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The relationship between seizures and damage to the hippocampus has been known since the early 1800s. In patients suffering from seizure disorders researchers noted neuronal loss and a hardening of the mesial temporal lobe, the area encompassing the hippocampus (Bouchet and Cazauvieilh, 1825). This hardened, or sclerotic, hippocampus is observed in 50 – 70% of patients and includes sizeable cell loss within areas CA1 and CA3 of the hippocampus (Margerison and Corsellis, 1966). In addition to the dramatic cell loss in those regions, a smaller amount of cell loss has been detected in the dentate gyrus, hilus and presubiculum (Corsellis and Bruton, 1983; Mathern et al., 1997). Within the last two decades, the abnormal growth of mossy fiber axons into the supragranular region of the dentate gyrus, as opposed to area CA3, has also been identified (Tauck and Nadler, 1985; Represa et al., 1987; Sutula et al., 1989; Houser et al., 1990; Babb et al., 1991; Sutula and Dudek, 2007). The aberrant formation of these axons is referred to as ‘mossy fiber sprouting’ and is now considered a hallmark of seizure disorders. In addition, these newly sprouted mossy fibers form functional synapses believed to create a recurrent excitatory network within the hippocampus (Sutula et al., 1988; Cronin et al., 1992; Wuarin and Dudek, 1996).

Several different animal models of epilepsy exist, each producing distinct anatomical alterations. However, the lithium-pilocarpine and pilocarpine models used in the proposed set of experiments remain the most relevant models to the human disease (Curia et al., 2008). The mechanism of SE induction by

pilocarpine is through activation of the cholinergic system, as pilocarpine is a muscarinic agonist and atropine, a muscarinic antagonist, blocks pilocarpine- induced SE (Clifford et al., 1987). However, once induced it is believed that the continuation and maintenance of SE is through a glutamatergic mechanism (Nagao et al., 1996; Smolders et al., 1997). As has been observed during the course of the current experiments a high dose of pilocarpine (390 mg/kg) does not always result in production of SE. To enhance the actions of pilocarpine, lithium chloride can be administered nearly 24 hours prior to pilocarpine treatment. In addition to increasing the effectiveness of pilocarpine, pre- administration of lithium chloride also significantly reduces the amount of pilocarpine needed to induce SE (Honchar et al., 1983; Clifford et al., 1987; Morrisett et al., 1987).

The prolonged SE that follows administration of pilocarpine (with or without administration of lithium chloride) brings with it an increased risk of mortality but it also initiates a severe and widespread cellular loss that persists for up to months following the event and eventually leads to tremendous neurodegeneration within the hippocampus and surrounding limbic regions. (Turski et al., 1983; Turski et al., 1984; Leite et al., 1990; Cavalheiro, 1995). In the present study, by 60 days post induction of SE, animals demonstrated nearly a 20% loss of both CA1 and CA3 pyramidal cells and a nearly 40% loss of hilar interneurons, while dentate granule cells remained essentially unaltered (Figure 1.5).

Figure 1.5. Neuronal cell loss in animals 60d post-SE. A.

Representative images of NeuN labeling from sham (left) and pilo-treated (right) animals 60 days post treatment. B. Optical density measurements from NeuN labeling in four hippocampal regions. 1. CA1: 81.5 ± 4.0% of control, n = 7 – 9 2.

CA3: 83.0 ± 2.9% of control, n = 7 – 9 3. DG: 93.3 ± 2.5% of control, n = 8 – 9 4.

Hilus: 62.7 ± 8.1% of control, n = 6 – 7. **p < 0.01; ***p < 0.001, Scale bar, 200 µM.

In addition to cell specific neuronal loss, the pilocarpine model also produces similar mossy fiber sprouting that is observed in patients with seizure disorders (Figure 1.6) (Turski et al., 1983; Turski et al., 1984; Leite et al., 1990; Cavalheiro, 1995; Sutula et al., 1998; Buckmaster and Dudek, 1999). The pilocarpine model is also associated with increased levels of astrocytes (Figure 1.7) (Garzillo and Mello, 2002; Binder and Steinhauser, 2006), a characteristic not as widely noted in epilepsy patients. Overall the anatomical alterations observed within the hippocampus of both humans and animals demonstrate the dramatic impact that seizures have on the network organization and functionality of the hippocampus.

The pilocarpine model provides an excellent animal model for studying status epilepticus and the development of epilepsy, however there are some disparities that should be acknowledged. One important consideration is the age in which the event occurs. As stated previously, SE is most common in children and precipitating injuries are also more common in childhood (Mathern et al., 2002). However the anatomical and physiological alterations associated with SE are difficult to reproduce in animals younger than about 21 days of age (Curia et al., 2008). Additionally, human epilepsy is often associated with more brief and focal seizures. These patients demonstrate limited, asymmetric brain damage and likely appear healthy in other neurological aspects. However the pilocarpine model of SE is composed of a prolonged generalized status epilepticus event. Animals exhibit more widespread, bilateral brain damage, both cognitive and behavioral alterations and frequent spontaneous seizures (Sloviter, 2005). A final

Figure 1.6. ZnT-3 labeling of mossy fiber sprouting in animals 60 and 200d post-SE. A., C. ZnT-3 labeling in a sham-treated animal at the 200 day time point, demonstrates robust labeling throughout mossy fibers and the hilar region. In animals 60d (D.) and 200d post-SE (B, E.) robust ZnT-3 labeling is observed within the inner molecular of the dentate gyrus (white arrows) indicative of aberrant mossy fiber sprouting. Scale bars, A, B, 200 µM; C – D, 100 µM.

Figure 1.7. Increase in GFAP labeling of astrocytes in animals 60 and

200 days post-SE. A., C. Minimal GFAP labeling in the hippocampus and area

CA3 in a sham-treated animal at the 200 day time point. In animals 60d (D.) and 200d post-SE (B, E.) robust GFAP labeling is apparent within area CA3. GFAP expression was significantly increased in area CA1, CA3 and DG at 5d, 60d and 200d post-SE. Scale bars, A, B, 200 µM; C – D, 100 µM.

consideration is the evidence that the site of spontaneous seizure generation in pilocarpine-treated animals is not the same as epilepsy patients (Mello and Covolan, 1996; Harvey and Sloviter, 2005; Sloviter et al., 2007). Differences in the site of seizure initiation may be due to differential neuronal damage or basic hippocampal circuitry. Despite the differences between the pilocarpine model and human epilepsy, this model is widely used and yields reproducible alterations sufficient for studying the epileptic condition.