Supplementary material S1

Animals

For the IAK model, male inbred C57BL/6N mice were obtained from Charles River (Sulzfeld,

Germany) at an age of 7 weeks. Some preliminary experiments were also performed in

C57BL/6J(OlaHsd) mice from Harlan UK (Blackthorn, UK). For the IHK model, male

C57BL/6J mice from either Janvier (Le Genest-Saint-Isle, France) or Charles River were

used. For the latter model, experiments were also performed in male CD-1 mice (Charles

River), because a previous study reported that outbred CD-1 mice behave similarly to inbred

C57BL/6 mice in the pilocarpine model of TLE,1 but it is not known whether the same is true

for the IHK model. Furthermore, for the IHK model, previously published data2 from male

NMRI mice (Charles River) were used for comparison with the CD-1 and C57BL/6 mouse

strains. Both CD-1 and NMRI are Swiss strain-derived multipurpose mouse outbred strains,3

which are widely used in epilepsy research.4 Animals were housed under controlled

conditions (ambient temperature 22-24°C, humidity 30-50%, lights on from 6:00 am to 6:00

pm). Mice were adapted to the laboratory conditions for at least one week before used in

experiments. Experiments were performed according to the EU council directive 2010/63/EU

and the German Law on Animal Protection (“Tierschutzgesetz”). Ethical approval for the

study was granted by an ethical committee (according to §15 of the Tierschutzgesetz) and the

governmental agency (Lower Saxony State Office for Consumer Protection and Food Safety;

LAVES) responsible for approval of animal experiments in Lower Saxony (reference number

for this project: 14/1659). All efforts were made to minimize both the suffering and the

number of animals. A total of 185 mice were used for the present experiments. All animal

experiments of this study are reported in accordance with ARRIVE guidelines.5

The intraamygdala kainate mouse (IAK) model

When we started to establish the model in our laboratory in 2013, we tried to replicate the
original protocol described by David Henshall and colleagues6,7 as closely as possible after a

personal visit in the lab of Dr. Henshall. In this protocol, kainate (0.3 µg in 0.2 µl of vehicle)

is injected into the basolateral amygdala (BLA) in freely moving C57BL/6J mice via a

previously implanted guide cannula. The EEG is recorded via skull-mounted recording

electrodes that are placed bitemporally and across the frontal cortex to record EEG. Forty

minutes following microinjection of kainate, mice receive intravenous lorazepam (6 mg/kg) to

terminate the kainate-induced SE. In our hands, however, using the C57BL/6J (OlaHsd) mice

from Harlan UK also used by David Henshall and performing the experimental protocol as

closely as possible, SE was not reliably induced by intraamygdala kainate and only few mice

developed spontaneous recurrent seizures (SRS).

We therefore tried to establish a slightly modified protocol that is used in the

laboratory of Annamaria Vezzani in Milan, Italy in male C57BL/6N mice.8 Again, two of us

(L.W., A.S.) spent several days in the lab of Dr. Vezzani to learn the details of the protocol.

Similar to the Henshall protocol, kainate (0.3 µg in 0.2 µl) is injected unilaterally in the BLA

of freely moving mice via a previously implanted guide cannula. The EEG is recorded via a

previously implanted electrode in the dorsal hippocampus, ipsilateral to injected amygdala.

Forty min after SE onset, mice receive diazepam (10 mg/kg i.p.) to increase their survival

rate. In our hands, SE yield by using this protocol was relatively low with only 10/18 mice

developing SE. Furthermore, most of the mice did not develop SRS after kainate.

Thus, we decided to modify the protocol and inject kainate into the BLA under

anesthesia to omit prior guide cannula implantation and potential problems with kainate

infusion in conscious mice. In a first series of experiments, we used isoflurane for anesthesia

and injected kainate at different doses (0.3 or 0.39 µg) in 0.2 µl vehicle into the BLA. When

SE was not suppressed by diazepam (10 mg/kg i.p.) 40 min after SE onset, all mice exhibited

poor health status following SE and had to be killed. Therefore, a second experiment was

performed with kainate (0.3 µg) injection under isoflurane anesthesia and diazepam (10
mg/kg i.p.) was injected 40 min after SE onset. All mice developed SE, but mortality was

40% and no clear latent period was observed in several of the surviving animals. When SRS

were monitored (24/7) over one week starting 4 weeks post-SE, the frequency of SRS was 4

seizures per day (range 1.3-8.1).

