Antiepileptic drug-resistant rats differ from drug-responsive rats in hippocampal neurodegeneration and GABAA receptor ligand binding in a model of temporal lobe epilepsy

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Abstract

The disabling seizures associated with mesial temporal lobe epilepsy (TLE) are often resistant to antiepileptic drugs (AEDs). The biological basis of this refractoriness is unknown but may include alterations in AED targets in the epileptogenic brain tissue, reduced AED penetration to the seizure focus, and neuropathological brain alterations such as hippocampal sclerosis typically found in patients with refractory TLE. In the present study, we used a rat model of TLE to examine whether AED responders differ from non-responders in their structural alterations and GABAA receptor characteristics in the hippocampal formation. In this model, spontaneous recurrent seizures develop after a status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala. The frequency of these seizures was recorded by continuous video/EEG monitoring before, during, and after daily treatment with phenobarbital, which was given at maximum tolerated doses for 2 weeks. Based on their individual response to phenobarbital, rats were grouped into responders and non-responders. The severity or duration of the initial brain insult (the status epilepticus) did not differ between responders and non-responders, indicating that the difference between the two subgroups is genetically determined. Subsequent histological examination showed a significant loss of neurons in the CA1, CA3c/CA4, and dentate hilus of non-responders, whereas responders did not differ in this respect from non-epileptic controls. The morphological alterations in the non-responders were associated with striking alterations in autoradiographic imaging of diazepam-sensitive and diazepam-insensitive GABAA receptor binding in the dentate gyrus with a significant shift to enhanced diazepam-insensitive binding. The present data indicate that neurodegeneration and associated GABAA receptor changes in the dentate gyrus are critically involved in the mechanisms underlying refractoriness of seizures in TLE.

Introduction

Mesial temporal lobe epilepsy (TLE) is the most common form of human epilepsy, characterized by recurrent complex partial seizures which are typically resistant to antiepileptic drugs (AEDs)(Engel, 2001). The mechanisms underlying this drug refractoriness of TLE are only poorly understood but may include the etiology of the syndrome, disease progression, alterations in AED targets in the epileptogenic brain tissue, reduced AED penetration to the seizure focus, and structural brain alterations (Regesta and Tanganelli, 1999, Kwan and Brodie, 2002, Löscher, 2002a). Hippocampal sclerosis is the most frequent pathological finding underlying the epileptogenic zone in patients with TLE undergoing temporal lobe surgery for medically refractory seizures (Cascino, 1995). Hippocampal sclerosis is characterized by marked (typically >50%) neuronal loss in the CA1 and CA3 fields of the hippocampus and the hilus of the dentate gyrus with relative preservation of neurons in the hippocampal CA2 field and granule cells of the dentate gyrus (Engel, 1996, Fisher et al., 1998). Accompanying this characteristic pattern of neuronal death in the hippocampal formation are circuit rearrangements, the most widely studied being the sprouting of dentate granule cell axons back onto the inner molecular layer of the dentate gyrus, termed mossy fiber sprouting (Nadler, 2003). Furthermore, cell loss in the hilus and elsewhere may be associated with persistent changes in the composition and function of receptors (e.g., Brooks-Kayal et al., 1998) or ion channels (e.g., Bender et al., 2003, Ellerkmann et al., 2003) in the hippocampal formation.

There is an old and still ongoing debate on whether these complex changes in the hippocampal formation are the cause or consequence of TLE (Fisher et al., 1998, Masukawa et al., 1999). Another clinically important question is whether and how hippocampal sclerosis is involved in the medical refractoriness of TLE (Theodore, 1992, Regesta and Tanganelli, 1999, Kwan and Brodie, 2002, Schmidt and Löscher, 2005). The fact that surgical resection of the epileptogenic hippocampus can turn a drug-resistant patient with TLE into a drug-sensitive patient is a strong argument for assuming that the structural and functional changes in the epileptogenic hippocampus alter the response to AEDs (Engel, 2001, Schmidt and Löscher, 2005). However, which changes in the hippocampus are important in this respect? This question is difficult to address in patients with TLE because of the lack of adequate controls.

In the present study, we examined which morphological changes in the hippocampal formation are associated with drug refractoriness of seizures in a rat model of TLE. Furthermore, we used autoradiographic imaging to characterize changes of GABAA receptors in drug-resistant rats. In the rat model used for these experiments, spontaneous recurrent seizures (SRS) develop after a latency period following a status epilepticus (SE) which is induced by prolonged electrical stimulation of the basolateral amygdala (BLA; Brandt et al., 2003). We have recently shown that daily administration of phenobarbital at maximum tolerable doses in epileptic rats of this model results in two subgroups, i.e., a responder subgroup with control of seizures and a non-responder subgroup without any significant reduction in seizure frequency (Brandt et al., 2004). We suggested that epileptic rats with such AED resistance offer unique approaches to the biological basis of refractoriness, particularly because pathological alterations in such rats can be directly compared with those of rats that respond to AEDs (Brandt et al., 2004). To our knowledge, the present study is the first to examine whether AED responders and non-responders differ in their structural and GABAA receptor alterations in the hippocampal formation.

Section snippets

Materials and methods

For the present study, we used brain sections from the same groups of epileptic rats that were described previously in terms of phenobarbital selection (Brandt et al., 2004) and expression of the multidrug transporter P-glycoprotein in the blood–brain barrier (BBB; Volk and Löscher, 2005). Therefore, the preparation and phenobarbital selection of these rats will only shortly be reported here except the EEG evaluation of the SE in subsequent AED responders and non-responders, which was not

Selection of responders and non-responders

Detailed results of selection with phenobarbital have been described recently (Brandt et al., 2004, Volk and Löscher, 2005), so that responders and non-responders will be only shortly characterized here. All rats received phenobarbital at maximum tolerated doses as indicated by the marked sedation which was observed in all rats during treatment. Analysis of plasma drug concentrations showed that drug concentrations within the therapeutic range (10–40 μg/ml) were maintained in all rats

Differences in neuropathology between responders and non-responders

We have previously reported that several but not all rats with SRS developing after a SE induced by prolonged BLA stimulation exhibited neurodegeneration in the hippocampal formation (Brandt et al., 2003). Even following the same type of generalized convulsive (type 3) SE, the hippocampus was variably affected, with several rats showing no damage despite development of SRS (Brandt et al., 2003). Thus, such neurodegeneration appeared to be not necessary for development of SRS in this model. This

Conclusions

It is important to consider that the different hypotheses of pharmacoresistance discussed above are not exclusive but may occur together in the same tissue or may even be interrelated. This is demonstrated by the present and previous data (Volk and Löscher, 2005) in phenobarbital responders and non-responders, showing that increases in the multidrug transporter P-glycoprotein, cell loss, and alterations in GABAA receptors occur together in the hippocampal formation of non-responders and may act

Acknowledgments

We thank Prof. Heinz Beck (Department of Epileptology, University of Bonn Medical Center; Bonn, Germany) for the helpful discussions during preparation of the manuscript and Dr. Dietmar Benke for the advice with the autoradiography. The technical assistance of Ms. Christiane Bartling is gratefully acknowledged. The study was supported by a grant (Lo 274/9) from the Deutsche Forschungsgemeinschaft (Bonn, Germany), the Studienstiftung des deutschen Volkes (Bonn, Germany), and a grant (1 R21

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