Elsevier

Epilepsy Research

Volume 62, Issues 2–3, December 2004, Pages 135-156
Epilepsy Research

Epilepsy induced by extended amygdala-kindling in rats: lack of clear association between development of spontaneous seizures and neuronal damage

https://doi.org/10.1016/j.eplepsyres.2004.08.008Get rights and content

Abstract

Most patients with temporal lobe epilepsy (TLE), the most common type of epilepsy, show pronounced loss of neurons in limbic brain regions, including the hippocampus, amygdala, and parahippocampal regions. Hippocampal damage in patients with TLE is characterized by extensive neuronal loss in the CA3 and CA1 sectors and the hilus of the dentate gyrus. There is a long and ongoing debate on whether this type of hippocampal damage, referred to as hippocampal sclerosis, is the cause or consequence of TLE. Furthermore, hippocampal damage may contribute to the progressive features of TLE. The present study was designed to determine whether development of spontaneous recurrent seizures (SRS) after extended kindling of the amygdala in rats is associated with neuronal damage. The kindling model of TLE was chosen because previous studies have shown that only part of the rats develop SRS after extended kindling, thus allowing to compare the brain pathology of rats that received the same number of amygdala stimulation but did or did not develop SRS. For extended kindling, rats were stimulated twice daily 3–5 days a week for up to about 280 stimulations. During long-term EEG/video monitoring, SRS were observed in 50% of the rats over the period of extended kindling. SRS often started with myoclonic jerks or focal seizures and subsequently progressed into secondarily generalized seizures, so that the development of SRS recapitulated the earlier kindling of elicited seizures. No obvious neurodegeneration was observed in the CA1 and CA3 sectors of the hippocampus, the amygdala, parahippocampal regions or thalamus. A significant bilateral reduction in neuronal density was determined in the dentate hilus after extended kindling, but this reduction in hilar cell density did not significantly differ between rats with and without observed SRS. Determination of the total number of hilar neurons and of hilar volume indicated that the reduced neuronal density in the dentate hilus was due to expansion of hilar area but not to neuronal damage. The data demonstrate that extended kindling does not cause any hippocampal damage resembling hippocampal sclerosis, but that SRS develop in the absence of such damage.

Introduction

Electrical kindling of limbic brain regions such as amygdala or hippocampus is one of the most widely used models of temporal lobe epilepsy (TLE), the most common type of epilepsy in humans (Löscher, 1999, McIntyre et al., 2002). Kindling is the progressive intensification of brain excitability (local, regional or global) upon repeated administration of initially subconvulsive electrical stimuli, which ultimately leads to the establishment of a permanent epileptic focus in the stimulated region (McIntyre et al., 2002). Fully-kindled rats are characterized by induction of complex-partial and secondarily generalized seizures upon brief electrical stimulation. In patients, TLE often develops after brain insults such as head trauma, status epilepticus (SE), complex febrile seizures, stroke, or brain infection, and is characterized by spontaneous recurrent complex-partial and secondarily generalized seizures which are often refractory to antiepileptic drugs (Browne and Holmes, 2001). One frequently observed feature of TLE in the majority of patients is a characteristic type of hippocampal damage, called hippocampal sclerosis, which is characterized by extensive (typically >50%) cell loss in the CA3 and CA1 sectors of the hippocampus and the hilus of the dentate gyrus (Engel, 1996, Fisher et al., 1998). In addition to hippocampal sclerosis, massive neurodegeneration is also seen in other anatomically linked limbic structures of the mesiotemporal lobe, including the amygdala complex and entorhinal region (Pitkänen et al., 1998, Yilmazer-Hanke et al., 2000). There is an old and still ongoing debate on whether mesial temporal sclerosis is the cause or consequence of TLE (Fisher et al., 1998, Masukawa et al., 1999). Furthermore, hippocampal cell loss, particularly in the hilus, is discussed as an important factor for the progressive features of TLE (Gorter et al., 2001, Kotloski et al., 2002, Pitkänen and Sutula, 2002).

