Abstracts

Rationale: Calbindin-D28k (CB) is an EF-hand calcium binding protein (CaBP) that plays an important role in neuronal excitability and neuroprotection/excitoprotection through its calcium buffering and anti-apoptotic properties. In the hippocampus, CB is widely expressed in granule neurons of the dentate gyrus and a subpopulation of CA1 pyramidal neurons, two regions that are known for their resistance to excitotoxic neuronal damage during epilepsy, but it is absent in CA3. The aim of this study is to examine whether seizures induced by kainic acid (KA) in newborn rats alter CB-immunoreactivity (IR) in granule cells of the dentate gyrus, and whether KA-induced CB changes are mediated by nitric oxide (NO). Methods: Ten-day-old Long Evans rats were divided into 3 groups of 6 animals each: I. Seizure-free control group - II. Seizure group treated with KA (2 mg/kg, intraperitoneally) and III. Group with KA-induced seizures pretreated with 7-nitroindazole (7-NINA, 1 mg/kg, intraperitoneally), a relatively selective neuronal NO synthase inhibitor. All animals had implantation of brain electrodes 24 hours before the experiments. Digital EEG monitoring was continuously recorded. Animals were euthanized 2 hours after KA injection. The cellular distribution of CB-IR was evaluated in the dentate gyrus by immunohistochemistry, using an affinity purified rabbit polyclonal antibody to calbindin-D28k (gift of Dr. Sylvia Christakos, Biochemistry/Mol Biol UMDNJ, Newark). The labeling index (LI) for each case was expressed as the percentage of immunolabeled CB neurons out of the total number of neurons counted in 10 non-overlapping high power/40x fields. Group means were compared using one-way analysis of variance (ANOVA, SPSS). Results: CB-IR was expressed in the dentate gyrus and CA1 sector of the hippocampus. Cellular distribution in all groups was neuronal and exhibited a dual cytoplasmic and nuclear compartmentalization. Increased somato-dendritic and nuclear CB staining was detected in granule cells of the dentate gyrus of KA-treated animals with seizures versus non-seizure controls (Table 1). No morphological evidence of neuronal necrosis (eosinophilic neurons with karyopyknosis/karyorrhexis) was seen.Conclusions: Our study shows a marked increase of CB-IR in the dentate granule cells following acute KA induced seizures, which is -in part- mediated by NO. These findings in neonatal seizures mirror the results previously reported in KA induced seizures in adult rats insofar as there is enhanced CB expression in the dentate gyrus. This early response supports the role of CB in excitoprotection/neuroprotection through nuclear and cytosolic calcium buffering and anti-apoptotic mechanisms. Further studies to elucidate the role of CB and its interaction with NO in epileptogenesis at different maturational stages are warranted.