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Introduction: Epilepsy was one of the first neurological disorders to be described, mentioned in Babylon circa 3,000 years ago. It is characterized by the occurrence of spontaneous, non-evoked seizure activities, and is widely distributed in the human population, affecting people of all ages, gender and social groups, with an incidence of about 1-3%. In 20% of the cases, seizures remain even with treatment, a condition known as intractable epilepsy. Temporal lobe epilepsy (TLE) is one of the most common forms of intractable epilepsy, which causes a specific kind of hippocampal damage, referred to as hippocampal sclerosis. Both gender and age influence the development, susceptibility and expression of epileptic seizures. Epidemiologic studies suggest that the youngest age groups have the highest age–specific incidence of epilepsy, and seizures develop in the immature brains more readily than in mature brain. Hormones, in particular sex-steroid hormones, influence the probability of seizure occurrence.
Motivation: Females are known to develop epilepsy or unprovoked seizures less frequently than males. In addition, female children experience a lower incidence of epilepsy or unprovoked seizures than do male children. However, in women, hormonal changes occurring in puberty and during the menstrual cycle, pregnancy or menopause may influence the frequency of occurrence of seizures. Several recent reports have evidenced a role for neuroactive steroid hormones in the occurrence of epileptic seizures. One of the most vulnerable brain regions is the hippocampal formation. Similar to other limbic structures, the hippocampus displays low thresholds for induction seizures and higher concentrations of glutamate and GABA receptors. A dense distribution of sex-steroid hormone receptors has also been described within this specific brain region. Neuroactive steroid hormones, such as progesterone and estrogen, have been shown to influence the occurrence and susceptibility to epilepsy. Progesterone exerts anticonvulsant effects in acute and chronic epilepsy models. In contrast, estrogen displays pro-convulsant effects, manifested as an exacerbation of seizures in women with epilepsy. Estrogen potentiates glutamate signaling, blocks GABA-mediated neurotransmission, and predominantly acts on neurons within the limbic system. Furthermore, several studies reported gender differences in the susceptibility to TLE, due to high levels of testosterone. In view of the multiple influences of sex hormones on brain activity, it would be very interesting to investigate whether such hormones play a role in the development of TLE, and further refine the effects of gender and age on susceptibility and vulnerability to epileptic events.
Development of a Marmoset Model of TLE: Historically the rodent has been extensively used in experimental models of TLE. However, because of the major interest of immediate applicability of findings to humans, the use of non-human primate models constitutes an important step towards translational studies devoted to improving the outcome of clinical treatment of diseases affecting the human brain. The marmoset (Callithrix jacchus) is a small New World monkey that is increasingly being used in the laboratory setting. The marmoset has a number of distinct advantages as a laboratory animal. It is a primate which is the size of a rat (300 – 500 g when adult) – but with a bigger brain – and has both practical and husbandry advantages over the larger but more commonly used primate species, the macaque. It breeds easily and frequently, and thus is widely available. NINDS presently has a large colony of marmosets, and another one is present in the NICHD facility in Poolesville. The marmoset is increasingly used as a model for human brain diseases, such as multiple sclerosis (MS) and Parkinson’s. In this project, we propose the development of a marmoset model of TLE, which we expect will be quite relevant and useful in advancing our current understanding of the etiology of TLE. To fully characterize the model, a comparative study will be performed to investigate the effects of gender, age, and sex hormones on the development, susceptibility and vulnerability of epileptic seizures in the marmoset. At later stages of the project we will investigate the role of sex hormones in altering neurogenesis and in modulation of the hippocampal network.
Experimental procedure: The pilocarpine model of temporal lobe epilepsy will be used. Marmosets will be implanted with a sensor for telemetry recordings of cortical EEG, temperature and general activity, and divided into 6 groups: infant females (GIF) and males (GIM); puberty females (GPF) and males (GPM); and adult (GAF) females and males (GAM) marmosets. These animals will be subjected to BRDU administration (4 times/day for four days) before pilocarpine injection. On the day of seizure induction, animals are initially injected with a solution of scopolamine methyl bromide (1 mg/kg, i.p.) 30 minutes prior to pilocarpine hydrochloride (Pilo- 250 mg/kg i.p.) in order to prevent the peripheral effects of the latter. Five minutes after the status epilepticus (SE) induction, the animals will be administered with diazepam (1.25 mg/kg, i.p.) for termination of the epileptic condition, and to allow the animals to survive for the assessment of neuropathology and behavior. The evaluation of the disease will be made by multimodal recording techniques. Electroencephalogram (EEG) recordings will be combined with functional MRI (fMRI) techniques to measure changes in brain hemodynamics elicited by SE. Our lab has extensive experience in the application of both techniques to study brain physiology in small animals. Combined recordings will be made in the hours immediately before SE (baseline period), during SE, and several months after SE induction (once per month for approximately 12-24 months) in order to evaluate the progress of the disease. Correlations between increases in EEG activity and functional markers of neuroactivity will be computed and analysed. At the end of two years the animals will be sacrificed and evaluated with markers for glial reaction (GFAP), synaptic reorganization (neo-Timm) and neuronal injury (NeuN, Nissl staining), GABA immunohistochemistry and BRDU staining (neurogenesis).
Conclusion: Our laboratory has a strong interest in understanding the cerebrovascular coupling in normal and abnormal brain states, and thus we feel the marmoset model of TLE will constitute a unique experimental platform to understand pathophysiological issues of the cerebrovascular coupling which may be invaluable in the advancement of the diagnosis and treatment of cerebrovascular diseases. In this specific project, we will work in close collaboration with Dr. William Theodore, of the NINDS Clinical Epilepsy Section (CES), who has been conducting research on the evaluation and treatment of uncontrolled epilepsy. Thus we feel this is a project of high feasibility, yet of high potential impact, with strong potential translational implications towards the current knowledge of epilepsy, and in particular, of TLE.
