Abstracts

Rationale: Focal cortical dysplasia (FCD) has been associated with mesial temporal lobe epilepsy. Even when extrinsic to the hippocampus (Hp), FCD seems to render the Hp vulnerable to epileptogenesis. However, it is unclear how FCD can remotely facilitate seizure development. Recently, Iyer et al. reported pro-inflammatory cytokine activation, such as interleukin-1beta (IL-1β), along with peripheral immune cell infiltration in FCD type II lesions resected from epileptic patients (Epilepsia. 2010; 51:1763-73). Therefore, we hypothesized that leukocyte extravasation corollary to FCD could lead to an immune response spanning adjacent non-dysplastic regions beyond the cortex (Cx), namely the Hp, hence altering Hp network excitability. We investigated potential pathogenic roles of FCD-induced inflammation via in vivo and in vitro experimental models. Methods: We induced focal cortical freeze lesions in Sprague Dawley rats at postnatal day 1 (P1) as previously described by our group. We also devised an in vitro co-culture model to recapitulate peripheral blood mononuclear cells (PBMCs) migration into dysplastic tissue: PBMCs extracted from P9/P10 Sprague Dawley rat spleen were primed with lipopolysaccharide (LPS) (1µg/ml) for 3 hours to induce pro-IL-1β synthesis, followed by overnight incubation with Nigericin (NIG) (10µM) to trigger IL-1β cleavage and release. Thereafter, immunogens were washed out and PBMCs were seeded on top of the Cx of 350µm organotypic brain slice cultures (OBSCs), which contain Cx and Hp tissue derived from the same rat (2.5-3.5×104 PBMCs/slice) (Figure 1). After 24 hours, we performed whole-cell patch-clamp recordings on pyramidal neurons in Cx layer II/III and Hp CA1. We analyzed inflammation markers in OBSCs as well as in brain tissue harvested from lesion and sham control rats at P3 using immunohistochemistry. Results: At P3, we detected co-labeling of IL-1β converting enzyme, Caspase-1, with CD3-positive T-cells in the paralesional zone of lesion rats (n=3) but not in sham controls (n=3). In the co-culture paradigm, recordings in current clamp revealed enhanced excitability in the Cx of OBSCs cultured with activated PBMCs (LPS+NIG+PBMCs-OBSCs; n=9), with Cx neurons manifesting significantly lower current injections needed for the cell to fire an action potential, i.e. rheobase, versus Cx neurons in naive OBSCs (n=11; p < 0.05), whereas the rheobase of Cx neurons of OBSCs cultured with LPS-primed PBMCs (LPS+PBMCs-OBSCs; n=7) seemed comparable to naive (p=0.81; One-way ANOVA). Likewise, LPS+NIG+PBMCs-OBSCs was the only group where the cytokine precursor NLRP3 was aberrantly elevated in the Cx (n=3). Strikingly, the rheobase was notably reduced in Hp neurons of both LPS+NIG+PBMCs-OBSCs (p < 0.01; n=19) and LPS+PBMCs-OBSCs (p < 0.05; n=17) as opposed to naive Hp neurons (n=24; One-way ANOVA). Furthermore, NLRP3 expression was ectopic in the Hp of LPS+NIG+PBMCs-OBSCs (n=3) and LPS+PBMCs-OBSCs (n=4) relative to naive OBSCs (n=5), thus suggesting that PBMC priming is sufficient to elicit inflammation in the Hp, which consequently results in neuronal hyperexcitability. Conclusions: Our results indicate that the lesion prompts T-cells to infiltrate the Cx. Yet, Hp neurons appear to exhibit higher susceptibility to peripheral inflammation compared to their counterparts in the Cx. Currently, we plan to target IL-1β maturation using Caspase-1 inhibitors, like VX-765, to rescue the physiological outcomes of lesion-induced inflammation. Funding: Canadian Institutes of Health Research (CIHR) grant MOP-123538