In recent years, the usefulness of kindling as a model of TLE has been questioned because rats do not have spontaneous recurrent seizures (SRS) at the time they reach kindling criterion (Pitkänen and Halonen, 1998). Furthermore, in contrast to most patients with symptomatic TLE, if at all there is only mild neuronal damage in the hippocampus or other limbic brain regions of kindled rats, which is thought to be a consequence of the evoked kindled seizures (Pitkänen and Halonen, 1998). However, extended kindling (“over-kindling”) with up to 150 evoked generalized seizures caused a marked (about 50%) decrease in the density of neurons in the hilus and CA1 subfield of the hippocampus in a pattern that was described to resemble hippocampal sclerosis (Cavazos et al., 1994). Furthermore, kindling induced SRS in different species, including cats (Wada et al., 1974, Gotman, 1984, Hiyoshi and Wada, 1992, Hiyoshi et al., 1993), monkeys (Wada et al., 1975, Wada et al., 1978, Wada and Osawa, 1976, Baba et al., 1986), dogs (Wauquier et al., 1979), and rats (Pinel et al., 1975, Pinel and Rovner, 1978a, Pinel and Rovner, 1978b, Michael et al., 1998, Sayin et al., 2003). It was, however, not studied whether neuronal loss is a prerequisite for development of SRS. Indeed, the conditions necessary for the development of SRS in the kindling model are incompletely understood, and little is known about their underlying mechanisms (Michael et al., 1998).

Compared to post-SE models of TLE, in which almost all rats develop SRS and neuronal damage after a chemically or electrically induced SE (Löscher, 1999), making it difficult to determine whether neuronal damage is necessary for development of SRS, extended kindling induces SRS in only part of the animals (Michael et al., 1998, Sayin et al., 2003). This model thus allows to compare neuronal damage in rats which have obtained the same number of electrical stimulations but differ in occurrence of SRS.

This prompted us to study development of SRS and alterations in hippocampal histology in the amygdala-kindling model in rats. Association between occurrence of SRS and morphological alterations was evaluated by comparing rats which did or did not develop SRS after the same number of amygdala stimulations. Age-matched normal rats, unstimulated (“sham-kindled”) rats implanted with electrodes, and conventionally (“fully”) kindled rats without extended kindling served as controls.

Section snippets

Animals

Fifty-two female Wistar rats were purchased at a body weight of 200–220 g (Harlan-Winkelmann Versuchstierzucht, Borchen, Germany). Female Wistar rats were used to allow direct comparisons with previous experiments of our group on the relationship between development of epilepsy and neuronal damage (Brandt et al., 2003a, Brandt et al., 2003b). Furthermore, previous experiments in this strain and gender have shown that the estrous cycle does not affect the seizure susceptibility upon repeated

Development of kindling

In the 31 rats that were kindled via electrical stimulation of the BLA, kindling criterion (the first stage 5 seizure) was reached after 8 ± 0.64 stimulations. No differences in kindling rate or other characteristics of kindling were seen in rats that were used as conventionally kindled rats (after 10 stage 5 seizures) and rats that were used for extended kindling.

Seizure development over extended electrical kindling

Twenty-two of the 31 kindled rats were used for extended kindling with up to a total of about 250 (range 129–269) evoked generalized

Discussion

As reported previously (Pinel et al., 1978a,b; Michael et al., 1998, Sayin et al., 2003), extended electrical kindling of the amygdala in rats resulted in the development of epilepsy with SRS in part of the animals. Since the animals’ EEG and behavior were not continuously monitored, we cannot rule out that spontaneous seizures occurred in the other rats, too. Had these seizures occurred with any frequency, however, they would likely have been detected by the extensive EEG-video monitoring used

Acknowledgements

The study was supported by a grant (project number 0310972) from the German Bundesministerium für Bildung und Forschung (BMBF) and by the Lower Saxony/Israel joint project ZN 1175. We thank Dr. M. Glien for help during the experiments and Ms. C. Bartling for technical assistance.

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    Present address: Abbott, Neuroscience Research, Ludwigshafen, Germany.

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