Abstracts

Rationale: Cytokines and nervous tissue inflammation has been shown to play a role in epileptogenesis. In this work the concentration of Interleukin 1-beta (IL-1?), C-C motif ligand 2 chemokine (CCL2), and Tumor Necrosis Factor-alpha (TNF-?) were measured in rats piriform cortex, hippocampus, neocortex, and cerebellum following status epilepticus (SE) induced by pilocarpine injection. Methods: Adult Sprague Dawley rats were treated with methylscopolamine (2 mg/kg) 30 min before pilocarpine i.p. injection (320 mg/kg). Ninety minutes after SE onset rats received diazepam (10 mg/kg). Control rats were injected with saline instead of pilocarpine. Animals were deeply anesthetized and decapitated 2h, 6h, 24h or five days (n=12 per timepoint) after diazepam treatment. The brain was quickly removed and the piriform cortex, hippocampus, neocortex and cerebellum were dissected and immediately frozen in liquid nitrogen. Cytokines tissue levels were assayed in tissue homogenates against a standard curve provided by the Milliplex MAP kit (Millipore). The fluorescence intensity was measured on the Luminex machine (Millipore) and the analyte concentration was determined using the BioRad software (BioPlex Manager). The assay was run in triplicate to confirm the results. The tissue concentration of the cytokines were normalized to the total protein concentration and presented as pg/?g of total protein SEM. Results: An example of the measured CCL2 concentrations in the piriform cortex, hippocampus, neocortes, and cerebellum can be observed in Figure 1. A large increase was observed in all time points. An increase of 3-4 times the basal levels (p<0.001) was already observed 2h after SE and this increase peaked 24h after SE with a ~100 fold increase in the hippocampus and ~200 fold increase in the piriform cortex (p<0.001 in both cases). A similar profile was observed in the neocortex but no significant alterations were observed in the cerebellum. Smaller increases were observed in IL-1? levels and no significant alterations of TNF-?. Conclusions: The large increase of CCL2 concentration on limbic and cortical structures involved with the later progression of spontaneous seizures and the absence of such alteration in the cerebellum indicate an important role for this chemokine in epileptogenesis. This chemokine can also be employed as a biological marker for inflammation of the nervous tissue.