Abstracts

Rationale: Chemoconvulsant-induced status epilepticus (SE) in rodents can lead to the development of temporal lobe epilepsy (TLE). This may be due to sustained microglial activation causing neuro-inflammation resulting in lipid peroxidation, reactive gliosis, hippocampal neurodegeneration, hyperexcitability, reorganization of neural circuits, and hyper-synchronicity. In our recent studies, we found Fyn, a non-receptor tyrosine kinase, upregulation in microglia in the mouse kainate (KA) model of TLE. Fyn KO mice or the WT mice pre-treated with Src family kinase inhibitor, saracatinib, were resistant/less responsive to KA-induced seizures confirming the role of Fyn/Src kinase in seizures. Therefore, we hypothesized that modulating Fyn/Src kinase after induction of SE can prevent/modify epileptogenesis. We tested the hypothesis in the rat KA model, implanted with telemetry device to observe seizures, and performed brain histology and multiplex assays. Methods: Adult male Sprague Dawley rats were implanted with a telemetry device 10 days prior to the induction of SE with KA [repeated low dose at 5 mg/kg, i.p., until convulsive seizures (CS) were observed]. The behavioral SE was terminated after 2h with diazepam. Saracatinib (25 mg/kg, oral) or vehicle treatment was initiated 2h post-diazepam (twice daily for the first three days followed by daily for the next four days). The rats were continuously vEEG monitored for four months. All the animals were euthanized with sodium pentobarbital (100 mg/kg, i.p.) at 4 months post-SE. The brain, serum, and cerebrospinal fluid (CSF) were collected and processed for IHC, Western blot and multiplex assay analyses. We quantified spontaneous seizures, epileptiform spikes, gliosis, and neurodegeneration. Results: Four months of continuous video-EEG study revealed that four out of seven saracatinib treated rats did not develop epilepsy, and in two rats the number of spontaneous CS were less than <9 in contrast to >120 seizures in the vehicle treated group. One rat did not respond to the treatment. Overall, there was a significant reduction in epileptiform spike frequency in the saracatinib group in contrast to the vehicle group. There was no significant difference in the initial SE severity between the groups. Immunohistochemistry (IHC) analysis of brain sections revealed a significant increase in gliosis. Intense CD68 and S100ß immunopositive microglia and astrocytes, respectively, were observed in CA1 hippocampus in the vehicle-treated group when compared to saracatinib group. We also observed a significant increase in neurodegeneration (FJB+NeuN positive cells) in the vehicle-treated group, which were reduced in the saracatinib treated rats. The rat cytokine 27-multiplex assay revealed a reduction in the proinflammatory cytokines such as LIX, IL-2, IL-6, IL-12, IL-13, RANTES, IL-1ß, Eotaxin and MIP-1a in serum and hippocampus, and IL-12, IL-1a and MCP-1 in CSF in saracatinib treated rats. Conclusions: Saracatinib treatment suppresses SE-induced neuroinflammation (reactive gliosis, proinflammatory cytokines and chemokines), neurodegeneration, epileptiform spike rate, and spontaneous recurrent seizures. These findings suggest that Fyn/Src kinase is a potential therapeutic target for disease modification in epilepsy. Funding: The work was funded by the startup funds to T. Thippeswamy from Iowa State University, Iowa, USA..