurons/mm3. Values obtained in different sections were averaged per rat and used for calculation of group values. Again, all neuron counts were performed by a person who was blinded with respect to the origin of the sections. Because SE may lead to hippocampal atrophy, we also measured the area of the hippocampus (including the dentate gyrus) and its septal, mid-septotemporal and temporal portions by image analysis, using the AxioVision system of Zeiss. In each rat, values were averaged from the six sections that were also used for neuronal counting, and average values were then used for calculating data per treatment group.

2.12. Statistics Depending on whether data were normally distributed or not, either parametric or nonparametric tests were used for statistical evaluation. Fisher‘s

exact test was used for calculation of significant differences in the incidence of spontaneous seizures in the vehicle and LEV-treated post-SE groups. The significance of differences in behavioral parameters between controls, and the vehicle and LEV-treated post-SE groups was calculated by analysis of variance (ANOVA), followed by post-hoc testing for individual differences by the t-test. If data were not normally distributed, ANOVA for nonparametric data (KruskaleWallis test), followed by post-hoc testing for individual differences by Dunn’s test were used. Within-group comparisons in the Morris water maze were performed by repeated measures ANOVA, followed by the paired t-test. Significant differences in the incidence of neuronal damage in the vehicle and LEV-treated groups were calculated by Fisher‘s exact test. The significance of differences in densities of neurons and hippocampal area between controls and the vehicle and LEV-treated post-SE groups was calculated by ANOVA, followed by post-hoc testing for individual differences by Student’s t-test. All tests were used two-sided; a P < 0.05 was considered significant.

3. Results Drug-associated adverse effects were not observed in the two experiments with LEV. Furthermore, the body weight gain was comparable in LEV-treated and saline-treated groups (not illustrated). Because of the differences in the two protocols used for evaluating the effects of LEV, all other findings will be separately described for the two experiments. 3.1. Experiment #1 The average daily doses of LEV administered via the osmotic minipumps was 257 (range 231e315) mg/kg over the 8 weeks of drug administration in this experiment. This resulted in plasma concentrations in the 10 rats of 35.9  2.9 mg/ml after 2 weeks, 25.8  1.9 mg/ml after 4 weeks, 27.7  1.5 mg/ml after 6 weeks, and 23.9  1.2 mg/ml after 8 weeks of continuous LEV administration, respectively. Analysis of these data by ANOVA indicated a highly significant difference between means (P ¼ 0.0008). Post-hoc analysis indicated a significant decrease in plasma levels of LEV at 4, 6 and 8 weeks of treatment compared to the drug level determined at 2 weeks (P < 0.05), while the values determined after 4, 6 and 8 weeks did not differ significantly from each other. During continuous treatment with LEV, except for one rat, all rats exhibited spontaneous seizures with a mean seizure frequency of 4.2  0.9 over 2 weeks (Fig. 3A). In the control group, all rats exhibited spontaneous seizures with a mean seizure frequency of 4.1  0.7 over 2 weeks (Fig. 3A). The absolute number of seizures during the treatment period ranged between no seizure and a maximum of 7 seizures in the LEV treated group and between 1 seizure and a maximum of 7 seizures in the control group. The latency to the first observed spontaneous seizure was 39.5  3.6 days on average following SSSE in the LEV treated group and 38.8  2.4 days following SSSE in the control group. However, although this seemed to indicate that LEV did not affect latency, these data were not based on continuous video-EEG recordings during the weeks following SE, so that they do not allow any definite conclusions about the duration of the latency period. The severity of spontaneous seizures did not differ between the LEV treated group and the control group (Fig. 3B). The spontaneous seizures were generally

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221 control group

A

LEV-treated group

B

10

213

*

5

9

mean seizure severity (score)

mean seizure frequency

8 7 6 5 4 3 2

4 3 2 1

1 0

C

During treatment

D

60

cumulative seizure duration [sec]

mean seizure duration [sec]

50 40 30 20 10 0

0

After treatment

During treatment

After treatment

During treatment

After treatment

300 250 200 150 100 50 0

During treatment

After treatment

Fig. 3. Spontaneous seizures recorded in experiment #1 following termination of SE in groups of rats that were either treated with vehicle (n ¼ 8) or LEV (n ¼ 10). Seizures were recorded during and after the period of treatment as illustrated in Fig. 1. All data are shown as means  S.E.M. As described in Section 3, all rats except one LEV-treated animal exhibited spontaneous seizures during the treatment phase. After treatment, all rats of both groups exhibited spontaneous seizures. In A, the frequency of spontaneous seizures recorded over 2 weeks during and after the treatment phase is illustrated. In B, the average severity of the spontaneous seizures is shown. C illustrates the average duration of spontaneous seizures and D the cumulative duration of all recorded seizures (which was recorded separately for each animal and than used for calculation of group means). No significant intergroup differences were observed for any of these seizure parameters. When data recorded during and after treatment were compared within each group, seizure severity was significantly higher in the LEV group after vs. during treatment (P ¼ 0.0078; indicated by asterisk).

