Regular articleNeuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model
Introduction
The induction of status epilepticus (SE) by pilocarpine or kainate in rodents leads to a series of neuropathological changes and the subsequent appearance of spontaneous recurrent seizures (epilepsy). Many features of the rodent models, such as hippocampal sclerosis and mossy fiber sprouting, resemble human temporal lobe epilepsy. The rat pilocarpine model is commonly used in epilepsy research and has been well characterized (e.g., Cavalheiro et al 1991, Mello et al 1993, Turski et al 1983. In contrast, the mouse pilocarpine model has received much less attention, although the availability of mice harboring mutations or deletions of many genes offers the opportunity to study the role of these genes in neurodegeneration and epileptogenesis. Neurodegeneration and development of spontaneous recurrent seizures (SRS) following pilocarpine injections in albino Swiss mice were first reported in 1984 (Turski et al., 1984) , and a few groups have studied the mouse pilocarpine model since then in different strains, such as albino (Cavalheiro et al., 1996), CD1 (Shibley and Smith, 2002), and C57BL/6 obtained from Harlan (Shibley and Smith, 2002) or from Charles River (Berkeley et al., 2002) . Both rats and mice show hilar and pyramidal cell loss, astrogliosis, and mossy fiber sprouting after pilocarpine-induced SE in the hippocampus. Moreover, severe cell damage was found in other brain regions, such as amygdala and thalamus (Turski et al., 1984).
Although neuronal cell loss, astrogliosis, and mossy fiber sprouting have been described in the mouse and rat pilocarpine model, many other neuropathological events in the latent period during epileptogenesis have not been studied to date. In the present study we focused on the time course of changes in activation of microglial cells, expression of neuropeptide Y (NPY), and axonal degeneration in CF1 and C56BL/6 mice to gain insight into potential mechanisms in the development of epilepsy. We report widespread microglial activation, persistent NPY upregulation in the mossy fiber pathway, and delayed axonal degeneration in the thalamus. We found unexpectedly large differences in pilocarpine-induced mortality in C57BL/6 mice obtained from different suppliers, a factor that may limit the usefulness of these mice for studies of epileptogenesis.
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Animals and treatment
Mice were obtained from Charles River (CF1, C57BL/6(Charles River)) or Jackson Laboratories (C57BL/6(JAX)) and housed under a 12-h light–dark cycle with food and water ad libidum. CF1 mice (6–10 weeks old, 30–42 g), C57BL/6 (JAX) (6–15 weeks old, 17–32 g, or >1 year old and 21–32 g), and C57BL/6 (Charles River) mice (10-weeks old, 21–25 g) were used. To minimize peripheral side effects of pilocarpine, mice were injected with methylscopolamine and terbutaline 15–30 min prior to pilocarpine (2
Behavior during SE and spontaneous seizures
Within a few minutes of pilocarpine injection in CF1 mice, immobility, staring, Straub tail, head bobbing, and occasional clonic seizures occurred, followed after 20–40 min by continuous clonic seizures. If continuous clonic seizures started earlier in CF1 mice, the animal invariably died. SE consisted mainly of continuous stage 3, 3.5, or 4.5 seizures and occasional isolated events of stage 5 and 6 seizures. Typically, seizures became less frequent about 5–6 h after pilocarpine injection. Due
Discussion
Our principal findings with the mouse pilocarpine model are (1) upregulation of NPY in the mossy fiber pathway beginning 1 day after pilocarpine and at 31 days in the supragranular layer, indicating mossy fiber sprouting; (2) widespread microglial activation detected by β2-microglobulin expression, evident 3 days after SE and persisting in some areas as late as 31 days, suggesting delayed neuronal damage; (3) delayed axonal degeneration visualized by APP-positive fibers particularly in thalamic
Acknowledgements
This study was supported by grants from the Emory University research council (K.B.), the Epilepsy Foundation (K.B.), and the NINDS (NS17771). We are grateful to Dr. David Rye for help with identification of thalamic nuclei and Robert Baul for excellent technical assistance.
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