Based on our previous positive experience with chloral hydrate anesthesia in the IHK

model,2,9 we decided to replace isoflurane by chloral hydrate. Because of the long-lasting

effect of chloral hydrate, we decided to skip SE interruption by diazepam, which otherwise

could interfere with pharmacological studies on antiepileptogenic drug effects.10 Direct

comparison with the data obtained with isoflurane anesthesia during kainate injection

substantiated advantages of chloral hydrate anesthesia in this model (less severe SE but

high frequency of SRS), so all subsequent experiments were performed with this anesthesia

as described in detail in the following.

In all experiments on the IAK model described above and in the following, the correct

location of the kainate injection in the BLA and EEG electrode in the hippocampus was

verified by preliminary experiments. During anesthesia with chloral hydrate (375 mg/kg i.p.),

kainate monohydrate (0.3 µg in 0.2 µl vehicle) was slowly injected over 60 s with a 0.5 μl

microsyringe into the right BLA using the following stereotactic coordinates (in mm to

bregma11: AP: -1.10; LL: -3.30; DV: -4.50. After injection, the needle of the syringe was

maintained in situ for additional 2 min to limit reflux along the injection track. A bipolar

EEG recording electrode was placed into the ipsilateral CA1 of the hippocampus using

the following coordinates: AP: -2.0; LL: -1.50; DV: -1.80. A screw, placed above the left

parietal cortex, served as the indifferent reference electrode. Additional skull screws,

superglue (Pattex® Ultra Gel; Henkel, Düsseldorf, Germany), and dental acrylic cement

(Paladur®; Kulzer GmbH; Hanau, Germany) anchored the entire headset. During all surgical

procedures and for about 1 h thereafter mice were kept on a warming pad to avoid

hypothermia. Furthermore, mice received electrolyte/glucose solution (Sterofundin® VG-5;

B. Braun Melsungen AG; Melsungen, Germany) subcutaneously to compensate for loss of

fluid and food during the day of surgery. Video/EEG monitoring was used to verify the onset,

type and duration of SE, to determine the latent period, and to monitor SRS following the

latent period.

The intrahippocampal kainate mouse (IHK) model

In this model, SE is induced by unilateral injection of kainate into the CA1 sector of the

dorsal hippocampus.12,13 For this purpose, mice were anesthetized with chloral hydrate (375

mg/kg i.p. in C57BL/6 mice; 500 mg/kg i.p. in CD-1 mice) and kainate monohydrate (0.21 μg

in 50 nl saline) was stereotaxically injected into the right CA1 area of the dorsal hippocampus

as described previously.9 Stereotaxic coordinates were AP, -2.0; LL, -1.5; and DV, -1.7 mm

from bregma for C57BL/6J mice and AP, -2.2; LL, -1.7; DV, -1.4 for CD-1 mice, using the

mouse brain atlas of Paxinos and Franklin.11 The correct location of the injection was

repeatedly approved in the different batches of mice used for the present experiments, and

coordinates were adapted if needed. Kainate was slowly injected over 60 sec with a 0.5 μl

microsyringe. After injection of kainate, the needle of the syringe was maintained in situ for

additional 2 min to limit reflux along the injection track. For EEG recordings, the animals

were immediately implanted with bipolar electrodes aimed at the site of kainate injection in

the ipsilateral CA1, using the same coordinates as for kainate injection (see Twele et al. 9). All

other details were as described above for the IAK model.