characterized by focal onset, in most cases culminating into a generalized convulsive stage 4/5 seizure. Duration of seizures (Fig. 3C) as well as the cumulative seizure duration (Fig. 3D) were about the same in the two groups of rats during treatment. During the post-treatment period, spontaneous seizures were observed in all rats of both groups. Average seizure frequency (Fig. 3A), seizure duration (Fig. 3C), and cumulative seizure duration (Fig. 3D) were about the same in both groups without any indication of an antiepileptogenic effect of LEV treatment. Furthermore, the severity of the seizures was not significantly reduced by LEV compared to controls (Fig. 3B). However, seizure severity observed in the LEV group was significantly higher after treatment than during treatment, which was not observed in the vehicle-treated group. None of the other parameters differed significantly within groups after treatment vs. during treatment. 3.2. Experiment #2 In the rats of this experiment, SSSE was interrupted after 4 h by diazepam. As in experiment #1, only rats with type 2 or type 3 SSSE were used for further experiments and randomly distributed to treatment groups. In all rats, the EEG was continuously recorded during the SE. Behavioral partial

and generalized seizure activity was associated with paroxysmal EEG activity, i.e., continuous ictal discharges recorded from the BLA. As described previously for self-sustained SE induced by electrical stimulation of either the lateral amygdala (Nissinen et al., 2000) or the BLA (Brandt et al., 2003a), a conspicuous feature of EEG during selfsustained SE is the occurrence of occasional bursts of high-amplitude and high-frequency discharges (HAFDs). In the BLA model used in the present experiments, these HAFDs typically last 5e20 s and have a frequency of about 3.5e7 Hz. Neither the number nor duration of these HAFDs differed significantly between treatment groups (Table 1). In between these HAFDS, there were continuous paroxysmal discharges with lower frequency in both subgroups without any obvious difference. Thus, these data indicate that the SE was comparable or equivalent in both treatment groups. Following the 5 days with 3 times daily i.p. administration of 200 mg/kg LEV, the average daily doses of LEV administered via the osmotic minipumps were comparable to those determined in the first experiment. This resulted in plasma concentrations in the 8 rats of 24.4  2.7 mg/ml after 2 weeks and 17.1  3.2 mg/ml after 4 weeks of continuous LEV administration (P > 0.05). The plasma level of LEV determined 8 h after the last i.p. administration of 200 mg/ kg was considerably higher (189  12.3 mg/ml).

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221

Group

Vehicle Levetiracetam

Characteristics of HAFDs in the EEG during SE Number

Frequency (Hz)

Average length (s)

111  23 118  26

4.54  0.081 4.14  0.16

15.3  1.4 12.5  1.2

Neither the number of HAFDs occurring during the 4 h of SE nor the average length of these HAFDs differed significantly between groups.

As shown in Fig. 4A, spontaneous seizures were observed in several rats of both groups (vehicle-treated, LEV-treated) during the treatment period. In the weeks after treatment (before the continuous video/EEG-recording period), SRS were also observed in several rats of both groups (‘‘after treatment’’ in Fig. 4). Five weeks after termination of drug treatment, the rats were video/EEG-monitored for 7 days (24 h/day). In both groups, 7/8 rats exhibited SRS. About 9 months later, rats were again used for observation of SRS, resulting in detection of SRS in all LEV-pretreated rats but only 4/8 saline-pretreated rats (P ¼ 0.0769). When data from all observation and recording periods were combined, the total percentage of rats with SRS in both groups was 100% (‘‘total’’ in Fig. 4A). The number of spontaneous seizures recorded in the various periods is shown in Fig. 4B. Seizure number tended to be higher in the LEV-group in several of the observation and recording periods, but this was mainly due to one rat (DS 168) with a very high SRS frequency. When the seizure frequency was calculated from the one week of continuous EEG-video recording, it was 3  1.3 (range 0e11; median 1.5) in the saline and 20.9  15.3 (range 0e127; median 7.5) in the LEV group, respectively (P ¼ 0.2921 by the ManneWhitney test). When seizure frequency of the LEV group was calculated without rat DS 168, it was 5.7  1.9 (median 6), which was also not significantly different from control. Most SRS were convulsive (stage 3, 4 or 5) seizures without any significant difference between the LEV and saline groups (not illustrated). Furthermore, there was no indication that seizure severity changed over the approx. 12 months of the experiment in any group. In addition to recording SRS, we used a battery of behavioral tests to evaluate behavioral differences between the saline- and LEV-treated rats and nonepileptic controls (Figs. 5e7). In the open-field, all three groups of rats stayed markedly shorter in the aversive internal ring and center of the field than in the external ring (Fig. 5A). No significant intergroup differences were observed. Furthermore, the locomotor activity of the rats, measured by the distance moved in the field, was not significantly different between groups (Fig. 5B). In the elevated-plus maze, saline-pretreated epileptic rats tended to stay longer on the aversive open arms than nonepileptic controls and LEV-pretreated rats, but the difference was not statistically significant (Fig. 5C). Similar to the open field, all groups of rats exhibited about the same locomotor activity (Fig. 5D).

