Abstracts

Rationale: Acquired epilepsy (AE) is characterized by spontaneous recurrent seizures as a result of long-term plasticity changes that occur in surviving neurons following an injury such as status epilepticus (SE). Our lab has focused on exploring the role of Ca2+ dynamics in the development of AE and the long lasting neuronal plasticity changes after brain injury. Studies have shown that a single episode of SE in both in vitro and in vivo models causes essentially permanent alterations in neuronal Ca2+ homeostasis. One major regulator of Ca2+ homeostasis is the calcium binding protein, calbindin D-28k which serves to buffer and transport Ca2+ ions within the neuron. This study was conducted to evaluate the long-term effects of SE on the protein levels of calbindin. Methods: This study employed the well-established rat pilocarpine-induced SE model of AE. Rats were confirmed epileptic by video monitoring and maintained in the animal housing facility. One and two years following the induction of SE, control and epileptic rats were sacrificed and brains were prepared for western blot or immunohistochemistry. Western blot analysis was carried out on whole hippocampal homogenates. Calbindin was detected using enhanced chemiluminescent analysis. Addtitionally, rats were fixed by transcardial perfusion and whole brains cryosectioned for immunohistochemical analysis of calbindin expression using 3-3’ diaminobenzidine.Results: Compared to control rats, the protein levels of calbindin from epileptic whole hippocampi were reduced by over 50% using western analysis. Immunohistochemical analysis of the distribution of calbindin demonstrated a significant decrease within the hippocampus. This decrease was most prevalent in the granule and molecular layers of the dentate gyrus (57.1% and 46.4%, respectively), hilus (59.7%), stratum lucidum of the CA3 (57.1%) and the pyramidal layer of the CA1 (53.8%). These changes were evident in animals for as long as one and two-years after SE. The results indicate that SE and the development of AE results in an essentially permanent decrease in calbindin expression in the hippocampus.Conclusions: Utilizing western blot and immunohistochemical analysis, the results of this study show a dramatic decrease in the hippocampal calbindin levels in epileptic animals at one and two years post SE. Calbindin is a major neuronal Ca2+ binding protein. Thus, the observed decrease in calbindin expression may contribute to the long term alterations in hippocampal neuronal Ca2+ homeostasis that has been observed in this model. Additionally, the decrease in calbindin expression in hippocampal CA1 and CA3 regions correlates with the regions most susceptible to damage following SE. This decrease in calbindin may be a major contributing factor underlying some of the plasticity changes that occur in epileptogenesis. (Supported by U01-NS058213-01, R01-NS052529-01, R01-NS051505-01)