Effects of Antiepileptic Drugs on Spontaneous Recurrent Seizures in a Novel Model of Extended Hippocampal Kindling in Mice

Abstract

Epilepsy is a common neurological disorder characterized by naturally-occurring spontaneous recurrent seizures and comorbidities. Kindling has long been used to model epileptogenic mechanisms and to assess antiepileptic drugs. In particular, extended kindling can induce spontaneous recurrent seizures without gross brain lesions, as seen clinically. To date, the development of spontaneous recurrent seizures following extended kindling, and the effect of the antiepileptic drugs on these seizures are not well understood. In the present study we aim to develop a mouse model of extended hippocampal kindling for the first time. Once established, we plan to evaluate the effect of three different antiepileptic drugs on the development of the extended-hippocampal-kindled-induced spontaneous recurrent seizures. Male C57 black mice were used for chronic hippocampal stimulations or handling manipulations (twice daily for up to 70 days). Subsequently, animals underwent continuous video/EEG monitoring for seizure detection. Spontaneous recurrent seizures were consistently observed in extended kindled mice but no seizures were detected in the control animals. The aforementioned seizures were generalized events characterized by hippocampal ictal discharges and concurrent motor seizures. Incidence and severity of the seizures was relatively stable while monitored over a few months after termination of the hippocampal stimulation. Three antiepileptic drugs with distinct action mechanisms were tested: phenytoin, lorazepam and levetiracetam. They were applied via intra-peritoneal injections at anticonvulsive doses and their effects on the spontaneous recurrent seizures were analyzed 10-12 h post-injection. Phenytoin (25 mg/kg) and levetiracetam (400 mg/kg) abolished the spontaneous recurrent seizures. Lorazepam (1.5 mg/kg) decreased motor seizure severity but did not reduce the incidence and duration of corresponding hippocampal discharges, implicating its inhibitory effects on seizure spread. No gross brain lesions were observed in a set of extended hippocampal kindled mice submitted to histological evaluation. All these data suggests that our model could be considered as a novel mouse model of extended hippocampal kindling. Some limitations remain to be considered.

Keywords: antiepileptic drugs; convulsion; electroencephalograph (EEG); epilepsy; hippocampus; kindling; mice; spontaneous seizures.

Figure 1

Experimental outline and diverse assessments of the implanted hippocampal electrodes. (A) Schematic diagram of the experiments. See text for details. (B) Representative hippocampal EEG traces collected from a mouse during baseline monitoring and following extended hippocampal kindling. Arrows show interictal spikes. (C) Frequencies of theta rhythms and irregular activities from mice in the control and the extended hippocampal kindled groups. (D) Verification of deep electrode site. Image obtained from a mouse following extended hippocampal kindling. Track denoted by a filled arrow. (E) Schematic illustrations correspond to coronal brain sections between 2.6 and 2.9 mm posterior to bregma. The solid squares indicate the correct localization of the electrode tips (blue represents 4 control animals and red represents 5 extended hippocampal kindled animals).

Figure 2

Stable SRS observed after termination of kindling stimulation. (A) The first EEG trace corresponds to a mouse on Day 1 after termination of extended hippocampal kindling. The second trace shows the EEG of the same animal but 8 weeks later. Filled circles denote movement artifacts. (B–D) SRS incidences, corresponding hippocampal discharge duration, and motor seizure stages measured within 3 days after termination of extended hippocampal kindling stimulation and 8–11 weeks later (n = 10). The values represent the mean ± S.E.M. *p = 0.002.

Figure 3

Effects of the AEDs on SRS and hippocampal interictal spikes. (A–D) Phenytoin (25 mg/kg), lorazepam (1.5 mg/kg) and levetiracetam (100 and 400 mg/kg) were applied via intra-peritoneal injections. SRS were measured 10–12 h post saline or AED injection. Interictal spikes were measured ~1 h post saline or AED injection. Recovered measures were done 24 h post AED injection. The values represent the mean ± S.E.M. *p = 0.002.

Figure 4

Effects of lorazepam on hippocampal EEG discharges. EEG traces collected from a mouse following a saline (A) or a lorazepam (1.5 mg/kg) (B) injection. Corresponding motor seizures presented in Supplementary Videos 3, 4.