A

Percent of rats with spontaneous seizures

100 90

Percent of rats with SRS

Table 1 Characteristics of high-amplitude and high-frequency discharges (HAFDs) occurring in the EEG recorded from the basolateral amygdala during selfsustained status epilepticus (SE) in rats that were randomly distributed into the vehicle and levetiracetam group in experiment #2

Saline LEV

80 70 60 50 40 30 20 10 0

B

During trmt

After trmt

EEG-video recording

During behav. tests

Total

Number of spontaneous seizures

240 220

Saline LEV

200 180

Number of SRS

214

160 140 120 100 80 60 40 20 0

During trmt

After trmt

EEG-video recording

During behav. tests

Total

Fig. 4. Spontaneous seizures recorded in experiment #2 following termination of SE in groups of rats that were either treated with vehicle or LEV after SE. In A, the percent of rats in which spontaneous seizures were observed during the different periods of the experiment as shown in Fig. 2 is illustrated. Group size was 9 vehicle controls and 8 LEV-treated rats. The period termed ‘‘during treatment’’ illustrates the incidence of spontaneous seizures during the 5 weeks of treatment with LEV or vehicle. The period termed ‘‘after treatment’’ illustrates the incidence of spontaneous seizures in the 5 weeks following termination of treatment. The period termed ‘‘EEG-video recording’’ shows the data from continuous EEG/video recording of spontaneous seizures 10 weeks following SE. Seizures were recorded 24 h/day for 7 consecutive days. Group size during EEG-video recording was 8 rats per group. Furthermore, the percentage of rats exhibiting spontaneous seizures during the behavioral tests about 9 months later is illustrated. The final pair of bars shows seizure incidence based on all spontaneous seizures that were recorded over all periods of the experiment, including both spontaneous seizures that occurred during handling (e.g., weighing, injection) of the animals or which were observed by coincidence in rats of both groups and seizures that were recorded by EEG/video. Based on these data, spontaneous seizures were detected in all rats of both groups. In B, the sum of all spontaneous seizures recorded per group in the different periods of the experiment is illustrated. No significant intergroup differences were observed for any of the data illustrated in A or B. Note that the increased number of seizures during the video-EEG recordings in the LEV groups was mainly due to one rat with a very high SRS frequency during this period. The median seizure number in the LEV group was 7.5 (vs. 1.5 in controls). When data recorded during and after treatment were compared within each group, no significant differences between different recording periods were determined.

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221

215

600

A

B

Control SE SE+LEV

400

Distance moved [cm]

Total duration [s]

500

300 200 100 0

3000 2000 1000

Center

20

10

Control

SE

SE+LEV

Control

D

1750

Distance moved [cm]

Time spent in open arms [s]

Internal ring

30

0

4000

0 External ring

C

5000

1500

SE

SE-LEV

1250 1000 750 500 250 0

Control

SE

SE+LEV

Fig. 5. Behavior of epileptic rats and nonepileptic controls in the open field (A,B) and elevated-plus maze (C,D). Three groups of rats were compared: rats treated with either vehicle (SE; n ¼ 8) or LEV after SE (SE þ LEV; n ¼ 5), and age-matched non-epileptic control rats (n ¼ 8). All data are shown as means  S.E.M. A illustrates the time that rats spent in the external ring and aversive internal ring and center of the open field. B illustrates the total distance that the rats moved during the 10 min of the open-field test. C illustrates the time that rats spent in the aversive open arms of the elevated-plus maze. D illustrates the total distance that the rats moved during the 5 min of the elevated-plus maze test. Analysis of data by ANOVA did not indicate any significant differences between groups for any of the parameters shown in the figure.

