Abstracts

Rationale: Status epilepticus (SE) increases the probability for subsequent seizures. For instance, prolonged febrile seizures are associated with a risk of subsequent temporal lobe epilepsy. Most experimental studies have focused on hippocampal epilepsy induced by SE following systemic drug administration or local injection of convulsants into limbic system structures. The mechanisms, by which focal neocortical SE induces chronic sequelae, including epileptogenesis, are not yet fully understood. In the present study we use immunohistochemistry (ICC) to assess structural changes after a focal episode of SE. We focused on gliosis (GFAP), thrombospondin-2 (TSP2), the calcium channel subunit α2δ-1, and on putative excitatory connectivity assessed by counting density of puncta of PSD95- and VGLUT-1- immunoreactivity (IR) in close apposition. Increases in these markers of epileptiform activity could indicate significant lasting effects of SE in involved neocortical structures. Methods: We induced a single episode of focal SE by epidural application of gabazine (50µM) and 4-AP (50µM) over the somatosensory cortex of anesthetized mice. Saline solution was applied to the same cortical area of control mice. Electrographic SE accompanied by contralateral focal myoclonic activity was recorded with epidural electrocorticogram. After ~1hr, seizures were terminated with intra peritoneal injection of diazepam (5-10 mg/Kg). Ten days later, standard techniques were used to determine GFAP, TSP-2, α2δ-1, PSD95 and VGLUT-1 immunoreactivity. A Zeiss confocal microscope was used to obtain images and counts of puncta in 40x40 µm grids (OD 1 µm, 63x) and areas of IR for GFAP, TSP-2 and α2δ-1 (single images 200 x 200 µm, 20x). Immunoreactive areas were measured as the ratio between immunoreactive pixels and the total number of pixels of every image. Puncta counts and immunoreactive areas were obtained with “Cell Profile” software. Results: Preliminary data from ongoing experiments indicate a trends towards an increase in numbers of PSD95 and VGLUT puncta in close apposition and increases in GFAP-, TSP-2-, and α2δ-1-IR 10 days after SE induction. Conclusions: Our preliminary results suggest that a single prolonged episode of acute SE may be sufficient to induce long-term morphological changes, observable ten days after recovery. The increases in PSD95 and VGLUT puncta in close apposition suggest an increase in putative excitatory synapses. Lasting increases in GFAP, TSPs and α2δ-1 are alterations that occur in other models of focal epileptogenesis and indicate that structural alterations, and likely plastic functional changes, occur in the neocortex involved in generation of SE. Results enable additional experiments to explore the chronic effects of SE on changes in cellular and network excitability and potential epileptogenesis in the post-SE neocortex, and potential treatments that would prevent these detrimental consequences. Funding: Supported by NIH grant R021NS090076 from the NINDS and the Pimley Research Fund.