Principles of animal welfare

To ensure principles of animal welfare, animals were closely observed during all experiments

for pain, distress, and discomfort using welfare score sheets for humane endpoints.14-16

Distress was rated from 0 (normal) to 3 (severe), using a distress scoring system.17,18. Score 1

(reduced food and water intake, loss of body weight of less than 10%, reduced locomotor
activity, normal response to environmental stimuli, normal grooming but dull coat) was

considered tolerable. Similarly, a transient score 2 (markedly reduced food and water intake,

loss of body weight of up to 20%, loss of spontaneous movement, reduced response to

environmental stimuli, reduced grooming) were considered tolerable. However, if score 2

persisted for more than 10 days, the experiment was interrupted. Similarly, reaching score 3

(no food and water intake, loss of body weight of >20%, loss of spontaneous movement, loss

of response to environmental stimuli, loss of grooming, red eye and nose exudates) for more

than 3 days led to immediate interruption of the experiment and cervical dislocation of the

animal. Animals that had to be killed in the IAK and IHK models because of issues of animal

welfare in the days after SE were included in data on SE mortality.

Video/EEG monitoring

Continuous (24 h/day) video/EEG monitoring was performed during SE and for at least one

week after SE as well as for further one week periods at different intervals (IAK 4 and 8

weeks, IHK in NMRI and CD-1 4 and 12 weeks, IHK in C57BL/6 4 and 6 weeks) following

kainate injection to record the different types of spontaneous electrographic and

electroclinical seizures developing after a latent period following intrahippocampal or

intraamygdala kainate in mice. For EEG-recording, mice were connected via a flexible cable

to a system consisting of 4 one-channel bioamplifiers (ADInstruments Ltd., Sydney,

Australia) and an analog-digital converter (PowerLab 8/30 ML870 or PowerLab 4/35

PL3504/P, ADInstruments). The data were recorded (sampling rate 200 Hz, time constant 0.1

s, low pass filter of >60 Hz, 50 Hz notch filter) and analyzed with LabChart 8 for Windows

software (ADInstruments). The EEG-recording was directly linked to simultaneous digital

video-recording of four mice per system using four infrared board cameras (Sony) merged by

one video quad processor (Monarcor TVSP-44COL). For video/EEG monitoring, mice were

housed singly in clear plexiglass cages. For monitoring during the dark phase, infrared LEDs
were mounted above the cages.

Types of spontaneous seizures

Three types of SRS were recorded in the two models. In both models, focal and secondarily

generalized electroclinical seizures were observed (see Results). Furthermore, as described

previously,2,19,20 in the IHK model highly frequent focal electrographic seizures occurred in

the EEG recorded from the kainate focus in the CA1. Based on their morphology, these

electrographic seizures were differentiated into high-voltage sharp waves (HVSWs) and

hippocampal paroxysmal discharges (HPDs) as described by us in detail recently.2 Such

electrographic seizures are not observed in sham controls.21

For comparing the frequency of electrographic and electroclinical seizures in vehicle-

and drug-treated mice, they were counted visually in the EEG of the video-EEG recording

periods. Electroclinical seizures, which were all associated with paroxysmal EEG activity in

the hippocampal recordings, were rated by a modified Racine scale22 as follows: stage 1,

behavioral arrest and stereotyped sniffing; stage 2, head nodding and mouth or facial

movements; stage 3, unilateral forelimb clonus; stage 4, rearing; stage 5, generalized tonic-

clonic seizures with loss of righting reflexes. Stage 1-3 seizures were considered focal and

stage 4 or 5 generalized convulsive seizures. For comparing the frequency of these

electroclinical seizures in vehicle- and drug-treated mice, they were counted visually in the

video/EEG recordings over the one-week periods of continuous recordings following SE. In

the drug studies described below, video/EEG monitoring was used to record SRS before,

during and after drug treatment.