For testing behavioral hyperexcitability of epileptic rats, the observational battery of behavioral tests described by Rice et al. (1998) was used as described in Section 2. In the finger-snap test, no differences between nonepileptic controls and the two epileptic groups were observed (Fig. 6C). In contrast, a clear difference was obtained in the approach-response, touch-response, and pick-up tests. Vehicle-pretreated and LEV-pretreated epileptic rats were significantly more touchsensitive than nonepileptic controls (Fig. 6B). In the approachresponse test, only LEV-pretreated rats exhibited a significantly enhanced response compared to controls (Fig. 6A). The opposite was true for the pick-up test, in which the saline-pretreated but not the LEV-pretreated rats significantly differed from controls (Fig. 6D). In the Porsolt swim test, no significant intergroup differences in duration of swimming, immobility or climbing were observed; in other words, the two groups of epileptic rats behaved in this test similar to nonepileptic controls (not illustrated). In contrast, clear intergroup differences were observed in the Morris water maze test (Fig. 7). Nonepileptic controls rapidly improved to locate the hidden platform (‘‘escape latency’’). ANOVA indicated highly significant differences between means over the 7 days of the trial (P < 0.0001). Significant improvement was seen between day 1 and days 3e7 (P < 0.01), but not between day 1 and day 2. Vehicle-pretreated epileptic rats also showed some learning upon the repeated trials

(ANOVA, P < 0.0001), but less rapid than controls (Fig. 7B,D). Thus, escape latencies determined on day 1 of the trial did not differ significantly from latencies of days 2e4, whereas latencies determined on days 5e7 were significantly shorter than those of day 1 (P < 0.05). However, at the end of the experiment, escape latencies of vehicle-pretreated epileptic rats were still significantly higher than those of nonepileptic controls (Fig. 7D). The reduced ability to locate the platform was not due to impaired motor performance, because the epileptic control rats did not swim slower than the nonepileptic controls (swim speed was 27.4  1.7 cm/s in nonepileptic controls vs. 28.7  1.4 cm/s in vehicle-pretreated epileptic rats). The epileptic rats of the LEV group also exhibited some learning over time (ANOVA, P ¼ 0.0337). However, post-hoc testing indicated a significant improvement of locating the platform versus day 1 only for day 6 of the experiment (P < 0.05). At the end of the experiment, escape latencies of LEVpretreated epileptic rats were significantly higher than those of nonepileptic controls (Fig. 7D). Interestingly, when analyzing the individual trials of each day of the experiment, the LEV group (Fig. 7C) seemed to learn better than the saline group (Fig. 7B) on days 4e7 of the experiment. However, when the averaged values per day (Fig. 7D) were used for comparison, the two groups of epileptic rats did not differ from each other in escape latencies at any time of the experiment. The swim speed of LEV-pretreated and vehicle-pretreated epileptic

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221

216

A

6

B

Approach-Response

7

Touch-Response

6

5

5

*

3

*

Score

Score

4 4 3

*

2 2 1

1

0

0

Control

C

SE

SE+LEV

D

Finger-Snap

3

Control 6

SE

SE+LEV

Pick-Up

5

4

Score

Score

2

3

*

2

1

1

0

0

Control

SE

SE+LEV

Control

SE

SE+LEV

Fig. 6. Behavior of epileptic rats and nonepileptic controls in an observational battery to test for behavioral hyperexcitability. Three groups of rats were compared: rats treated with either vehicle (SE; n ¼ 8) or LEV after SE (SE þ LEV; n ¼ 5), and age-matched non-epileptic control rats (n ¼ 8). Data (means  S.E.M.) are shown for the following tests: (A) approach-response test; (B) touch-response test; (C) finger-snap test; and (D) pick-up test. Significant differences to controls are indicated by asterisk (P < 0.05). LEV-pretreated rats did not differ significantly from vehicle-pretreated epileptic rats in any of these tests.

rats did not significantly differ (24.0  1.8 vs. 28.7  1.4 cm/s, respectively). In the spatial probe on day 8 of the trial, in which the platform was removed and the crossings of the former platform position during a single trial were recorded over 60 s, the three groups of rats showed the expected behavior. Non-epileptic controls swam more rapidly to the former location of the platform than epileptic rats, stayed longer in the platform zone compared to other areas of the maze, and more often swam to this zone than epileptic rats (not illustrated). 3.3. Degeneration of the hippocampus in epileptic rats Fig. 8 illustrates thionin-stained sections of the temporal hippocampal formation of nonepileptic controls and the two groups of epileptic rats. Visual inspection of these sections indicated neuronal damage of CA1, CA3a and CA3c and/or granule cell dispersion in the dentate gyrus in 6 of 8 vehicletreated epileptic rats (Fig. 8B). The most severe damage was seen in rat DS 155 with an almost complete bilateral loss of

