Rationale:
Chemical nerve agent (CNA) exposure at high concentrations can trigger status
epilepticus (SE) which can eventually develop into epilepsy. Following SE, an upregulation of Fyn-PKCδ signaling have been indicated to drive epileptogenesis with an increase in oxidative stress and proinflammatory cytokine production (PMID: 34087381) (PMID: 33363455). Saracatinib (SAR), a Src Tyrosine Kinase Inhibitor has been shown to effectively reduce gliosis, nitrooxidative stress and proinflammatory cytokine production following an organophosphate nerve agent exposure (PMID: 35052568). In this study, we investigated the long-term effects of SAR fed in diet, as a disease modifying strategy in the rat soman model.
Methods:
Sprague Dawley rats (eight weeks) were administered with Soman (132µg/kg, i.m.) or vehicle immediately followed by atropine sulfate (2mg/kg, i.m.) and HI-6 (125mg/kg, i.m.) to control the peripheral effects of AChE inhibition. Following soman injection, behavioral SE severity was scored on a modified Racine for 60 minutes. Midazolam (MDZ, 3 mg/kg, i.m.) was administered one hour post-soman to limit mortality. Animals were then randomly allotted to experimental groups with equal number of male and females (n=12-13/sex) with matched SE severity, and treated with vehicle or SAR (5-20 mg/kg; oral gavage or in-diet). Neurobehavioral tests and brain MRI scans were performed eight to ten weeks post soman. Telemetry devices were implanted and continuous video-EEG was acquired for two months. Animals were either perfused with 4% paraformaldehyde or snap frozen the fresh tissue. Brains were processed for immunohistochemistry (neurodegeneration and gliosis), and the serum was used for nitrooxidative and cytokine assays. Statistical differences were calculated using unpaired t-test, one-way or two-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test, or a non-parametric Mann Whitney or Kruskal Wallis post-hoc test. Data was presented as mean ± SEM.
Results:
1. SAR significantly reduced the number of microglia, astroglia, and FJB positive neuron in soman exposed animals in different regions of the brain.
2. Exposure to soman, induced a significant cognitive and motor deficits, reduced fear in open spaces and altered the sense of olfaction. SAR significantly restored a normal response to open spaces and motor functions.
3. SAR significantly reduced nitrooxidative markers (nitrite, ROS, GSH:GSSG) and proinflammatory cytokines/chemokine in serum (MCP1, TNFα, IL-17A, IL-6, IL-18, and IL-1α).
4. Upon MRI analysis, SAR improved the functional hippocampal connectivity and structural integrity in various region of the brain following soman exposure.
5. SAR reduced average epileptiform spikes per minute in soman exposed animals.
Conclusions:
A tapering dosing regimen of SAR (20-5 mg/kg) for long-term (~ 3 months) significantly reduced gliosis, nitrooxidative stress, pro-inflammatory cytokines and restored key neurobehavioral responses. SAR treatment also restored functional and structural MRI changes in the brain.
Funding:
Funding from the NIH (U01NS117284, CounterACT program). SAR was supplied by the AstraZeneca.