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We have recently demonstrated the role of the Fyn-PKCδ signaling pathway in status epilepticus (SE)-induced neuroinflammation and epileptogenesis in experimental models of temporal lobe epilepsy (TLE). In this study, we show a significant disease-modifying effect and the mechanisms of a Fyn/Src tyrosine kinase inhibitor, saracatinib (SAR, also known as AZD0530), in the rat kainate (KA) model of TLE. SAR treatment for a week, starting the first dose (25 mg/kg, oral) 4 h after the onset of SE, significantly reduced spontaneously recurring seizures and epileptiform spikes during the four months of continuous video-EEG monitoring. Immunohistochemistry of brain sections and Western blot analyses of hippocampal lysates at 8-day (8d) and 4-month post-SE revealed a significant reduction of SE-induced astrogliosis, microgliosis, neurodegeneration, phosphorylated Fyn/Src-419 and PKCδ-tyr311, in SAR-treated group when compared with the vehicle control. We also found the suppression of nitroxidative stress markers such as iNOS, 3-NT, 4-HNE, and gp91phox in the hippocampus, and nitrite and ROS levels in the serum of the SAR-treated group at 8d post-SE. The qRT-PCR (hippocampus) and ELISA (serum) revealed a significant reduction of key proinflammatory cytokines TNFα and IL-1β mRNA in the hippocampus and their protein levels in serum, in addition to IL-6 and IL-12, in the SAR-treated group at 8d in contrast to the vehicle-treated group. These findings suggest that SAR targets some of the key biomarkers of epileptogenesis and modulates neuroinflammatory and nitroxidative pathways that mediate the development of epilepsy. Therefore, SAR can be developed as a potential disease-modifying agent to prevent the development and progression of TLE. [Display omitted] • SAR treatment for a week reduced spike rate and SRS in a 4-month video-EEG study. • SAR mitigated SE-induced pSrc-416 and pPKCδ-Ty311, and reactive gliosis. • SAR suppressed nitroxidative stress mediators- iNOS, 3-NT, 4-HNE, and gp91PHOX. • SAR reduced SE-induced IL1β and TNFα in both hippocampus and serum at 8d post-SE. • SAR prevented SE-induced neuroinflammation and neurodegeneration. [ABSTRACT FROM AUTHOR]

Chemical nerve agents (CNA) are increasingly becoming a threat to both civilians and military personnel. CNA-induced acute effects on the nervous system have been known for some time and the long-term consequences are beginning to emerge. In this study, we used diisopropylfluorophosphate (DFP), a seizurogenic CNA to investigate the long-term impact of its acute exposure on the brain and its mitigation by an inducible nitric oxide synthase (iNOS) inhibitor, 1400W as a neuroprotectant in the rat model. Several experimental studies have demonstrated that DFP-induced seizures and/or status epilepticus (SE) causes permanent brain injury, even after the countermeasure medication (atropine, oxime, and diazepam). In the present study, DFP-induced SE caused a significant increase in iNOS and 3-nitrotyrosine (3-NT) at 24 h, 48 h, 7d, and persisted for a long-term (12 weeks post-exposure), which led to the hypothesis that iNOS is a potential therapeutic target in DFP-induced brain injury. To test the hypothesis, we administered 1400W (20 mg/kg, i.m.) or the vehicle twice daily for the first three days of post-exposure. 1400W significantly reduced DFP-induced iNOS and 3-NT upregulation in the hippocampus and piriform cortex, and the serum nitrite levels at 24 h post-exposure. 1400W also prevented DFP-induced mortality in <24 h. The brain immunohistochemistry (IHC) at 7d post-exposure revealed a significant reduction in gliosis and neurodegeneration (NeuN+ FJB positive cells) in the 1400W-treated group. 1400W, in contrast to the vehicle, caused a significant reduction in the epileptiform spiking and spontaneous recurrent seizures (SRS) during 12 weeks of continuous video-EEG study. IHC of brain sections from the same animals revealed a significant reduction in reactive gliosis (both microgliosis and astrogliosis) and neurodegeneration across various brain regions in the 1400W-treated group when compared to the vehicle-treated group. A multiplex assay from hippocampal lysates at 6 weeks post-exposure showed a significant increase in several key pro-inflammatory cytokines/chemokines such as IL-1α, TNFα, IL-1β, IL-2, IL-6, IL-12, IL-17a, MCP-1, LIX, and Eotaxin, and a growth factor, VEGF in the vehicle-treated animals. 1400W significantly suppressed IL-1α, TNFα, IL-2, IL-12, and MCP-1 levels. It also suppressed DFP-induced serum nitrite levels at 6 weeks post-exposure. In the Morris water maze, the vehicle-treated animals spent significantly less time in the target quadrant in a probe trial at 9d post-exposure compared to their time spent in the same quadrant 11 days previously (i.e., 2 days prior to DFP exposure). Such a difference was not observed in the 1400W and control groups. However, learning and short-term memory were unaffected when tested at 10-16d and 28-34d post-exposure. Accelerated rotarod, horizontal bar test, and the forced swim test revealed no significant changes between groups. Overall, the findings from this study suggest that 1400W may be considered as a potential therapeutic agent as a follow-on therapy for CNA exposure, after controlling the acute symptoms, to prevent mortality and some of the long-term neurotoxicity parameters such as epileptiform spiking, SRS, neurodegeneration, reactive gliosis in some brain regions, and certain key proinflammatory cytokines and chemokine. This study was designed based on the hypothesis that DFP exposure induces hyperexcitability of neurons to cause seizures/SE and activates glia to produce excessive inducible nitric oxide synthase (iNOS/NOS-II) and proinflammatory cytokines and chemokines to cause long lasting brain injury. Therefore, inhibiting iNOS with a highly selective pharmacological inhibitor, 1400W, we demonstrate a significant suppression/reduction in: iNOS, 3-NT, and serum nitrite at 24 h; mortality in <24 h post-exposure; gliosis and neurodegeneration at both 7d and 12 weeks in certain brain regions; key proinflammatory cytokines and chemokine in the hippocampus, and; SRS and epileptiform spikes during 12 weeks of continuous monitoring. Unlabelled Image • 1400W, a selective iNOS inhibitor, suppresses DFP-induced brain iNOS and 3-NT at 24 h. • 1400W prevents DFP-induced mortality in <24 h post-exposure. • 1400W suppresses DFP-induced SRS and epileptiform spiking during 12 weeks. • 1400W suppresses DFP-induced gliosis and neurodegeneration at 7d and 12 weeks. • 1400W reduces DFP-induced key proinflammatory cytokines/chemokine and serum nitrite. [ABSTRACT FROM AUTHOR]