Authors :
Presenting Author: Suraj Sundara Vasanthi, M.V.Sc – Iowa State University
Nyzil Massey, PhD – Postdoctoral Research Associate, Biomedical Sciences, Iowa State University; Christina Meyer, MS – Graduate Research Assistant, Biomedical Sciences, Iowa State University; Thimmasettappa Thippeswamy, PhD – Professor, Biomedical Sciences, Iowa State University
Rationale:
Exposure to organophosphate nerve agents (OPNA) results in severe neurological dysfunctions that leads to status epilepticus (SE) with the development of epilepsy. During epileptogenesis, an upregulation in Fyn-PKCδ pathway with a consequent rise in proinflammatory cytokines, reactive gliosis (neuroinflammation) and neurodegeneration have been observed (PMID: 34087381; PMID: 33515429). Saracatinib (SAR, also known as AZD0530), is a non-receptor Src/Fyn tyrosine kinase inhibitor that has been shown to be effective in modifying epileptogenesis by decreasing proinflammatory cytokines, oxidative stress, reactive gliosis (neuroinflammation) and neurodegeneration (PMID: 34087381; PMID:35052568). In this study, we tested the efficacy of different doses of SAR by incorporating it in the drug in the rat diet and fed for four weeks.Methods:
Sprague Dawley rats (eight weeks old) were either administered with DFP at the dose rate of 4 mg/kg (s.c.) or vehicle immediately followed by 2 mg/kg ATS (i.m.) and 25 mg/kg 2-PAM (i.m.) to reduce the peripheral effects of AChE inhibition. Post DFP injection, the animals were observed and scored for behavioral SE severity using a modified Racine scale for an hour. Midazolam (MDZ, 3 mg/kg, i.m.) was administered after 1h of DFP injection. Animals were randomized and allotted to experimental groups based on behavioral SE severity with equal number of male and females (n=5-6/sex). During first week all the rats were administered vehicle or SAR (10 or 20 mg/kg orally) followed by SAR-in-diet in two tapering dosing regimens (20-10 mg/kg or 10-5 mg/kg for three weeks). At the end of the study all animals were euthanized and perfused with 4% paraformaldehyde. Immunohistochemistry analysis was performed to quantify gliosis and neuroinflammation. Serum samples were processed for nitrooxidative stress markers and cytokine analysis. The data is presented as mean±SEM and appropriate statistical tests were applied.Results:
1. No significant differences in SE severity score were observed between males and females following DFP exposure.
2. SAR significantly mitigated nitrooxidative markers (nitrite, ROS, GSH: GSSG) and proinflammatory cytokines/chemokine in serum (IL-1α, TNFα, IL-6, MCP1, IL-17A and IL-18) in both dosing regimens.
3. SAR reduced the total number of microglial and astroglial cells as well as reactive glial cells, FJB positive neurons compared to DFP+vehicle group in the hippocampus, amygdala, piriform cortex and different thalamic nuclei at both dosage regimens.
Conclusions:
Our results suggest that long-term SAR-in-diet was effective in mitigating the neurotoxic effects of OPNA which we could not achieve with oral dosing for a week in our previous study (PMID: 35052568). Also, SAR-in-diet approach has a translational value and involves less handling-stress to animals.
Funding:
Funded by the NIH/NINDS CounterACT (U01 NS117284-01) and W. E. Lloyd Endowment fund (SG2200008). AstraZeneca supplied SAR under the Open Innovation.