Experiments with carbamazepine (CBZ)

In the IHK model, the anti-seizure effect of CBZ was studied in C57BL/6 and CD-1 mice as

previously described for NMRI mice.2 Starting 12-16 weeks after SE, the effect of CBZ (20 or
40 mg/kg i.p.) on epileptic EEG activity (HVSWs and /or HPDs) was studied in groups of

mice. Drug injections were always performed at the same time of day, i.e. between 9:30 a.m.

and 11:00 a.m. As previously reported,23 administration of drug vehicle had no effect on

epileptic EEG activity, so that we did not repeat such vehicle experiments. Video/EEG

monitoring was started about 24 hours prior to each drug experiment. For i.p. drug injections,

mice were disconnected from the cables and reconnected immediately afterwards. CBZ was

administered at doses of 20 and 40 mg/kg i.p., dissolved in 30% polyethylene glycol (PEG)

400. For evaluation of drug effects on epileptic EEG activity, the number of HVSWs and

HPDs were visually assessed during four consecutive 0.5-h periods before and after drug

injection, respectively. Each animal was thus used as its own control, with a delay of at least

4-7 days between two drug experiments. The effect of CBZ on the relatively infrequent

electroclinical seizures was not evaluated in these experiments.

In the IAK model, CBZ had to be administered over several days to allow testing of

drug activity on electroclinical seizures, which were much less frequent than the

electrographic seizures in the IHK model (see Results). For this purpose, starting 6 weeks

after SE, epileptic mice were video/EEG monitored (24 h/day) over 5 consecutive days

(Monday to Friday) for predrug control recordings, followed by a five-day period of drug

treatment (Monday to Friday), during which CBZ was administered i.p. three times daily with

30 mg/kg at 7:00 a.m., 30 mg/kg at 1:00 p.m., and 45 mg/kg at 7:00 p.m., respectively. The

higher dose in the evening was given because of the 12-hr interval between the evening and

morning treatments. Following termination of drug treatment, postdrug SRS monitoring was

performed over three consecutive days (Saturday to Monday). The experiment was

subsequently repeated in the same mice with vehicle injection instead of CBZ administration

starting 10 weeks after SE.

Histology
Following the last video/EEG recording period, the mice were anesthetized and transcardially

perfused with paraformaldehyde. Series of coronal brain sections (40 μm) were prepared for

histology as described previously24 and stained with thionin (for details see Polascheck et

al.25). Brain sections between -0.90 and -2.30 from Bregma were scanned in a meander form

and each of the subregions of the left and right hippocampal formation (CA1, CA2, CA3a,

Ca3c, and dentate hilus) as well as the amygdala were assessed for neurodegeneration.

Furthermore, the brain sections were assessed for granule cell dispersion (GCD) in the dentate

gyrus.

Drugs

CBZ (Sigma-Aldrich®, Steinheim, Germany) was dissolved in 30% polyethylene glycol 400.

Drug solutions were prepared freshly twice a day; the injection volume was 10 ml/kg. Vehicle

controls obtained drug vehicle with the same injection volume and number of injections as the

drug-treated groups. Kainate monohydrate (Sigma-Aldrich®) was dissolved in isotonic NaCl,

divided into 25 µl portions in vials and stored at -80 °C. On the day of kainate injection, one

of these portions was thawed and kept on ice during the experiments. Portions were not

reused to minimize degradation of kainate.

Statistics

In all experiments, mice were randomly assigned to the drug- and vehicle-treated groups and

experiments were performed in a blinded fashion. Depending on whether data were normally

distributed or not, either parametric or nonparametric tests were used for statistical evaluation.

In case of more than two groups, analysis of variance (ANOVA) with post-hoc testing was

used. Depending on data distribution, either the ANOVA F-test, followed posthoc by

Dunnett’s multiple comparison test, or the Kruskal-Wallis test followed posthoc by Dunn’s

multiple comparisons test were used. Paired data were compared by Wilcoxon matched-pairs
signed rank test or paired t-test. For comparison of frequencies in a 2 x 2 table, Barnard’s

unconditional test26 was used, because this test preserves the significance level and generally

is more powerful than Fisher’s exact test for moderate to small samples.27 Except Barnard’s

unconditional test, all statistical analyses were performed with the Prism 8 software from

GraphPad (La Jolla, CA, USA). All tests were used two-sided; a P ≤ 0.05 was considered

significant.

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