Abstract
Astrocyte and microglial activation occurs following seizures and plays a role in epileptogenesis. However, the precise temporal and spatial response to seizures has not been fully examined. The pilocarpine model of temporal lobe epilepsy was selected to examine glial changes following seizures because morphological changes in the hippocampus closely mimic the human condition. Astrocytic and microglial changes in the hippocampus were examined during the first 5 days after pilocarpine-induced seizures in rats by analyzing GFAP, Iba1 and S100B-immunolabeling in CA1, CA3, and the hilus. Also, 3-dimensional reconstructions of microglial cells from the hilus and granule cell layer were analyzed. Lastly, astrocyte hypertrophy was examined in the hilus using electron microscopy. At 1 day after seizures and continuing throughout the 5 days examined, hypertrophied Iba1-labeled microglial cells and glial fibrillary acidic protein (GFAP)-labeled astrocytes were observed. At 1 and 2 days after seizures, significantly greater Iba1 immunolabeling was observed in CA1, CA3, and the hilus. In addition, both the area of Iba1 labeled processes and the number of their endings were increased in the hilus beginning at 1 day after seizures. S100B-immunolabeling was significantly elevated in CA3 at 1 day, in CA3 and CA1 at 2 days, and in all three hippocampal regions at 3 days after seizures. Electron microscopy confirmed astrocytic hypertrophy and demonstrated astrocytic cell bodies in the location where glial endfeet normally appear on capillaries. The differential response patterns of astrocytes and microglial cells following pilocarpine-induced seizures may signify their detrimental role in neuroinflammation after seizures.
Highlights
The Iba1 cells at the border between the subgranular zone and the base of the granule cell layer (GCL) were the most intensely immunolabeled and displayed bushy/ramified branching patterns. This population of Iba1-labeled microglial cells was not observed in the control rats
At the 1 day time point, there were relatively fewer glial fibrillary acidic protein (GFAP)-labeled cells compared to the control rats
GFAP-labeled astrocytes had a hypertrophied appearance with thickened processes
Summary
Animals Adult male Sprague-Dawley rats (N = 24) were used for this study. All protocols and experiments were carried out in accordance with the Institutional Animal Care and Use Committee at the University of California, Irvine. Pilocarpine (320–340 mg/kg, i.p.) was used to induce seizures as previously described (Shapiro et al, 2007a). At the appropriate time points after seizures, the rats were deeply anesthetized with euthasol and transcardial perfusions were carried out using 250 ml of 0.9% sterile saline, followed by 4.0% paraformaldehyde. The brains were allowed to postfix in the skull for 24 h, after which they were dissected out and placed in 4.0% paraformaldehyde for 24 h. The brains were transferred to phosphatebuffered saline (PBS) and 50 μm sections were cut using a vibratome
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