neurons in CA1, CA3a and CA3c in the temporal and, less marked, mid-septotemporal and septal parts of the hippocampus (Fig. 8B), reminiscent of severe hippocampal sclerosis in patients with TLE. As illustrated in Fig. 8B, damage in the hippocampus was associated with marked enlargement of ventricles. In addition, astrogliosis was observed in several parts of the hippocampal formation, including the hilus. A similar extent of hippocampal damage and/or granule cell dispersion was observed in the LEV-treated epileptic rats (Fig. 8C,D). Thus, all six rats which could be examined in this respect exhibited neuronal damage of CA1, CA3a and CA3c and/or granule cell dispersion in the dentate gyrus. As shown in Fig. 8, sections were indistinguishable from sections of vehicle-treated epileptic rats. Statistical comparison of the frequency of hippocampal damage in vehicle-treated and LEV-treated epileptic rats by Fisher‘s exact test did not indicate any significant intergroup difference in hippocampal damage. Granule cell dispersion was observed in 75% of the vehicle group vs. 100% of the LEV group (P ¼ 0.4725; Fisher’s exact test).

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221

Nonepileptic controls 60

B Escape latency [s]

Escape latency [s]

A

50 40 30 20 10 0

50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

Individual trials

Individual trials

Escape latency [s]

SE + LEV group 60

Escape latency [s]

SE-saline group 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

D C

217

50 40 30 20 10

*

60

*

50

* o

o

40

*

30

Controls SE SE+LEV

o o

20 10 0

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

0

1

Individual trials

2

3

4

5

6

7

Day of trial

Fig. 7. Acquisition learning of platform location by epileptic rats and nonepileptic controls in the Morris water maze. Three groups of rats were compared: rats treated with either vehicle (SE-saline group; n ¼ 8) or LEV after SE (SE þ LEV group; n ¼ 5), and age-matched non-epileptic control rats (n ¼ 6). Each day, the rats were subjected to four 1-min trials in the maze. The data from these four daily trials, resulting in a total number of 28 trials over the 7 days of water maze testing, are illustrated individually for each group in A, B, and C. In D, the data of the four trials per day were averaged for each rat for calculating intergroup differences. Statistical analysis of intergroup differences by ANOVA indicated significant differences for day 2 (P ¼ 0.019), day 3 (P ¼ 0.006), day 5 (P ¼ 0.015), day 6 (P ¼ 0.038), and day 7 (P ¼ 0.003). Significance of individual differences between vehicle-pretreated epileptic rats and controls are indicated by asterisk (P < 0.05), while significant differences between LEV-pretreated epileptic rats and controls are indicated by circle (P < 0.05). LEV-pretreated epileptic rats did not differ significantly in learning from vehicle-pretreated epileptic rats at any day of the trial. Within-group comparisons by repeated measures ANOVA indicated significant differences for controls (P < 0.0001), vehicle-treated epileptic rats (P < 0.0001), and LEV-treated epileptic rats (P ¼ 0.0337), suggesting spatial learning in all three groups (for discussion of differences between groups see text).

During visual inspection of the dentate hilus, loss of hilus neurons was seen in several vehicle-treated and LEV-treated epileptic rats. This was confirmed by counting of neurons in the dentate hilus (Fig. 9). As described in Section 2, two different methods of cell counting were used (termed ‘‘method 1’’ and ‘‘method 2’’ in Fig. 9). Both methods yielded about the same differences between treatment groups, substantiating previous experiments of Xu et al. (2004) with biased and unbiased counting techniques in the kainate model. In nonepileptic controls, the neuronal density (neurons per unit area) was higher in the temporal vs. mid-septotemporal or septal parts of the dentate hilus (Fig. 9B,C,E,F). In rats treated with vehicle after SE, a reduction of neuronal density was observed in the hilus, particularly in its temporal part, when neuronal counts were compared to those of nonepileptic rats. Treatment with LEV after SE did not prevent the alterations in the hilus. The more marked reduction of neuronal density in the temporal hippocampus of LEV-treated rats (Fig. 9B,C,E,F) was not secondary to alterations in the hilus area, because the area of the hilus of the temporal hippocampus of both groups of epileptic rats did not significantly differ from controls (not illustrated). We also measured the area of the hippocampus, including the dentate gyrus, in all rats (Fig. 9A,D). No

significant intergroup differences were observed, indicating the absence of any significant hippocampal atrophy in the epilepsy groups. 4. Discussion In contrast to our expectations, LEV did not exert any significant effect on the long-term consequences of SE, i.e., the development of spontaneous seizures, behavioral alterations and hippocampal damage in this experimental model. In contrast, independent of whether rats were treated with saline or LEV after SE, all rats developed SRS without significant intergroup differences in frequency, severity or duration. Furthermore, hyperexcitability and impairment of spatial learning were observed in both groups compared to nonepileptic controls. The histological examination of the hippocampal formation revealed morphological alterations, particularly in the temporal part, without any indication of a neuroprotective effect of LEV treatment. It should be considered that the SE model used in this study was titrated so that all rats in the vehicle group developed epilepsy. Consequently, a worsening of the condition by drug treatment would not have been detected, which may be regarded as a limitation of the study.

