Abstracts

Rationale: We have recently demonstrated the disease modifying effects of a highly selective and potent inhibitor of inducible nitric oxide synthase (iNOS), 1400W, from a continuous video-EEG study for six months, in the rat kainate model of temporal lobe epilepsy (TLE) (1). The 1400W treatment (20 mg/kg, i.p., twice daily for first 3 days post-SE) after the induction of SE by kainate caused a significant reduction in: epileptiform spike frequency, blood-brain-barrier (BBB) damage, extracellular albumin, reactive astrogliosis and microgliosis, and neurodegeneration at 7d post-SE, and mossy fiber sprouting and ultimately spontaneous recurrent seizures (SRS) in the long-term. Based on these observations, we hypothesized that nitro-oxidative stress and pro-inflammatory cytokines and chemokines levels that are known to be upregulated during the post-SE period are modulated by the 1400W treatment. Methods: We tested the hypothesis in the Sprague Dawley male rat kainate model using the same protocol described in our recent publication (Neurobiology of Disease, 2016, 93:184-200), and the animals were euthanized at 7d post-SE, i.e., 4 days after the 1400W treatment regimen. The SE was induced by repeated low dosing method to achieve a relative consistency in seizure severity between the animals and also to reduce mortality. Two hours after the established SE, the behavioral SE was terminated with diazepam (10 mg/kg, i.m.). Further two hours later, they were treated with 1400W (20 mg/kg, i.p., twice daily for three days) or the vehicle (distilled water). The animals were euthanized at 7d post-SE with pentobarbitone (100 mg/kg, I. p). We collected the serum, cerebrospinal fluid (CSF), hippocampal tissue lysates, and brain sections for various analyses. We performed multiplex assay for cytokines and chemokines, and WB for other proteins analysis. We processed the brain sections for immunohistochemistry using the standard method previously published from our laboratory (2). Results: We demonstrate that the 1400W, when compared with the vehicle treated group at 7 day post-SE, significantly reduced: i) the nitro-oxidative stress markers such as the membrane associated NADPH oxidase-2 (NOX2) subunit, gp91phox; the lipid peroxidation marker, 4-hydroxyanonenal (4-HNE); and the 3-nitrotyrosine (3-NT) in both tissue lysates (by Western blotting) and the brain sections [immunohistochemistry (IHC)] (Fig.1) , ii) proinflammatory markers such as IL-1 (not in serum), IL-2, and IL-6 in the CSF, serum, and tissue lysates by multiplex assay (Fig.1), and iii) the M1 type reactive microglia by IHC and morphometric analysis (Figs. 1 and 2). Furthermore, the 1400W, in contrast to the vehicle, significantly increased: i) the anti-inflammatory cytokines/hormones such as IL-4 and leptin (Fig. 1); ii) the HSP70 content in the hippocampus marginally (WB); and iii) the M2 type alternative state microglia cells as evident from increased arginase-1 IHC, and interestingly the arginase-1 immunostaining was also found to increase in neurons of the CA region and the hilus, the regions vulnerable to neurodegeneration at 7d post-SE. Conclusions: Overall, these observations suggest that the 1400W's disease modifying properties (1) during epileptogenesis are mediated by selective modulation of anti- and pro- convulsive and neuroprotective molecules in the epileptic foci of the brain, which had the long-term disease modifying effect in the rat model of temporal lobe epilepsy (1). Funding: This work was supported by the start-up funds, to T. Thippeswamy, from Iowa State University, Iowa. USA. References: 1). Neurobiology of Disease, 2016, May 18; 93:184-200. 2). Front Biosci (Elite Ed), 2016, Jun 1;8:390-411.