Abstracts

Rationale: It is known that the epileptogenic zone in neocortical epilepsy has the highest interictal spike frequency. There appears to be a correlation between interictal spike frequency and the activation (phosphorylation) of CREB in layers 2 and 3 of the human neocortex. Similar studies have not been conducted in patients with lesional epilepsy due to brain tumors. Methods: Long-term extraoperative intracranial EEG was recorded utilizing subdural grids and/or hippocampal depth electrodes, placed over or through the tumor and surrounding cortex, as dictated by neuroimaging, scalp EEG and seizure semiology. Nine consecutive patients who underwent 2-stage surgery for resection of tumor and epileptogenic zone were studied. Two to four 10-minute interictal awake EEG samples taken ≥ 6 hours from the last clinical seizure were analyzed using automatic spike detection software (Stellate Systems) and reviewed by a single observer. Artifacts identified as spikes were discarded whereas spikes missed by the software were included. These corrected files were used to calculate the average spike frequency for each electrode. The surgical approach was tailored to remove the tumor as well as the epileptogenic zones (seizure onset and early spread). Cortical tissue blocks (1 x 1 cm) corresponding to identifiable electrodes with varied spike frequencies were sampled; part was flash-frozen; the remainder was fixed in 4% paraformaldehyde, impregnated with sucrose and frozen in OCT for sectioning. Frozen sections from each sample were examined by immunofluorescence. Antibodies used were anti-NeuN, anti-CREB, anti-phosphoCREB. Secondary antibodies were Alexa Fluor 488 and 647 conjugates; DAPI was used to stain nuclei. Sections were examined using epifluorescence microscopy and images captured with a monochrome digital camera. Color composite images were generated and analyzed for the presence and localization of neurons containing phosphorylated CREB. Results: Neuronal CREB phosphorylation was identified in all 9 patients, in those samples corresponding to the highest interictal spike frequencies. In contrast, cortex corresponding to low or absent spiking showed no CREB activation. In some cases clear boundaries were identifiable between phospho-CREB and non-activated CREB along the cortex examined on a single section. Conclusions: The activation of CREB within neuronal populations of the cortex appears to correlate with high interictal spike frequency. These novel findings may help to elucidate further the mechanisms underlying intractable neocortical seizures in patients with lesional epilepsy.