218

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221

Fig. 8. Representative thionin-stained horizontal sections of the hippocampal formation of a non-epileptic control rat (A), an epileptic rat (DS 155; B) that was treated with vehicle after SE, and an epileptic rat (DS 169; C,D) that was treated with LEV after SE. Both the vehicle-treated and LEV-treated epileptic rats showed severe neuronal damage in various parts of the hippocampal formation, reminiscent of hippocampal sclerosis in medial temporal lobe epilepsy. Furthermore, granule cell dispersion as shown in D was observed in several rats of both groups. In addition, marked astrogliosis and enlarged ventricles were seen in these rats. Calibration bar in panel D (for AeD) ¼ 500 mm.

Apart from the lack of any antiepileptogenic or diseasemodifying effect of LEV in our experiments, LEV did also not exert any clear anticonvulsant effect on spontaneous seizures during the treatment period in the two experiments. We have previously shown that LEV does exert anticonvulsant effects on SRS in the pilocarpine model of TLE when given over 2 weeks once rats have developed SRS (Glien et al., 2002). However, some rats were resistant to the anticonvulsant effect of LEV and other developed tolerance (i.e., loss of anticonvulsant efficacy) in the second week of treatment (Glien et al., 2002). Thus, both resistance and tolerance could be involved in the lack of any obvious anticonvulsant activity of LEV in the present experiments. In the first experiment with LEV, in which treatment was started 24 h after onset of SE, we may have missed a ‘‘therapeutic window’’, i.e., a limited time domain after SE to intervene in the epileptogenic process, which was the reason to change the timing of treatment for the second experiment, in which LEV was administered immediately after termination of SE by diazepam. However, this change in the treatment protocol did not improve the ability of LEV to prevent development of SRS, but all rats developed epilepsy in both experiments. A further explanation for the lack of antiepileptogenic or neuroprotective efficacy of LEV might be that too low doses were administered. LEV is much more rapidly eliminated in rats

than in humans (Lo¨scher et al., 1998; Doheny et al., 1999), which was the reason to administer LEV by continuous infusion via implanted minipumps. In the first experiment, we used a loading dose of 200 mg/kg LEV together with onset of infusion. In the second experiment, we treated rats for 5 days with three times daily 200 mg/kg LEV before onset of infusion. In this way, we wanted to guarantee that high brain levels of LEV are achieved during the critical period following the SE. During the subsequent period of LEV infusion, average plasma levels of LEV ranged between 17e36 mg/ml, which is in the range of ‘‘therapeutic plasma concentration’’ (23e43 mg/ml) reported for this drug in patients with epilepsy (Patsalos, 2002). It might be that these levels are too low for achieving any antiepileptogenic or neuroprotective effect in the rat model chosen for the present experiment, but plasma levels over the first 5 days after SE in experiment #2 were much higher (190 mg/ml and above) than therapeutic drug levels in patients. With respect to the lack of any effect of LEV in the present study, there are at least two critical issues. First, is it at all possible to prevent or modify epileptogenesis and brain damage by a pharmacological treatment after SE? And, second, is there a critical time window to achieve such an effect after SE? When reviewing the literature in this respect, it is important do differentiate between drug effects resulting from

C. Brandt et al. / Neuropharmacology 53 (2007) 207e221

B Neurons/mm2

Area (mm2)

6 5 4 3 2 1 0

D

Septal

Medial

Temporal

Total

E

Hippocampal area, right 7

Neurons/mm2

Area (mm2)

6 5 4 3 2 1 0

Septal

Medial

Temporal

Total

Neuronal density, left (method 1) 1,600 1,400 1,200 1,000 800 600 400 200 0

C

* *

*

*

*

Neuronal density, left (method 2) 35,000 30,000 25,000

*

20,000

*

15,000

*

10,000 5,000

Septal

Medial

Temporal

F

* *

*

* Septal

Medial

Control

Temporal

SE

0

Total

Neuronal density, right (method 1) 1600 1400 1200 1000 800 600 400 200 0

Neurons/mm3

Hippocampal area, left 7

*

Total

Neurons/mm3

A

219

Septal

Medial

Temporal

Total

Neuronal density, right (method 2) 30,000 25,000 20,000 *

15,000 10,000 5,000 0

Septal

Medial

Temporal

Total

SE + LEV

Fig. 9. Hippocampal area and density (neurons per unit area) of polymorphic neurons (i.e., mossy cells and interneurons) in the hilus of the dentate gyrus. Data are shown for nonepileptic control rats (n ¼ 8) and epileptic rats either treated with vehicle (n ¼ 8) or LEV (n ¼ 6) after SE. All data are shown as means  S.E.M. Data shown in panels AeC are from the left (contralateral) hemisphere, while data shown in panels DeF are from the right (ipsilateral) hemisphere. Data for subregions of the hippocampus are based on two sections per subregion, while the data shown for ‘‘total’’ are averaged from the three subregions, i.e., from 6 sections of the hippocampus. The term ‘‘medial’’ is used for the mid-septotemporal subregion of the hippocampus. Neuronal density was estimated by two different methods, termed ‘‘method 1’’ (estimation of all neurons in the hilus per section level by a biased counting technique) and ‘‘method 2‘‘ (estimation of hilar neurons by a stereological method) in the figure. Both methods yielded similar differences between groups. Statistical analysis of data by ANOVA (separately for each hemisphere) indicated that the three groups of rats differed significantly (P < 0.05) in the density of neurons in the temporal part of the hilus in both hemispheres, independent of the counting method used. Additional differences were seen in the septal hippocampus and ‘‘total’’ hippocampus by method 1, and the ‘‘total’’ hippocampus of the left hemisphere by method 2. Individual differences from post-hoc analysis are indicated by asterisks (P < 0.05 vs. controls).

‘‘initial insult modification’’ and effects representing ‘‘true’’ antiepileptogenic or neuroprotective drug efficacy (Lo¨scher, 2002; Pitka¨nen, 2002a,b). Initial insult modification means that the long term consequences of the insult can be diminished by reducing the severity or duration of the initial brain insult, such as SE. This has, for instance, been demonstrated by reducing the duration of SE by phenobarbital, the Nmethyl-D-aspartate (NMDA) antagonist MK-801 (dizocilpine), pregabalin or diazepam in SE models in rats (Prasad et al., 2002; Andre´ et al., 2003; Pitka¨nen et al., 2005). In electrical models of SE, a SE duration of at least 3 h is needed to induce epileptogenesis in the majority of rats, so that any reduction of this duration by AEDs will result in a modification of the longterm consequences of the SE in such a way that fewer rats develop epilepsy or that the epilepsy that develops is milder (Lo¨scher, 2002; Pitka¨nen, 2002a,b, 2004). Thus, in such SE models the antiepileptogenic or neuroprotective potential of a drug should be tested by administering this drug after a SE of at least 3 h duration (Lo¨scher, 2002). In chemical models of SE, such as the pilocarpine or kainate model, the critical duration of SE for induction of epileptogenesis and brain damage is considerably shorter, i.e., about 60e90 min (Lo¨scher, 2002). There are numerous studies that tested drugs after such critical duration of SE for effects on epileptogenesis, brain damage and/or cognitive alterations in rats (cf., Lo¨scher, 2002; Pitka¨nen, 2002a,b, 2004). For instance, we

found that one single administration of a low dose (0.1 mg/ kg) of the NMDA antagonist MK-801 after a kainate-induced SE of 90 min was capable of preventing most of the brain damage occurring in this model, but this treatment did not prevent the development of SRS (Brandt et al., 2003b). A similar finding was obtained more recently by starting prolonged treatment with valproate after 4 h of an electrically induced SE, which completely prevented any hippocampal damage, including cell loss in the hilus, but did not prevent development of SRS (Brandt et al., 2006). Interestingly, although treatment with valproate after SE did not exert an antiepileptogenic effect, it did prevent most of the behavioral alterations developing after SE in rats (Brandt et al., 2006). To our knowledge, there is no incontrovertible evidence supporting the idea that AEDs administered during the latency period following SE prevent the development of epilepsy, although some studies indicated that development of epilepsy may be delayed or the severity of spontaneous seizures may be reduced by such treatment (Capella and Lemos, 2002; Pitka¨nen et al., 2004). Such effects were not observed in the present study with LEV. Whereas the lack of antiepileptogenic efficacy of LEV in a post-SE rat model is in line with the inefficacy of most AEDs previously examined in this regard (Lo¨scher, 2002; Pitka¨nen, 2002a,b, 2004), the lack of any neuroprotective activity of LEV in this model is surprising. Hanon and Klitgaard (2001) reported that LEV reduced infarct volume in the rat middle

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cerebral artery occlusion model of focal cerebral ischemia. This effect was seen after bolus injection of 44 mg/kg followed by continuous 24 h infusion with 10 mg/kg per hour (i.e., 240 mg/kg per 24 h), which corresponds to the daily dose that was achieved by continuous infusion in the present study. Hanon and Klitgaard (2001) concluded that LEV possesses neuroprotective properties, which may be relevant for its antiepileptogenic action. In a subsequent study by Rekling (2003) using rat hippocampal slice cultures exposed to oxygen/glucose deprivation, cell death was significantly reduced by several AEDs, including phenobarbital, phenytoin, ethosuximide, midazolam, carbamazepine, felbamate, lamotrigine, tiagabine, and oxcarbazepine, whereas gabapentin, valproate, retigabine and LEV were not neuroprotective at concentrations up to 300 mM. When LEV (25, 50 or 100 mg/kg) was administered before kainate in rats, it did not inhibit the kainate-induced SE nor the hippocampal damage, whereas neuroprotective effects were obtained with nefiracetam, carbamazepine, diazepam, and ethosuximide (Kitano et al., 2005). However, the doses of LEV used in the study of Kitano et al. (2005) may have been too low, because Gibbs et al. (2006) recently reported that injection of LEV (200 or 1000 mg/kg) during and after SE induced by electrical stimulation of the perforant pathway in rats protected against mitochondrial dysfunction in the hippocampus, but only at a dose of 1000 mg/kg. Thus, overall LEV seems to exert neuroprotective effects in some models but, at least in part, such effects only occur at very high doses. Despite the effect that we used daily doses of up to 600 mg/kg LEV in the present study, no indication of neuroprotective activity was obtained in the post-SE TLE model used. Assuming that antiepileptogenic drug effects exist, the lack of such efficacy in a drug study may be a consequence of the model, study design, or compound that does not work (Pitka¨nen, 2002a). Perhaps, as suggested by Pitka¨nen (2002a), SE-induced epileptogenesis is not an optimal model for antiepileptogenesis studies. At present, however, no studies have investigated the antiepileptogenic effects of AEDs after other epileptogenic insults, such as stroke or head trauma. The use of kindling as a model to study antiepileptogenic drug effects has been criticized (Pitka¨nen and Halonen, 1998), because rats do not have spontaneous seizures at the time they reach kindling criterion (i.e., a fully kindled stage 5 seizure), so that it is difficult to conclude that kindling-preventive drugs inhibit the processes that ultimately lead to epilepsy. In a recent study, Yan et al. (2005) used the spontaneously epileptic rat (SER: zi/zi, tm/tm) for evaluating whether LEV exerts antiepileptogenic efficacy in this genetic animal model of idiopathic epilepsy. The SER is a double mutant exhibiting both absence-like seizures and generalized tonic convulsions (Serikawa et al., 1991). Yan et al. (2005) administered LEV in these rats from postnatal weeks 5e8, i.e., before the appearance of any seizure expression, at a daily dose of 80 mg/kg. A significant reduction of absence-like seizures and tonic convulsions was observed up to 5 weeks following termination of treatment with LEV, which led the authors conclude that LEV possesses antiepileptogenic properties (Yan et al., 2005). However, because all of the LEV-treated rats ultimately

developed seizures, a more likely interpretation of these data is that LEV exerted a disease-modifying effect rather than a true antiepileptogenic effect. This would also correspond to our previous data in the kindling model, in which LEV did not prevent kindling development, but reduced the duration of the kindled seizures, which persisted after termination of treatment (Lo¨scher et al., 1998). In conclusion, in contrast to our recent study with valproate in the same rat model (Brandt et al., 2006), the present study on LEV was largely negative without any observable drug effects on development of spontaneous seizures, behavioral alterations or hippocampal damage. This does not mean that LEV is not a potentially interesting agent in the treatment of SE. For instance, in rats in which SE was induced by electrical stimulation of the perforant path, treatment with LEV during the maintenance phase of SSSE diminished (at 200 mg/kg) or aborted seizures (in doses of 500 or 1000 mg/kg), while diazepam (5 mg/kg) was ineffective (Mazarati et al., 2004). When treatment with LEV and diazepam was combined, this resulted in a marked over-additive anticonvulsive effect on SE (Mazarati et al., 2004). First clinical studies suggest that LEV may be useful for termination of benzodiazepine-refractory SE (Patel et al., 2006; Rossetti and Bromfield, 2006). Thus, based on the concept of initial insult modification (see above), LEV may help to reduce the long-term consequences of SE by allowing to terminate SE as early as possible.

Acknowledgements We thank Maria Hausknecht, Christiane Bartling and Michael Weissing for technical assistance in the histology. The study was supported by UCB Pharma (Braine l’Alleud, Belgium).

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